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Page 1: ... of Genes Encoding Cyanamide Hydratase and Antifungal Proteins Thesis submittedto the Pondicherry University for the Degree of Doctor of Philosophy by S ISAAC KlRUBAKARAN Department of Biotechnology School of Life Sciences Pondicherry University Puducherry 605 014 INDIA MAY m ...

Page 2: ...yanamide hydrahue and antifungal proteins ir I record CISresearch work done by h ccandldulc Mr S Isaac Kirubakaran durlng the perlod of h rrtudy In lhc Dcpalinlent of Blchechnolopy Pondicherry llni err ty Puduchcrry under my aupervlrion atid thnl it has not formed prevlourly the bakik lor the awud of any degree dlplorna asrnciateship or fellowrh p Puducherry Date I I I eq I b Y w L L N Sakthivel P...

Page 3: ...k e n c rriedour by me under the guidance and supervision of Dr N SAKTHIVEI Dcpann cnlof Biotechnology School of Life Sciences Pondicherry Clniversily Puducherry b0 5 014 and this work ha not been whrnitted elsewhere for any other degrce Puducherry Date 11 O C 2 ...

Page 4: ...DEDICATED TO MY BELOVED PARENTS ...

Page 5: ...ful to Dr V Arul Dr S Sudharani Dr N Arurnugarn Dr Lata Shukla and Dr K Prashant for their co oprition I wish to record my slncere thanks to the doctoral committee members Pruf S Jayachandran and Dr N Parthasarathy I express my deep sense of gratitude to Prof S Muthukrishnan Kansas State University Department of Biochemistry Kansa USA for providing me the harley and wheat cDNA clones and cnh and c...

Page 6: ...nk our office staffs Mr Iiul krishnan Mr P Rnn alinguni Mr Rarnarnuorlhy Mr S Kannuyiram hlrs S Valli and Mrs Kalivnnl for their co operation and help My speci lthanks goes to all the non teaching staff menlhcrs of Pondicherry University who helped me throughout the study I gratefully acknowledge Department of Science and Technology 1 ST New Delh for providing me with Junior Research Fellowship La...

Page 7: ...CONTENTS CHAPTER 1 INTRODUCTION 2 REVIEW OF LITERATURE 3 MATERIALS AND METHODS 4 RESULTS 5 DISCUSSION 6 SUMMARY 7 REFERENCES Page No ...

Page 8: ...min complementmy DNA centimeter deoxyrihonucleic acid Dithiothreitol 2 deoxynucleotide S trlphosphate ethylenediaminetetraaceticarid 5 enol pyruvyl shikimate phosphate synthase endoplasmic reticulum N acetyl D glucosamine gerrnin like protein hour hygromycin phosphotransferase indole 3 acetic acid isopropyl P D thiogalactoside jasmonic acid kilo base kilo Dalton kilo hertz kilo volt Luria Bertani ...

Page 9: ...ycin phosphorri n icr non specific lipid rr in lcr protein optical density phosphinothricin N acetyl rr insferase phosphate huffer saline polynierase chain reaction potato dextrose agar pathogenesis related root mean square dev alion reactive oxygen species revolulions per mlnute salicylic acid SA rehponsive element sodium dodecyl sulphate sodium dodecyl sulphate polyacrylamidc gel electrophoresis...

Page 10: ... Uv v v WGA YEB thaumatin like protein unweighted pair gmup of cuithl lcticmean analysis l l l t l i l l t vcrlt volil ncby vc lumc weight hy volume vhc rt gcrm glutiriin yeast extract hrorh microgram microlitre ...

Page 11: ...INTRODUCTION ...

Page 12: ... concern towards the toxicity and biomagnification potential of chemical fungicides in agriculture current practices based on molecular biology techniques which involve the development of transgenic resistance in crop plants merit attention Schickler and Chet 1997 In response to developmental signnls and pathogen attack plants evolve a number of defense responses Bell 1981 and synthesize an array ...

Page 13: ...s producing herbicide resistant weeds Vasil 1994 Arriola and Ellstrand 1996 In comparison to other selectable markers cyanamide hydratose cah gene is being considered as a unique marker because of its ability to convert calcium cyanamide into urea a useful nitrogen compound which can be considered as a fertilizer source to plants Maier Greiner et ul 1991a Sahrawat et al 2003 Recombinant DNA techno...

Page 14: ...ves of the present study have been formulated as follows I Cloning and expression of chitinase and LTP genes in E coli and evaluation of their antimicrobial potential towards phytopathogens 2 Site directed mutagenesis cloning and expression of cuh gene in E coli 3 Construction of gene cassettes with cah marker and antifungal gents encoding chitinase and LTP 4 Transgenic expression of cah marker an...

Page 15: ...REVIEW OF LITERATURE ...

Page 16: ...grams that can accelerate the development of elite varieties with disease resistance and good agronomic characteristics Muthukrishnan er ol 2001 2 2 Pathogenesis related proteins Biological activity and function Plants synthesize a variety of compounds when exposed to biological stress viz fungi bacteria viruses insects and herbivores Collinge and Slusarenko 1987 Linthorst 1991 The inducible defen...

Page 17: ...s were determined The PR 2 proteins has an endo P 1 3 glucanase activity Kauffmann et 01 1987 whereas the PR 3 PR 4 PR 8 and PR I I proteins were shown to have endochitinase activity Legrand el ul 1987 Most fungi contain P 1 3 glucans or chitin in their cell walls Bartnicki Garcia 1968 Thus exposure of fungal pathogens to these hydrolytic enzymes could result into the release of cell wall fragment...

Page 18: ... and Nyman 1996 and some PR 10 proteins have been shown to have an RNAse activity which is important to cleave viral RNA s in antiviral pathway Somssich et ul 1986 Moiseyev et al 1994 The PR protein classificution has been extended to include other inducible proteins namely defensins PR 12 thionins PR 13 and lipid transfer protein PR 14 Van L m n and Van Strien 1999 Germins and germin like protein...

Page 19: ...proteases Van Loon 1999 The structure of a PR 1 family member tomato PRl b was solved by nuclear magnetic resonance NMR and was found to represent a unique molecular architecture The protein contains four a helices and four P strands arranged antiparallel between helices The tight packing of the a helices on both sides of the central P sheet a asandwich structure results in a compact bipartite mol...

Page 20: ...inase Chitinase type Ill Tobacco peroxidase Lignin forming peroxydase Parsley PRI Ribonuclease like Tobacco chitinase Chitinase type I Radish Rs AFP3 Defensin Arabidopsis THI2 1 Thionin Barley LTP4 Lipid transfer protein Barley OxOa germin Oxalate oxidase Barley OxOLP Oxalate oxidase like Tobacco PRp27 Unknown Antoniw er 111 1980 Antoniw rr 111 1980 Van Loon 1982 Van Loon I982 Van Loon 1982 Green ...

Page 21: ...poplast whereas basic tobacco PR I accumulates in the vacuole Bol ef al 1990 This may be valid for one PR family PR I in a host plant such as tobacco but cannot be generalized as a differential localization feature of acidic and basic proteins in plants Presently PR proteins are established in all plant organs such as leaves stems roots and flowers Van Loon 1999 and found particularly abundant in ...

Page 22: ...oel and Portnoy 1998 Pathogen derived elicitors are potent PR protein inducers Well characterized elicitor includes glucan and chitin fragments derived from fungal cell walls fungus secreted glycoproteins peptides and proteins of elicitin family Miinch Garthoff er ul 1997 HonCe er ul 1998 Zhou 1999 Kombrink er ul 2001 Edreva et ul 2002 Protein products of avirulence genes in fungi and bacteria are...

