|
|
|
Enhanced resistance to Botrytis cinerea and Rhizoctonia solani in transgenic broccoli with a Trichoderma viride endochitinase gene |
YU Ya, ZHANG Lei, LIAN Wei-ran, XU Feng-feng, LI Shuang-tao, XIANG Juan, ZHANG Guo-zhen, HU Zan-min, ZHAO Bing, REN Shu-xin, GUO Yang-dong
|
1、College of Agriculture & Biotechnology, China Agricultural University, Beijing 100193, P.R.China
2、Department of Biology Science & Technology, Wuhan Institute of Bioengineering, Wuhan 430415, P.R.China
3、Institute of Genetics & Developmental Biology, Chinese Academy of Sciences, Beijing 100101, P.R.China
4、School of Agriculture, Virginia State University, Petersburg VA23806, USA |
|
|
摘要 A endochitinase gene (Tch) from the fungus Trichoderma viride was introduced into broccoli (Brassica oleracea var. italica) by Agrobacterium-mediated transformation. Sixty-eight putative transformants were obtained and the presence of the Tch gene was confirmed by both PCR and Southern blot analysis. RT-PCR analysis showed an accumulation of the transcript encoding the endochitinase protein in the transgenic plants. Using real-time quantitative PCR, the expression profiling of endochitinase gene was analyzed. Primary transformants and selfed progeny were examined for expression of the endochitinase using a fluorometric assay and for their resistance to the pathogenic fungi Botrytis cinerea and Rhizoctonia solani. The endochitinase activities in T0 in vitro plants, T0 mature plants and T1 mature plants were correlated with leaf lesions, and the transgenic line T618 had high endochitinse activities of 102.68, 114.53 and 120.27 nmol L–1 MU min–1 mg–1 protein in the three kinds of plants, respectively. The endochitinase activity showed a positive correlation with the resistance to the pathogens. Most transgenic T0 broccoli had increased resistance to the pathogens of B. cinerea and R. solani in leaf assays and this resistance was confirmed to be inheritable. These findings suggested that expression of the Tch gene from T. viride could enhance resistance to pathogenic fungi in Brassica species.
Abstract A endochitinase gene (Tch) from the fungus Trichoderma viride was introduced into broccoli (Brassica oleracea var. italica) by Agrobacterium-mediated transformation. Sixty-eight putative transformants were obtained and the presence of the Tch gene was confirmed by both PCR and Southern blot analysis. RT-PCR analysis showed an accumulation of the transcript encoding the endochitinase protein in the transgenic plants. Using real-time quantitative PCR, the expression profiling of endochitinase gene was analyzed. Primary transformants and selfed progeny were examined for expression of the endochitinase using a fluorometric assay and for their resistance to the pathogenic fungi Botrytis cinerea and Rhizoctonia solani. The endochitinase activities in T0 in vitro plants, T0 mature plants and T1 mature plants were correlated with leaf lesions, and the transgenic line T618 had high endochitinse activities of 102.68, 114.53 and 120.27 nmol L–1 MU min–1 mg–1 protein in the three kinds of plants, respectively. The endochitinase activity showed a positive correlation with the resistance to the pathogens. Most transgenic T0 broccoli had increased resistance to the pathogens of B. cinerea and R. solani in leaf assays and this resistance was confirmed to be inheritable. These findings suggested that expression of the Tch gene from T. viride could enhance resistance to pathogenic fungi in Brassica species.
|
Received: 29 August 2014
Accepted:
|
Fund: This work was partly supported by the 863 Program (2012AA100A03), 973 Program (2012CB113900) and Beijing Agricultural Industry Project, China (BLVT-03). |
Corresponding Authors:
GUO Yang-dong, Tel: +86-10-62734845,E-mail: yaguo@cau.edu.cn; ZHAO Bing, Tel: +86-10-62734095,E-mail: zhaobing@cau.edu.cn
E-mail: yaguo@cau.edu.cn;zhaobing@cau.edu.cn
|
About author: * These authors contributed equally to this study. |
Cite this article:
YU Ya, ZHANG Lei, LIAN Wei-ran, XU Feng-feng, LI Shuang-tao, XIANG Juan, ZHANG Guo-zhen, HU Zan-min, ZHAO Bing, REN Shu-xin, GUO Yang-dong.