Page 23: ...mation has largely heen recognized Anderson et ul 1998 Surplus et ol 1998 Grant and Loake 2000 Schultheiss et al 2003a Many of these secondary signals such as salicylic acid SA jasmonic acid JA and ethylene are well known inducers of PR protein expression Dumer et 01 1997 Surplus et 01 1998 Anderson et ol 1998 Zhou 1999 Poupard et ol 2003 Cross talks are common between signaling pathways mediated ...

Page 24: ...ngal pathogens The simultaneous expression of tobacco P 1 3 glucanase and chitinase genes in tomato plants results in increased resistance to fungal pathogens Melchers et al 1998 Resistance is locally or systemically induced in plants when PR proteins are accumulated In tobacco specific P 1 3 glucanases from alfalfa barley tobacco and soybean have been shown to suppress fungal diseases Bucher ef a...

Page 25: ...I PR 2 and PR 3 transgenes in potato plants enhanced their resistance against Phytophthora infeslans Bachmann er ul 1998 Similar results about the effectiveness of the co expression of chitinase and P 13 glucanase in plant disease resistance was also reported by Kombrink er al 2001 Brussicu nupus transgenic plants constitutively expressing a chimeric chitinase gene display field tolerance to funga...

Page 26: ...e of family 18 chitinases Class I chitinases are basic and contain a cysteine rich N terminal domain with putative chitin binding properties They are usually localized in the vacuole and are potent growth inhibitors in vitro of many fungi Mauch and Staehelin 1989 Keefe et al 1990 Class 11 chitinases are generally acidic and extracellular and can b e detected in the apoplastic fluid or culture medi...

Page 27: ... class Ib 11 Ill IV 2nd VI are secreted to the apoplast Arie era 2000 Chitinaes EC 3 2 1 14 ye classified as glycosyl hydrolases and catalyze the degradation of chitin an insoluble linear P 1 4 linked polymer of N acetyl D glucosamine GlcNAc Chitin is a major component of the exoskeleton of insects of crustacean shells and of the cell wall of many fungi According to the glycosyl hydrolax classific...

Page 28: ...oted hypothesis that chitinases act as a defense mechanism against pathogens 2 3 1 Structure distribution and mode of action of chitinase Multi domain proteins include several chitinases and chitin binding protein domains The 3D structures of representatives of the major classes of these proteins have been determined by X ray diffraction H a r tet ul 1993 or NMR spectroscopy Andersen et ul 1993 St...

Page 29: ...cidic and basic extracellular proteins with an N terminal hevein domain Araki el 01 1992 Huynh er ul 1992 They have the shortest chain lengths with four internal deletions The primary structure of class IV chitinases is 35 50 identical with those of class I and class I1 ones and more than 60 among themselves The primary structures of class I and class I1 chitinases are 60 65 identical while there ...

Page 30: ...ngal activity against the growth of Trichoderma reesei Alternoria solani and Fusarium oxysporum Roberts and Selitrennikoff 1986 isolated two antifungal proteins from barley grains Antifungal chitinases isolated from the grains of wheat barley and maize functioned as endochitinases and inhibited hyphal elongation of test fungi Roberts and Selitrennikoff 1988 Plants are known to produce antifungal c...

Page 31: ...al 1998 Number of PR proteins that belong to PR 8 protein Metmux er al 1988 and PR I I protein Melchers er al 1994 have been reported from cucumber and tobacco respcctively Different plant chitinases reported against phytopnthogens are listed in Table 2 ...

Page 32: ...rytis cinereu R solani Punja and Raharjo 1996 Cercospra arachidicolu Rohini and Sankwa Rao 2001 Rice chitinase R solani Lin et al 1995 Datta et ul 2000 2001 Sphuerotheca humuli Asao el al 1997 Diplocarpon rosue Marchant er 01 1998 B cinerea Tabei et al 1998 Takatsu et ul 1999 F graminearum Chen et al 1998 Magnaporthe grisea Nishizawa et al 1999 Uncinula necator Elisinoe ampelina Yamamoto el al 200...

Page 33: ...icle bombardment and studied the inheritance of uansgene in TI plants Chen et al 1998 introduced a rice chitinase gene chill into a wheat cultivar Bobwhite by bombarding immature embryos A rice chitinase cDNA RCC2 driven by CaMV 35s promoter was introduced into cucumber Cucumis sativa L through Agrobacterium mediated transformation Tabei et al 1998 The cucumber plants expressing chitinase exhibite...

Page 34: ...e causative agent of brown patch disense Transgenic rice plants expressing chill chitinase gene exhibited enhanced resistance against the sheath blight pathogen R solani Lin et al 1995 Xu et al 1996 studied the regulation expression and function of a new basic chitinase gene RC24 in transgenic rice Nishizawa er al 1999 introduced rice chitinase genes chit2 and chit3 into japonica rice varieties Ni...

Page 35: ...athogenesis related proteins that have potent antifungal and antibacterial activities Molina et ul 1993 Elicitins and LTP are small cysteine rich lipid binding proteins secreted by oomycetes and plant cells respectively they share some structural and functional properties Elicitins are known elicitors of plant defense and recent research demonstrates that elicitins and LTPs share the same biologic...

Page 36: ... so named because of their ability to stimulate the transfer of broad range of lipids between membranes in vitro Arondel and Kader 1990 Yamada 1992 2 4 1 Structure distribution and mode of action The ns LTPs are a family of plant peptides previously thought to be involved in lipid shuttling between organelles Kader 1996 and have been recently implicated in plant defense Lipid transfer proteins hav...

Page 37: ...trong structural homologies as revealed by sequence alignment and hydrophobic cluster analysis Tchang er al 1988 Desormeaux et al 1992 However it was found that LTPs from mammalians and plants do not show any sequence homology Winz 1991 Plant LTPs differ from oleosins which are hydrophobic proteins associated with oil bodies in seeds Qu and Huang 1990 Tertiary structure of LTPs has been extensivel...

Page 38: ...shed structures of wheat maize barley and rice ns LTPs Structural comparisons of free and bound wheat ns LTP reveal that the binding of prostaglandin B2 hardly affects the global fold of the protein Tassin Moindroter 01 2000 2 4 2 Biological roles and antimicrobial activity of LTP Based on their in vitro properties in lipid transfer and acyl chains binding the LTPs could be involved in several fun...

Page 39: ...TPs in plants The relative activities of different LTPs vary between pathogens suggesting that they have some degree of specificity While certain LTPs were much more active than thionins against the bacterial pathogen Clavibucrer michigunensis sub sp sepedonicus the opposite was true against the fungus Fusurium solani indicating that these families of antimicrobial peptides might complement each o...

Page 40: ...gens P syringue pv tabaci and P syringae pv tomato The defense role of LTPs is supported by the observation that transgenic tobacco and Arabidopsis plants overexpressing barley LTP showed drastic reduction of disease symptoms after inkction of the leaves with the bacterial pathogen P syringue Molina and Garcia Olmedo 1997 Are AMP1 has been successfully engineered into scented geranium Bi et ul 199...

Page 41: ...n Sclerorium roysii Onlon Fusarium sp Puccinia sp Cammue er al 1995 Ace AMP1 Bacillus megatarium Sarcina lutea Botrytis cinerea Bi et a 1999 Sphaerotheca pannosa Li et a 2003 M grisea Rhizocronia soluni Patkar and Chattoo 2006 Xanthomonas oryzae pv oryzae Blumeria graminis f sp tritici Roy Barman et a 2006 Peat1 Millet seeds Trickodema viridae Velazhahan er al 2001 Pennisetumglaucum L R solani Ric...

Page 42: ...e been made and their expressions in transgenic plants have been achieved Grison et al 1996 Shah 1997 Punja 2001 Although several methods such as electroporation D Halluin et al 1992 microinjection Neuhaus et al 1987 or delivery by virus Boevink et al 1996 have also been exploited the most common methods for introduction of DNA into plant cells use Agrobacterium rumrfaciens bacteria or rapidly pro...

Page 43: ...ganisms that could be used as vehicles for DNA lead researchers to the genus Agrobacterium A tumefaciens is capable of infecting almost any plant tissue Because of its uniqueness A tumefaciens has become the primary choice for many plant transformation experiments A tumefaciens is a soil dwelling pathogenic bacterium first described in 1943 Braun 1943 Some strains of Agrobacterium are capable of i...

Page 44: ...gle stranded DNA molecule called T strand Thus T DNA enters the plant as a protein nucleic acid complex composed of a single VirD2 molecule attached to a single stranded T DNA Apart from this several other vir genes and host coded proteins are also involved in the T DNA transfer process Once the T DNA enters the host nucleus T DNA genes encode enzymes involved in the production of plant growth reg...

Page 45: ...te the phenotype of transformed tissues from non transformed tissues Therefore the probability to recover transgenic plants in the presence of a selective agent is greater than in its absence especially because the transformation rate is always low 10 to 10 Moreover only in rare cells the introduced gene is correctly integrated and expressed Approximately fifty marker genes were used for transgeni...

Page 46: ... bacterial genes that confer resistance to the antibiotics kanamycin and hygromycin or to the herbicide glufosinate ammonium As these have been the target of concern among environmental authorities scientists have been encouraged to develop alternative selection strategies ACNFP 1994 although such concerns may prove unfounded Flavell et al 1992 The development of safer systems for environment that...

Page 47: ...Msliga rc 01 1988 Hllle rr ol 1986 Peru rr 01 1989 Gurrlnevu el 01 1990 Iclcnrk a sr 01 20 XJ DeBlnck et ol 19R4 DcRlwkulol 1985 Herbicides Pharph nolhrictn pol bar Phosphinolhncin acetyl Strepromyrrs hy rorr p cusDeBlock rr 01 1989 tranrfcrase Ci phos te EPSPsynrharr 5 Enolpyruvylshikimale 3 Perunu hybndo Zhuu el 01 I995 phosphate synlhase b o mays Howc rtol 2002 oroA S EnolpyruvylrhiUmale 3 h i ...

Page 48: ...b cunr Anlhranilate synlhssc Tohilcco mutat Anthranilate synthdse Rice Thrrnnine dcnmtnase E mli Cpochmme P450 Humnn monooxygenase Unin 1996 Daniel1rr 01 2001 Per1tral I993 K o i c l rlul 1981 Goddljn n 01 1993 Hmen Ertnrlla rr 01 I983 Eichholtr rr ol 1987 lrdan t101 I998 Cho nul 2 W Kobnyarht rr 01 2MlS Ehme rr rtol 2 W lnut PIUI 2W5 hrflura on fluridone pds Phyiwnedcsaturax Hydnila vmiollolo Ana...

Page 49: ...Davies 1973 2 6 1 2 nptII or neo gene The nptll or neo gene was isolated from the transposon Tn5 of E coli and it encodes for the enzyme neomycin phosphotransferase I1 NPT 11 E C 2 7 1 95 also known as aminoglycoside 3 phosphotransferase I1 APH 3 II Bevan et al 1983 Fraley et a 1983 Herrera Estrella e r al 1983 The active aminoglycoside antibiotic inhibits the protein synthesis in prokaryotic cell...

Page 50: ...version of glutamate to glutamine removing the toxic ammonia from the cell This enzyme plays as essential role in the regulation of nitrogen metabolism and ammonia assimilation When the GS is inhibited it results in ammonia accumulation and as associated disruption of chloroplast structure that leads to inhibition of photosynthesis and to death of the plant cell Lindsey 1992 2 6 2 2 aroA or epsps ...

Page 51: ...o seed germination The enzyme has extremely narrow substrate specificity The use of cyanamide hydratase as a selectable marker has been demonstrated in wheat Weeks et al 2000 tobacco Maier Greiner et al 1991b potato tomato rice and Arabidopsis Damm 1998 2 6 3 Marker genes for positive selection Some marker genes for positive selection make possible the identification and selection of genetically m...

Page 52: ...nnose 6 phosphate by endogenous hexokinase Therefore when mannose is added to the culture medium it could minimize the plant growth due to mannose 6 phosphate accumulation Even though most of the plant species are sensitive to mannose some species especially dicotyledonous have shown a considerable insensibility to this sugar including carrot tobacco sweet potato and leguminous crops Other species...

Page 53: ...election was much greater than for the nptll gene and the regeneration of shoots was significantly faster It was suggested that the enzyme from S ruhiginosus posed no biosafety issues as it is used in the food industry and considered safe Haldrup el 01 1998a 2 7 Transgenic expression of tobacco as model plant Tobacco played an important role in the development of plant tissue culture methods and m...

Page 54: ...MATERIALS AND METHODS ...

Page 55: ...riphosphates dNTPs and 1 kb DNA ladders were purchased from Promega Madison WI USA Nitrocellulose membrane used for immunoblotting was purchased from Advantech MFS Inc CA USA The unique site elimination USE mutagenesis kit was purchased from Pharrnacia Biotech Inc Piscataway NJ USA Solvents and reagents were purchased from Sisco Research Laboratories Mumbai India 3 1 2 Microorganisms plasmids and ...

Page 56: ... coli strain Genotype NM 522 mirrS s i p E hi 1 lor proAB zcrB hsflSlSM S rK m K 7 F proAB Iuc lqZ MIS JM 109 e l4 McrA recA1 rndA I gyrA rhi l h s t R 17 r mr slrpE44 relA I A 10c ProAB F rraD36proAB Iacf Z AM 151 BL 2 l DE3 F ompT gal durn Ion h idSn rs mn X DE3 ...

Page 57: ...C8 backbane pCAMBlA 1300 Veclor slze 8958 bp hprll R gene planls Kan R gene bacteria pUC18 polylinker T DNA s i e 2728 bp MSV veclor family Borlchi cDNA clone from a library of Erysiphe graminis infected barley Llp 3FI EDNAclone from a library of Fusorium grarnineurum infecled wheat Antiserum Cah Cyanamide hydralase antiserum Antiberum Bar2chi Barley chitinase antiserum Antisemm LTP Wheal lipid tr...

Page 58: ...a Tea Clover Sugarcane Mango Microbial Culture Collec ion MCC Pondicherry University lndia Microbial Culture Collection MCC Pondicherry Universily lndia Microbial Culture Collection MCC Pondicherry University India Microbial Culture Collection MCC Pondicherry University India Microbial Culture Collection MCC Pondicherry University lndia Microbial Culture Collection MCC Pondicherry University India...

Page 59: ...uclense enzymes All restriction endonucleases and T4 DNA iigase were purchased from Promega Madison WI USA and other molecular biology buffers and reagents were obtained from Bangalore Genei Bangalore India Table 8 List of antibiotics chemicals and their concentrations used in this study Antibioticsf Stock solution Working concentration Chemicals mdml pdml Ampicillin 100 in water Kanamycin 100 in ...

Page 60: ...1 5 agar was added to LB broth before autoclaving Potato dextrose broth Atlas 1993 Potato infusion 200g Glucose Distilled water Potato dextrose agar medium 1 5 agar was added to potato dextrose broth before autoclaving Yeast extract broth YEB medium Van Larebeke et al 1977 Peptone l o g Yeast extract l o g NaCl 5 g pH adjusted to 7 4 with NaOH and the final volume was made to 1 O O O ml using doub...

Page 61: ...04 7H20 8 6 mg 86 0 mg H BOJ 6 2 mg 62 0 mg KI 0 83 mg 8 3 mg Na2Mo04 2H20 0 25 mg 2 5 mg CuS04 5HzO 0 025 mg 0 25 mg CoC12 6HzO 0 025 mg 0 25 mg FeEDTA 40 0 mg 1 0 g L O ml Nicotinic acid 0 5 mg 12 5 rng Pyridoxine 0 5 rng 12 5 rng 10 ml Thiamine HC1 0 1 rng 2 5 mg Glycine 2 0 mg 50 0 mg Myoinositol 100 0 mg Sucrose 30 0 g Added as solid PH 5 8 Adjusted with 0 1 N KOH and 0 1 N HCI ...