2015.
Enhanced resistance to Botrytis cinerea and Rhizoctonia solani in transgenic broccoli with a Trichoderma viride endochitinase gene. Journal of Integrative Agriculture, 14(3): 430-437.
|
Anderson N A. 1982. The genetics and pathology of Rhizoctoniasolani. Annual Review of Phytopathology, 20, 329-347Baranski R, Klocke E, Nothnagel T. 2008. Chitinase CHIT36from Trichoderma harzianum enhances resistanceof transgenic carrot to fungal pathogens. Journal ofPhytopathology, 156, 513-521Bezirganoglu I, Hwang S Y, Fang T J, Shaw J F. 2013.Transgenic lines of melon (Cucumis melo L. var.makuwa cv. ‘Silver Light’) expressing antifungal proteinand chitinase genes exhibit enhanced resistance to fungalpathogens. Plant Cell Tissue and Organ Culture, 112,227-237Bolar J P, Norelli J L, Wong K W, Hayes C K, Harman G E,Aldwinckle H S. 2000. Expression of endochitinase fromTrichoderma harzianum in transgenic apple increasesresistance to apple sacb and reduces vigor. Phytopathology,90, 72-77Cao J, Earle E D. 2003. Transgene expression in broccoli(Brassica oleracea var. italica) clones propagated in vitrovia leaf explants. Plant Cell Reports, 21, 789-796Charenopornwattana S, Thara K V, Datta S K, PanbangredW, Muthukrishnan S. 1999. Inheritance, expression, andsilencing of a chitinase transgene in rice. Theoretical andApplied Genetics, 98, 371-378Chen L F O, Lin C H, Kelkar S M, Chang Y M, Shaw J F. 2008.Transgenic broccoli (Brassica oleacea var. italica) withantisense chlorophyllase (BoCLH1) delays postharvestyellowing. Plant Science, 174, 25-31Choquer M, Fournier E, Kunz C, Levis C, Pradier J M, SimonA, Viaud M. 2007. Botrytis cinerea virulence factors: Newinsights into a necrotrophic and polyphageous pathogen.Fems Microbiology Letters, 277, 1-10Collinge D B, Kragh K M, Mikkelsen J D, Nielsen K K,Rasmussen U, Vad K. 1993. Plant chitinases. The PlantJournal, 3, 31-40Distefano G, Malfa S L, Vitale A, Lorito M, Deng Z N, Gentile A.2008. Defence-related gene expression in transgenic lemonplants producing an antimicrobial Trichoderma harzianumendochitinase during fungal infection. Transgenic Research,17, 873-879Gokul B, Lee J H, Song K B, Rhee S K, Kim C H, PandaT. 2000. Characterization and application of chitinasesfrom Trichoderma harzianum-A review. BioprocessEngineering, 23, 691-694Gooday G W. 1999. Aggressive and defensive roles forchitinases. In: Jalles P, Muzzarelli R A A, eds., Chitin andChitinases. Birkhauser Publishing, Switzerland.Guo Y D, Yli-Mattil T, Pulli S. 2003. Analysis of timothy (Phleumpretense L.) genetic variation with RAPD and UP-PCR.Hereditas, 138, 101-113Harman G E, Hayes C K, Lorito M, Broadway R M, DiPietro A. 1993. Chitinolytic enzymes of Trichodermaharzianum: purification of chitobiosidase and endochitinase.Phytopathology, 83, 313-318Hu C H, Wei Y R, Huang Y H, Yi G J. 2013. An efficient protocolfor the production of chit42 transgenic Furenzhi banana(Musa spp. AA group) resistant to Fusarium oxysporum. InVitro Cellular & Developmental Biology-Plant, 49, 584-592Lorito M, Harman G E, Hayers C K, Broadway R M, Tronsmo A,Woo S L, Di Pietro A. 