Page 62: ...adjusted to 8 0 and the final volume was made to 100 ml using double distilled water Solution 11 NaOH 0 2 M SDS 1 Double distilled water Solution 111 Sodium acetate 3 M Double distilled water pH adjusted to 5 2 with acetic acid RNase A 2 5 mg of RNase A was dissolved in I ml of 10 rnM Tris HCI pH 7 6 and boiled at 70 C for 10 min ...

Page 63: ... was discarded the phenol was again extracted with 0 1 M Tris HCI to get pH 8 0 and then 8 hydroxyquinoline 0 1W was added Phenol Chloroform Distilled phenol 50 ml Chloroform isoarnylalcohol 24 1 50 ml Agarose gel electrophoresis buffers TAE buffer SOX Tris HC1 242 g Glacial acetic acid 57 11111 EDTA pH 8 0 0 5 M 100ml pH adjusted to 7 2 and the final volume was made to 1 0 0 0rnl using double dis...

Page 64: ...so4 0 1g Double distilled water lOm1 Solution C 2 sodium potassium tartarate Sodium potassium tawrate 0 2 g Double distilled water lOml Prior to use 49 ml of solution A was mixed with each 0 5 ml of solution B and C Folin Ciocalteau reagent The commercially available Fohn reagent was diluted with equal volume of water or in the dilution ratio as specified by the manufacturer SD Fine Chern Ltd Murn...

Page 65: ...29 2 g Methyl bisacrylamide 0 8 g Double distilled water 100ml The acrylamide solution was deionized with Amberlire I d l 0 0 ml tiltcrcd through Whatman No 1 paper and stored in brown hottle at 4 C Separating gel 5 mi 10 Double distilled water 1 9ml Acrylamide Bisacrylamide 30 1 7 ml 1 5 M Tris HCl pH 8 8 1 3ml 10 SDS 0 05 ml 10 Ammonium persulphate 0 05 ml TEMED 0 002 ml ...

Page 66: ...0 8 Ammonium persulphate 0 02 m1 TEMED 0 002 ml Gel running buffer or tank buffer 50 X Tris HCI 25 mM 47 g Glycine 250 rnM pH 8 3 94 g SDS 0 1 W V l o g Double distilled water 1OOOml 2X Sample loading buffer Tris HCI pH 6 8 0 125 M 2 5 ml 10 SDS 4 ml Glycerol 2 ml fhnercaptoethanol I ml Bromophenol blue 0 15 15mg Double distilled water IOml ...

Page 67: ...ue was first lissolved in methanol hefore adding acetic ncid and double distilled water Destaining solution I 100 ml Methanol 50 Acetic acid 10 Double distilled water Destaining solution 11 100 ml Methanol 5 5 ml Acetic acid 7 7 ml Double distilled water 88 ml Western blotting buffers Towbin transfer buffer Glycine Tris HCI pH 8 4 Methanol ...

Page 68: ...8 0 20 mM NaCI 1 50 mM Tween 20 0 05 Blocking solution Gelatin or skim milk powder 370 Tris huffered saline l00ml 3 1 7 Oligonucleotide primers Oligonucleotide primers for chitlnase lipid transfer protein LTP cyanamide hydratase genes mutagenic primers and selection primers used in site directed mutagenesis are listed in Table 9 ...

Page 69: ... ATC TAT ATT GG 3 Wheat LTP LTP3F1 F 5 AGG ATC CTT GAT CGA GAT GGC CCG TT 3 LTP3Fl R 5 CAA GCT TGG AGT GGA AGA ACA ACC 3 Cyanamide hydratase CAHX F 5 CGC CCA TGG CTT CAG AAG TCA AAG C 3 CAHX R 5 CGG GAT CCG AGT TAC TCC CAA GGC TTC 3 Target mutagenic primers Lysine 5 GAC TCC CTG GGA AAA CTT GGT GAT 3 Leucine 5 GAC TCC CTG GGA AAG aGGT GAT 3 fiuI site selection primers 5 G7T GGG AAG GG C AAT TGG TGC...

Page 70: ...gal cultures were maintained on PDA slants in sterile tuhes at 4 C 3 2 2 Sterilization All the media buffers reagent and glasswares used in this study were sterilized at 15 lbslinch2 for 20 min unless otherwise specified All heat labile hemicals and antibiotics stock solutions were filter sterilired using 0 22 pm filter Millipore Molsheim France 3 2 3 Molecular techniques Standard molecular biolog...

Page 71: ...The tube was then stored on ice for 5 min To this 150 p1 of ice cold solution 111 5 M potassium acetate glacial acetic acid and water was added and vortexed gently by inverting for a few seconds to disperse solution 111 through the viscous bacterial lysate The tube wa5 stored on ice for 5 min Then it was centrifuged at 12 000 g for 5 min at 4 C The supernatant was transferred lo a fresh tube and a...

Page 72: ...The gel was documented using photo gel dwumentarion system Vilber Lourmat France 3 2 3 3 Agarose gel electrophoresis Agmse gels were prepared by adding required amounl of qarnse in required volume of 0 5 X TAE buffer melted using a microwave oven for 1 2 min It was allowed to cool ethidium bromide solution 10 pdml way added mixed and poured into the gel casting tray fixed with the combs to fonn we...

Page 73: ...bility of the trees were measured by bootstrap analysis with 1500 trials and the phylogenetic trccs were edited using molecular evolutionary genetics analysis and sequence alignment tool MEGA v 3 1 Kumar et 01 2004 3 2 3 3 2 Molecular modeling of chitinase and lipid transfer protein Protein Data Bank code ICNS barley seed chitinase and PDB code lAFH maize ns LTP were used as the templates respecti...

Page 74: ...d using PCR DNA purification resin Promega Madison WI USA The expression vector PET 28a and PCR products were double digested with specific restriction enzymes Ndrl and BumHI ligated and transformed into E coli BL 21 DE3 cornpelent cells The recombinant clones were streaked onto LB agar plate containing kanamycin 50 pg ml and the plate was incubated at 37 C for 16 h A single colony was used to ino...

Page 75: ...he supernatant The pellet containing inclusion bodies after sonication was resuspended in wash buffer I0 mM Tris HCI pH 7 5 300 mM NaCI I mM EDTA 1 Triton X 100 and I M urea Washing was done 5 times at 12 000 g for 10 min at 4 OC follvwed by extensive washing with sterile double distilled water to remove EDTA To renature the protein properly from inclusion bodies the pellet was dissolved in 10X vo...

Page 76: ...model Unimax 1010 1000 Heidolph Germany One ml of overnight culture was used to inoculate 100 ml of LB broth containing ampicillin 100 pglml in a 250 ml culture flask and the culture was grown at 37 T with vigorous agitation When cells reached an optical density O D of 0 6 at 600 nm isopropyl P D thiogalactoside IPTG 0 3 mM was added After 5 h of induction at 37 C cells were harvested by centrifug...

Page 77: ...tant was discarded The precipitate was collected and dissolved in phosphate buffered saline PBS pH 7 2 and dlalyzed against PBS pH 7 2 The dialyzed protein snmple was subjected to gel filtrntion chromatography on a Sephadex G 75 column equilibrated with the same buffer Protein elution was carried out with PBS pH 7 2 with the flow rule of 0 3 mV rnin and the eluate was monitored by measuring the ab...

Page 78: ... Coomassie brilliant blue R 250 dissolved in 1070 acetic acid and 50 methanol in water The gels were destained for 3 h in 5 6 methanol and 7 acetic acid in distilled water and documented using photo gel documentation system Vilber Lourmat France 3 2 3 3 10 Western blotting After SDS PAGE gels were soaked in the tranafer huffer 25 rnM Tris 192 mM glycine 30 methanol pH 8 4 and electmtransferred at ...