1993. Chitinolytic enzymes producedby Trichoderma harzianum: Antifungal activity of purifiedendochitinse and chitobiosidase. Phytopathology, 83,302-307Lorito M, Woo S L, Fernadez I G, Colucci G, Harman G E. 1998.Genes from mycoparasitic fungi as a source for improvingplant resistance to fungal pathogens. Proceedings of theNational Academy of Sciences of the United States ofAmerica, 95, 7860-7865Kern M F, Maraschin F S, Endt D V, Schrank A, Vainstein MH, Pasquali G. 2010. Expression of a chitinase gene fromMetarhizium anisopliae in tobacco plants confers resistanceagainst Rhizoctonia solani. Applied Biochemistry andBiotechnology, 160, 1933-1946Mora A A, Earle E D. 2001. Resistance to Alternaria brassicicolain transgenic broccoli expressing a Trichoderma harzianumendochitinase gene. Molecular Breeding, 8, 1-9Pappinen A, Defefu Y, Syrjälä L, Keinonen K, Weissenberg K V.2002. Transgenic silver birch (Betula pendula) expressingsugarbeet chitinase 4 shows enhanced resistance toPyrenopeziza betulicola. Plant Cell Reports, 20, 1046-1051Prasad K, Bhatnagar-Mathur P, Waliyar F, Sharma K K. 2013.Overexpression of a chitinase gene in transgenic peanutconfers enhanced resistance to major soil borne andfoliar fungal pathogens. Journal of Plant Biochemistry and Biotechnology, 22, 222-233Qin Y, Li H L, Guo Y D. 2007. High-frequency embryogenesis,regeneration of broccoli (Brassica oleracea var. italica) andanalysis of genetic stability by RAPD. Scientia Horticulturae,111, 203-208Sahai A S, Manocha M S. 1993. Chitinase of fungi and plants:their involvement in morphogenesis and host-parasiteinteraction. Fems Microbiology Reviews, 11, 317-338Schäfer T, Hanke M V, Flachowsky H, König S, Peil A, KaldorfM, Polle A, Buscot F. 2012. Chitinase activities, scabresistance, mycorrhization rates and biomass of own-rootedand grafted transgenic apple. Genetics and MolecularBiology, 35, 466-473Shah J M, Raghupathy V, Veluthambi K. 2009. Enhancedsheath blight resistance in transgenic rice expressing anendochitinase gene from Trichoderma virens. BiotechnologyLetters, 31, 239-244Tabei Y, Kitade S, Nishizawa Y, Kikuchi N, Kayano T, Hibi T,Akutsu K. 1998. Transgenic cucumber plants harboring arice chitinase gene exhibit enhanced resistance to graymold (Botrytis cinerea). Plant Cell Reports, 17, 159-164Vishnevetsky J, White T L, Palmateer A J, Flaishman M, CohenY, Elad Y, Velcheva M, Hanania U, Sahar N, Dgani O, PerlA. 2011. Improved tolerance toward fungal diseases intransgenic Cavendish banana (Musa spp. AAA group) cv.Grand Nain. Transgenic Research, 20, 61-72Xian H Q, Li J R, Zhang L Q, Li D C. 2012. Cloning and functionalanalysis of a novel chitinase gene Trchi1 from Trichotheciumroseum. Biotechnology Letters, 34, 1921-1928Yu Y, Liu L S, Zhao Y Q, Yang P, Zhao B, Guo Y D. 2010. Ahighly efficient in vitro plant regeneration and Agrobacteriummediatedtransformation of Brassica oleracea var. botrytis.New Zealand Journal of Crop and Horticultural Science,38, 1-11 |
No Suggested Reading articles found! |
|
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
|
Shared |
|
|
|
|
|
Discussed |
|
|
|
|