Page 79: ... pH 5 4 was incubated at 50 C for 60 min Termination of the reaction was done by adding 2 ml of dinitrosalicylic acid reagent and heating in boiling water for 5 min The reaction wuq then cooled to room temperature and centrifuged ill 6 MK g for 10 min The upernntant was subjected to spectrophotornetric measurement at 530 nm One unit of chitinilse activity was defined as the amount of enzyme that l...

Page 80: ...r LTP served as control The plates were further incubated at 28 C until thc n ycelial growth has enveloped the peripheral well containing the control and had produced crescents of inhibition around the wells loaded with purified protein Antifungal assays were repeated twice to confirm the results 3 2 3 3 14 Microscopic analyses Deformed mycelia from the zone of inhibition were observed under scann...

Page 81: ...taining PET 28a or pMAL p2x without insert chitinase or LTP served as control The treated and control spores were stained with lactophenol cottonblue and examined under a light microscope Novex Holland 400 In order to determine whether purified protein has fungistatic or fungicidal activity 12 h treated and control spores were plated on PDA and incubated at 28 C for 4 days Similarly the germinatio...

Page 82: ...AA CTT GGT GAT 3 and 5 GAC TCC CTG GGA AAG CTC GGT GAT 3 respectively were used In the primers the underlined codons replaced the original AAG and C7T codons for lysine and leucine respectively in the cah gene Designed to introduce a mutation into each of the two PvuI sites at positions 387 and 1283 in the pUC8 the selection primers had the nucleotide sequences 5 GTT GGG AAG GG C AAT TGG TGC GGG C...

Page 83: ...o acids were amplified using gene specific primers CAHX F and CAHX R listed in Table 9 The wild and mutant type cah genes from pCAM pCAM MutK and pCAM Mu11 plasmids encoding cah genes were used as template The conditions for PCR were 94 C I rnin 61 C 1 min 72 C 1 min and 72 C for 10 min for 30 cycles The PCR amplified DNA fragments were purified double digested with NcoI and BumH1 ligated into Nco...

Page 84: ...n was washed with buffer B containing 20 mM imidazole and enzyme was eluted with 300 mM imidazole in buffer B The flow rate was 0 25 ml min and fractions of 0 5 ml were collected in sterile microcentrifuge tubes The fractions were analyzed by SDS PAGE dialyzed using PBS buffer pH 7 2 and concentrated by lyophilization The concentrated protein samples were taken for activity assay 3 2 3 4 4 Cyanami...

Page 85: ...Hindlll and EcoRl fragment Ubil promoter mutated coh and nos terminator of pCAM MutK plasmid was subcloned into plant transformation vector pCAMBIA 1300 and transformed into E coli JM109 This new plant transformation vector pCAMBIA cah containing mutated cah was used to construct gene cassettes for genetic transformation in tobacco 3 2 3 5 2 Construction of gene cassettes with cah marker and chi o...

Page 86: ...nts far leaf disc transformation in tobacco 3 2 3 5 3 2 Agrobaclerium mediated transformation of gene cassette in tobacco 3 2 3 5 3 2 1 Freeze thaw method The gene cassettes pCAMBI 4 cah Ltp 3FI and pCAMBIA cah Bar2chi were transformed into Agrobacteriurn strain LBA 4404 using the freeze thaw method as described Chen et al 1994 Briefly Agrobacleriurn strain LBA 4404 helper Ti plasmid were grown in...

Page 87: ...A coh Bar2chi was used for tobacco plant transformation The bacterium was grown overnight in liquid YEB medium supplemented with 100 mgA rifampicin and 100 mg l kanamycin to an OD of 0 8 at 550 nm and then used in co cultivation experiments Plant transformation was done following the leaf disc method as described by Horsch et al 1985 Briefly leaf pieces originating from 8 week old in vitro tobacco...

Page 88: ...4 Cyanamide hydratase assay Tobacco leaf tissues 100 mg were frozen in liquid nitrogen homogenized with a pestle and mortar diluted with 100 pl of 5 rnM sodium phosphate buffer pH 8 0 vortexed for 5 min and centrifuged at 8 000 g for 5 min The supernatant was transferred 2 p1 of 1 M cyanamide was added to the solution and incubated at 37 C for 12 h To the 60 p1 of incubation mixture 40 pl of sodiu...

Page 89: ...of chloroform isoamyl alcohol 24 l was added mixed thoroughly and kept on ice for 15 min The content was centrifuged at 10 000 g for 10 min at 4 C The aqueous phase was transferred to a new mjcrocentrifuge tube and mixed with equal volume of ice cold isopropanol and kept at 20 C for 20 30 min After 30 min tubes were centrifuged at 10 000 g for LO min at 4 C The supernatant was discarded pellet was...

Page 90: ...template of transformants The amplification conditions includes initial denaturation at 94 C for 2 min 30 cycles of denaturation at 94 C for 1 min annealing at 61 C for 1 min and extension at 72 C for 1 min with a final extension at 72 C for 10min 3 2 3 5 3 2 5 3 Southern blot analyses of transformants Southern blot analyses were conducted to confirm the integration of genes cah Chi and Lrp in the...

Page 91: ...bottom side of the gel was facing upwards A nylon membrane Hybond Boehringer and Mannheim UK cut to the size of the gel was first wetted in sterile distilled water then soaked in 2X SSC and placed over the gel Three Whatman No 3 sheets cut to the size of the gel were moistened with 20X SSC and placed over the nylon membrane and one layer of dry Whatman No 3 sheet was kept on the top Further stacki...

Page 92: ...d at 65 O C for 15 20 h in a hybridization oven After the completion of hybridization solution was discarded and the membrane was rinsed briefly with 30 ml of 2X SSC and 1 hSDS Then the blot was washed twice with 2X SSCIO I hSDS followed by one wash with 0 5X SSC O I SDS for 30 min at 65 C After the final washing the membrane was removed from the hybridization bottle the excess moisture was remove...

Page 93: ... using a Mighty Small Unit I1 system model SE 250 Pharmacia Biotech Asia Pacific Ltd Hongkong as described by Laemmli 1970 Western blot analyses of proteins were performed using Cnh or Chi or LTP antibody 1 1000 dilution as described earlier Towbin er al 1979 3 2 3 5 3 2 5 5 Transgenic resistance of tobacco leaves To test the antifungal resistance of transgenic tobacco expressing Chi or Ltp genes ...

Page 94: ...was recorded at appropriate time and the difference in chitinase or LTP expressing transgenic plants was observed as described Bhargava et ul 2006 ...

Page 95: ...RESULTS ...

Page 96: ...ng domain which is separated from the catalytic domain by a proline and glycine rich hinge region Phylogenetic tree constructed on the basis of amino acid sequence alignment revealed the similarity of barley chitinase with other plant chitinases The predicted amino acid sequence of barley chitinase BarZchi Hordeum vulgare PI 1955 has 95 3 homology with rye chitinase Secale cereale Q9FRV1 79 8 with...

Page 97: ...chitinases Secole cereule Q9FRV1 AAG53609 Triticum aestivum CAA53626 Poa prurrnsi AAF044541 Oryzu r lriva CAA60590 CAA82849 AAL34318 Musa purudisiaca ABD47583 and POdiploperennis AAT40027 The conserved amino acid residues are highlighted lth asterisks Conserved and semiconserved substitutions are represented by colons dnd dots respectively ...

Page 98: ...7 P i s u s d t i v u AMU W Dalcga oriantdlis P11955 Hordeua wlgare Bar2ohi AALY3lB 0r od sativa AAR18735 Blrbma oldhail Fig 2 Phylogenetic tree of barley chitinase Bor2chi Hordeum vulgare P11955 on the basis of amino acid sequence homology with other plant chitinases ...

Page 99: ... variable loops The position of the eight cysteine residues are conserved where the third and founh cysteines are consecutive in the polypeptide chain and the fifth and sixth cysteines are separated by only one residue Phylogenetic tree constructed on the basis of amino acid sequence alignment revealed the similarity of wheat LTP with other plant LTPs The predicted amino acid sequence of Ltp 3F1 T...

Page 100: ... 1a0 Fig 3 Amino acid sequence alignment of wheat lipid transfer protein Ifp 3FI Trificurn aestivum EF432573 with other monocot LTPs Sorghum hii olor ICAA50660 Zea muys AAB06443 Oryzu surivo AABl8815 Hordeurn vulgrrre ICAA91436 Triticum arstivum AAV28706 Seturiu irulico AAL30846 The conserved amino acid residues are highlighted with asterisks Conserved and srm conservedsubstitutions are represente...

Page 101: ...846 Setaria itslica AM18815 Oryra sativa AAMGSO Sorghun bicolor CAME61 Sorghn bicolor CAA4ZB32 a r d e a vulqare 1m CAA15210 R i t i c u tatqim Fig 4 Phylogenetic tree of wheat lipid transfer protein Llp 3F1 Triticum aestivum EF432573 on the basis of amino acid sequence homology with other ...

Page 102: ...i et al 1993 The predicted 3D structural model of barley chitinase is presented in Fig JA The structure of wheat chitinase revealed the presence of 13 a helices 5 P strands and 28 loop turns and the presence of 6 conserved cysteine residues that are responsible for the formation of 3 disulphide bridges Cys98 CyslM Cys172 Cys180 and Cys279 Cys311 The active site residues Glu142 and Glu164 responsib...

Page 103: ...chi A 3D tructural n odcl 1 the 3 5 kDa barley chit nasewss propo cd w t h13 11 hel ce 5 j trand and 2X 1 q turns B The catalytic glutam ca c drevdue Glu142 and GIu164 re pon hle chrt nase activity were identified in the loop sequence connected lo 3 trand ...

Page 104: ...PROCHECK Ramachandran Plot Plu depees Plot St tlitlC5 Fig 5 C Ramachandran plot of barley chitinase structure ...

Page 105: ...dicted 3D structural model of wheat LTP is presented in Fig 6A The structure of wheat LTP revealed the absence of P strands and presence of 6 a helices and 9 loop tums and the presence of 8 conserved cysteine residues that are responsible for the formation of 4 disulphide bridges Cys29 77 Cys39 54 Cys55 97 Cys75 111 The active site residues Gly30 Pro5O Ala52 and Cys55 are responsible for catalyzin...

Page 106: ...d transfer protein 3F1 A 3D tructural model of the 9 kDa wheat LTP was proposed with 6 a helices and 9 loop urns 6 The active site catalytic residues GIy30 Pro50 Ah52 and Cys55 are t ponsiblefor catalyzing the reaction involved in lipid binding ...

Page 107: ...PROCHECI Ramachandran Plot Fig 6 C Ramachandranplot of wheat LTP structure ...

Page 108: ...y chitinase gene is presented Fig R When the wheat cDNA clone Ltp 3FI was used as template gene specific primer uccessfully amplified wheal lipid transfer protern gene of 348 hp size Fig 9 The nucleotide and ammo acid sequence of wheat lipid transfer protein gene is presented Fig 101 Fig 7 Amplification of DNA corresponding to the sequence encoding barley chitinase Agarose gel Lane 1 I kb DNA ladd...

Page 109: ......

Page 110: ...GAG CTC TG CTT CA CCC CTG CAT V T E 1 4 S Q r i 4 1 l l CTC CTA TGC ACG CGG CAA CGG CGC CAG CCC ATC TGC GCC CTG CTO CAI C T TAG GAG 4 H A P s 4 H 4 TCT AGT CAG CTC AGC CCG GAG CAC CGC TGA CAA CCA AGC GGC GTG CAA GTG CAT CAA A I S 4 A H S 1 I1 K Q A h I K 4 CCC TGC TGC TGG GCT CAA CGC TGC CAA GGC CGC CGG CAT CCC CAC AAA GTG CCG CGT TAG I 4 K A A G I P T h 4 CGT CCC TTA CGC CATCAG CTC TTC GGT CGA CT...

Page 111: ...o the expression vector PET 2Ra Fig I I The i verexpression of the recombinant plasmid PET Barlchi wah achieved at 30 C in LB medium with kanamycin SO pg ml and induced for 5 h with IPTG 0 25 mM SDS PAGE analysia revealed the accumulation of 35 kDa chlt nnse only in the IPTG nduced culture The uninduced control did not express ch tinahe k g 12 A Fig 11 Construction of expression vector PET BarZchi...

Page 112: ...0 mM NaCI 1 mM EDTA 1 Triton X 100 and 1 M urea was effective and yielded high purity of recombinant protein In order to solubilize the recombinant protein urea was used as denaturant After incubation of inclusion bodies with 8 M urea solution the chitinase protein was recovered in the supernatant fraction To keep the protein in monomeric form P mercaptoethanol 2 5 mM was added to the urea solutio...

Page 113: ...Bar2chi Lane 3 total cell proteins from nduced E c oli BL 21 containing PET Bar2chi Lane 4 supernatant from centrifugation of induced sonicated cells Lane 5 pellet from centrifugation of Induced sonicated cells Lane 6 purified chitinase from inclusion bodies B Western blot of purified chitinase after incubation with barley chitinase antiserum The arrow Indicatesthe chitinase enzyme 35kDa ...

Page 114: ...ant plasm dpMAL LTP wa achleved at 37 C in LB rned uniw l h n p cillin 100 pglml and induced for 5 h ulth IPTG 0 3 mM1 SDS PAGE nalyr srevealed the accumulation 01 51 kDa LTP MBP 1 1 LDa LTP 40 LDa IBP fu on protein only in the IPTG induced culturc The unlnduced cc nlml d d not rpre z the LTP MBP fu ion proteln Fig 11 fig 13 Construction of expression vector pMA1 I TP for recombinant lipid transfe...

Page 115: ... from uninduced E coli BL 21 DE 7 containing pMAL LTP L anc 2 total cell protein from induced E roli BL 21 DE3 conta ningpMAL LTP Lane 3 pellcl frcrm ccntnfugation of induced son catedcella Lane 4 rupernatant I rom centrifugauon of Induced sonicated cells Lane 5 purified LTP MBP I ur onprotein The arrow Indicates the MBP LTP fusion protein 51 kDa ...

Page 116: ...s p i e d Fig 15 The protein was filnher concentrated hy lyophilization The yield of purified LTP fusion protein w u 20 tngl 4 8 Determination of protein solubility and Factor Xa cieavnge n s y When Wilkinvon and Harrison model 1991 for theoretical calculation was employed wheat Lrp 3FI showed 82 7 chance of insolubility when overexpressed in E coli Therefore pMAL p2x expression system wllr used t...

Page 117: ...and concentrated 4 9 Antifungal assays of purified chitinase Antifungal assays were performed using major phytopathogenic fungi The purified chitinase showed a broad spectrum antifungal activity at a concentration of 100 pg 0 42 U md 300 pg 1 2 U against phytopathogenic fungi such as Rorrytis cinereo blight of tobacco Pestulotia rheue leaf spot of tea Bipoloris oryzue brown spot of rice Alternuria...

Page 118: ...ons such as lysis and fmgmentation whereas the mycelium from the control plate was normal and intact without any distortion Fig 18 Similarly the treated mycelia of B cinererr appeiued to have uppression of spomlalion and deformations where as the contn l hliowed hpxul ion and intact mycelia Fig 19 4 10 Effect of purified chitinase on germination olsclerotia Sclerotial germination says revealed the...

Page 119: ...fied chitinase towards 1 Bolryds cinerea 11 Pestolotio theoe 111 Bipolaris oryzae IV ANernaria sp A Control 3 X pg af protein extracts of induced E roli containing pET28 a without chitinue insert B 700 pg chitinase and C 100pg chitinare ...

Page 120: ...elia showing lysis and fregnientat onwith poor myccl lrldeveli plncnt due to chitinase treatment A B Y 1 f iiLJ 4 0 0 i e a b lip j i F Fig I8 Light microscopic observation of control and treated fungal mycelin of Bipoloris oryzae A Control B oryzae showed normal branched mycelium B Chitinase treated hyphae showed deformed mycelium with lysis and fragmentation ...

Page 121: ...mal well developed ntycel urn u th spomlation 9 Chitinahe treated hyphae showed deforniot onIn thc rnycrlial fragment without sporulation Fig 20 EtTect of purified chitinase on germination of sclerotia of Rhizoclonia rohni A Control sclerotia germinated in 48 h B Chitina e treated sclerotia did not eerminate even after 4 days ...

Page 122: ...oduced by purified LTP fusion protein was examined under light microscope 400 the hyphne appeared to have clear cut mycelial deformations such as poorly developed mycelium with swollen margin whereas the mycelium from the control plate was normill branched well developed and intact Fig 22 A and B 4 12 Effect of LTP on germination of spores Spore germination assays revealed the fungistatic effects ...

Page 123: ...Fig 21 Inhibitory activity of LTP fusion protein towards Rhizocfonia solani A1 Control containing 300 pg maltose hinding protr n Bl 300 pg of LTP fus on protein and C 100 pg of LTP fusion proteln ...

Page 124: ...ro utaand B Alternoria sp Control hhowed normal well developed rnycelia wtth porulation and treated mycelia bowed por rlydevcl pedmycclium wirh hwollen niugin without sporulation Effecl of LTP fusion prote non germination of fungal pores Light microscopic observation of control and treated spores of IC C urvulurio lrrr uruand D AIternoriu sp Control spores showed gerrnination and treated pores d d...

Page 125: ... E coli Recombinant plasmids pQ cah wild type pQ rahMutK and pQ i ohMutL mutant type were constructed through cloning of amplified mutated rah genes into pQE 60 Fig 24 IPTG induction of E coli carrying wild type pQ ruh as well as mutants pQ cahMutK and pQ cahMutL showed overexpression of rah as his tag fusion protein 4 13 3 Purification of enzyme and cyanamide hydratase assay Cell free crude enzym...

Page 126: ...C Ac CA T A I I h a I t I h I r 4 I I J t 121 CT GTT GCC GAG CCC ATC ATT CCT CAT CA CAT TC CC TA AT AA AT A A TTl I 4 I I I H I 1 b 1 1 1 I I I t 481 I C GCT CAC TTG ATC CAC CTC GCT ACC CTT TAT AC AAT TC C C TA AT G AI T I I I 0 I 1 r I I I I I 541 AT CAT TTT GGT AGC TCG GTT CAT CAC ACC AC4 C AA ACT ATC AA A A TT C A h l I F 5 M I I I I U I I t 1 Mll A CAT GCT TGG TGT TCT TGC TTT CCC TG AC TT CCT ...

Page 127: ...Col El L j Col El k Fig 24 Construction of recombinant plasmids A pQ cah wild type 1 B pQ cahMutK and C pQ cahMutL for overexpression of rub in E ccheric hiu roli ...

Page 128: ... pQ rrrlzMutK i ndpQ c rllMutL inutants grown only in thc presence of IPTG I mM showed the expression of Cah cnzyme Fig 25 Tlie purified enzyme showed 27 7 kDa Cah enzyme Fig 26 A tnd the Western hlol analysis of the purified enzyme prepatations with urh antihody 1 1000 dilution indicated the presence of 27 7 kDa Cah proteins Fig 26 B ...

Page 129: ...I Protein molecular weight marker Genei Bangalore India Lane 2 total ccll protclns from uninduced E roli containing pQ cub Lanea 3 5 total ccll proteins from induced E i oli containing pQ cab pQ cuhMutK and pQ cuhMutL The arrow indicates the Cah enzyme 27 7 kDa ...

Page 130: ... Cnh A Lanes 1 3 purified cah protein from overexpressed Q L I I pQ c uhMutK and pQ c ulrMutL Bl Western blot after incubation with the cah antiserum Lanes 1 3 purified protein of pQ cuh pQ cahMutK and pQ cahMutL The arrow ndicatesthe Cah enzyme 27 7 kDa ...

Page 131: ...s The recombinant plasmid pCAMBIA cuh Lrp 3FI Fig 27 B was successfully constructed by three fragment ligation of the vector pCAMBIA cuh digested with HindIII Ubil promoter Hindlll BarnHI fragment and Ltp 3F1 gene BamH1 Hind111 fragment Similarly pCAMBIA cah Bur2chi Fig 27 C was also constructed by three fragment ligation of vector CAMBIA cuh digested with HbldIIl Ubil promoter HindIII BamH1 fragm...

Page 132: ...Fig 27 Genetic map of gene cassettes A pCAMBIA wh B pCAMB1A cuh Ltp 3F1 and C pCAMB1A cah Bur2chi ...

Page 133: ...ndltl 5 3 5 3 J HindIll I ClAP HindIll BarnHI Three FragmentLigation HlndIll H i n d I L EcoRl 1 EcoRl pcmnein w PCAMBIA cahL 3F1 Bw2chi Fig 28 Construction of gene cassettes for plant transformation Three fragment ligation was performed using pCAMBlA 1300 to construct the gene cassettes pCAMBIA cah Ltp 3FI and pCAMBI 4 cah BarZchi ...

Page 134: ...cyanamide failed to regenerate and revealed 5 m M cyanamide as LDsoand 10 mM as LD Of 150co cultivated leaf discs 77 leaf discs produced shoots after 2 weeks on MS medium amended with 5 mM cyanamide Shoots were then transferred to 10 mM cyanamide to further eliminate the escapes if any Hence about 6 5 10 6 regeneration was achieved after selection using 10 mM cyanamide and about 46 6 66 6 were ide...

Page 135: ...ection and regeneration of transformants A Shooting of transformed leaf disc on shoot election medium amended with 5 mM cyanamide B Regenerated shoot transferred on rooting medium containing 10 mM cyanamide C Transgenic tobacco plant with normal phenotype ...

Page 136: ...ed within 72 h of cyanamide treatment The treated plant grew to maturity and set heeds U I O U I any pparentside effects due to cyanamide treatment T h e e results nd ci ted the fact that cyanamide can be employed as a solution or in granulc l nrrn f or effcct vcmean of drntifyingtransgenic plants from the non transgenic plants In a populat onof egregating progeny plants Fig 30 Fig 30 Cyanamide to...

Page 137: ...trol Tnhle 6 Table 6 Cyanamide hydratase enzyme activity of transgenic lobacco plants Reduction in absorbance at 530 nm in colorimetric assays containing 0 5 mM cyanamide salt with leaf extracts from tobacco plants Experiments with extracts 0 5mM cyanamide 100 rng tissue Reduction in O D 530nm over control due to enzyme activity Blank Control Untransformed plant Cah expressing transgenic plants Pl...

Page 138: ... analysis was carried out on a11 the tronsfur uantsusing cah coding sequences as probes Two transformants plant I and 3 displayed one hybridizable band and 4 transformants plant 2 4 5 and 6 exhibited two hyhridizable bands for rah probe from the total DNA of transformants indicating the slahle integration of marker gene rah in transgenic tobacco plants Fig 31B Transgenic tobacco plants were studie...

Page 139: ...ol and Lane 8 plasmid pCAMBiA cull pos tivecontrol B Southern blot analysis of cuh gene in transfbrrnants Lane C positive control pCAMBIA cuh Lanes 1 6 transgenic plants 1 6 Plant I lane I and 3 lane 3 bowing one hybridizable band Other plants 2 4 6 lanes 2 4 6 showing two hyhridizable bands for cah gene Lane 7 Untransformed control plant C Western blot showed expression of cyanamide hydratase 27 ...

Page 140: ... out on all the transformants using Chi coding sequences as probes Three transform nts plant 1 3 and 5 displayed one hybridizable band and 2 transformants plant 2 and 4 exhibited two hyhridiz hle hands for Chi probe from the total DNA of transformants indicating the stable integration of marker gene Chi in transgenic tobacco plants Fig 328 Transgenic tobacco plants were studied for the expression ...

Page 141: ... Bar2chi positive control B Southern blot analysis of Chi in transformants Lane C positive control pCAMBIA r uh Lrp 3FI Lanes 1 5 transgenic plants Plant I lane 1 plant 3 lane 3 and plant 5 lane 5 showing one hybridizable band and other transformants plant 2 and 4 lanes 2 and 4 showing two hybridizable bands for Chi gene Lane 6 untransformed control plant C Western blot showed expression of chjtin...

Page 142: ... on all the transformants using Ltp coding sequences as probes Two transformants plant 2 and 4 displayed one hybridizable band and 6 transformants plant 1 3 5 8 exhibited two hybridirtble bands for cah probe from the total DNA of transformants indicating the stable integration of marker gene cah in transgenic tobacco plants Fig 338 Transgenic tobacco plants were studied for the expression of cyana...

Page 143: ...MBIA cuh Lrp 3F1 positive control B Southern blot analysis of 1 1 1 in transformants Lane C positive control pCAMB1A cuh Lfp 3FI Lanes 1 8 transgenic plants plant 2 lane 2 and plant 4 lane 4 showing one hybridizahle band and other 6 transformants plant 1 3 5 8 lanes I 3 5 8 showing two hybr dizable hands for Lrp gene Lane 9 untransformed control plant C Western blot showed expression of wheat LTP ...

Page 144: ...g tobacco plants to fungi Fig 34 When observations were made 5 10 days afier inoculation of fungi Bipolcrris oryzrre Cylindrocladium scopnrium and Alfernaria sp the leaves from both non transgenic control plants and pCAMB1A cah transgenic plants showed similar level of severe infectibn diameter of lesion 2 3 cm with more yellowing around the inoculated mycelial plug The leaves from chitinase or li...

Page 145: ...in Up I Detached leaf from control tobacco plant showing spreading lesions due to A Bipolaris oryue B Cylindrorladium scoparium and C Allernaria sp infection 11 Detached leaf from transgenic tobacco plant showing limited infection and smaller lesion Observation was made 10days after inoculalion ...

Page 146: ...plant Fungus Di tn stcr of lesion cm S E Control Bipoluris oryzae 3 0 0 2 Transgenic Chi B oryzae 1 2 0 2 Control Cylindrocludium scoporium 2 5 i 0 3 Transgenic Llp Cy scopurium 1 3k 0 3 Control Alrernuri sp 2 0 0 2 Transgenic Ltp Alrernariu sp 0 8 i 0 2 Data represents the average of three replications ...

Page 147: ...DISCUSSION ...

Page 148: ...s with cyanamide hydratase cuh maker and antifungal protein genes and to study their heterologous expression and antifungal activity In order to achieve this a site directed mutation was performed in cah gene The cuh gene devoid of Hind111 ha been cloned into pCAMBlA 1300 vector Barley chitinase Bar2chi and wheat lipid transfer protein Ltp 3FI genes have been characterized expressed and evaluated ...

Page 149: ...henylalanine and conservation of eight cysteine residues that could form a network of disulfide bridges necessary for the maintenance of the tertiary structure of the molecule together with the central helical core while the variable loops would provide the sequences required for the specific functions of the proteins Jose Estanyol et al 2W Phylogenetic analysis indicated the high level homology o...

Page 150: ...o I m M and low temperatures 16 OC 20 OC were used in this study as reported earlier Schein 1989 Shirano and Shibata 1990 However BarZchi was expressed as inclusion bodies and the solubility of recombinant protein was not affected either by changing the IPTG concentration or reducing the temperature Though the overexpressed chitinase protein has 6x His tag the presence of P mercaptoethanol in the ...

Page 151: ...otease activity that co purified with the fusion protein Similar problems were already encountered with other investigators Maina et al 1988 Antifungal potential of plant chitinases has been well documented According to Roberts and Selitrennikoff 1988 chitinases isolated from the grains of wheat barley and maize functioned as endochitinases and inhibited hyphal elongation of fungi Huynh er al 1992...

Page 152: ...e been established as the members of PR protein family Van Loon and Van Strien 1999 Some members of the nonspecific lipid transfer proteins ns LTP that facilitate in vitro transport of lipids also showed antimicrobial activity in vitro Nielsen et al 1996 reported two novel nearly identical antifungal proteins IWFI and IWF2 from sugar beet leaves Velazhahan el 91 2001 reported an antifungal protein...

Page 153: ...erns the outcrossing through sexual transmission to wild relatives producing herbicide resistant weeds Vasil 1994 Arriola and Ellstrand 1996 cah gene is considered as a unique marker because of its ability to convert calcium cyanamide into urea Maier Greiner et al 1991a Sahrawat el ul 2003 Earlier studies also supported the use of calcium cyanamide fertilizer to control soil borne diseases Mattusc...

Page 154: ...ublic concern about the possible transfer of these genes to unintended species such as microbial pathogens and weeds Mike and Mc Hugh 2004 For the first time present study described the expression of mutated cah gene devoid of Hind111in tobacco plants Agrohacterium method was found to be efficient biological method of gene transfer The non transformants did not form roots exhibited yellow necrosis...

Page 155: ...estern blot analyses of transgenic tobacco confirmed the expression of cah Bar2chi and Ltp 3FI genes in their respective transgenic lines In the present investigation plant transformation cassettes with cah marker and antifungal genes Bar2chi and Ltp 3F1 have been constructed Site directed mutation of cah gene cloning and expression of cah Bar2chi and Ltp 3FI genes were achieved Varying degree of ...

Page 156: ...SUMMARY ...

Page 157: ...The predicted amino acid sequence of Ltp 3F1 has 80 homology with barley and other LTPs of monocots BarZchi gene was cloned into PET 28a and overexpressed in E coli The recombinant chitinase was purified from the inclusion bodies of bacterial pellets SDS PAGE and Western blot analyses with an antiserum against barley chitinase confirmed the production of 35 kDa recombinant chitinase in E coli Ltp ...

Page 158: ...af spot of clover 6 oryzae brown spot of rice Cylindrocladium scoparium root necrosis of banana and Sarocladium oryzae sheath rot of rice even at a concentration of 100 pg When the mycelia from the periphery of the zone of inhibition were examined under light microscope the hyphae appeared to have lysis and fragmentation whereas the control showed normal well developed and inlact mycelia without a...

Page 159: ...ution confirmed the presence of 27 7 kDa Cah protein The mutated cah gene devoid of HindIII site was successfully cloned along with Ubil promoter and nos terminator into pCAMBIA 1300 vector and the recombinant plasmid pCAMBIA cah was constructed The gene cassettes harboring cah marker chitinase and LTP were constructed by three fragment ligation of the dephosphorylated vector pCAMBI 4 cah digested...

Page 160: ...ression of cnh Chi and Ltp in the transformants was further confirmed by Western blot analysis Transgenic tobacco plants expressing chitinase or LTP showed resistance against phytopathogenic fungi The cah marker gene may be considered as a unique marker because of its ability to convert calcium cyanamide into urea a useful nitrogen fertilizer source to plants Plant transformation cassettes harbori...

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