Cloning And Charactization Of Chitinase V Gene And Other Genes From Trichoderma Harzianum

In the sustainable development circumstance of the world,awareness of sustainable development and environmental protection improved the important bio-technology, which got the focus of the international society. Using the high-effect, broad and cheap biology pesticide instead of chemical pesticide could decrease the environment pollution for green produce and green environmental protection.Trichoderma Harzianum, an important plant biocontrol fungus with wide application, had important applied value in the biocontrol of plant diseases, especially diseases infected through soil. T. harzianum can produce many kinds of antibiotics and ergosterols against plant fungal pathogens based on different mechanisms, such as the production of antifungal metabolites, competition for space and nutrients and mycoparasitism. It was widely used in biocontrol of plant disease. But because of various adverse suvivial conditions, the antimicrobial efficacy of T. harzianum was restricted and badly damaged, so it was necessary to enhance the antimicrobial efficacy and the adversity resistance of T. harzianum in order to realize its function of biocontrol.We used the vector of pBI121,pCAMBIA1301 and clone vector of pUC18 to successfully construct express vector pCA-GChiâ…¤and pCA-GSod for filamentous fungi transformation mediated by Agrobacterium tumefaciens. Including the Chiâ…¤and Sod gene, hygromycin resistance gene as selected marker and CaMV35S promoter with section of T-DNA the vector pCA-GChiâ…¤and pCA-GSod could be used for genetic transformation of mycelial fungus. The construction of vector pCA-GChiâ…¤and pCA-GSod, suggests a stratergy for the enhancement of Chiâ…¤and Sod expression in T. harzianum and improvement of T. harzianum'environmental resistance. The best conditions of this system to transformate T. harzianum was got by analysis every transformation factors.Positive transformant was harvested by the selected method, southern blot analysis, RT-PCR and passage culturing.Antifungal activities of the Chiâ…¤and Sod transformants of T. harzianum was tested by means of dual culture with three kinds of plant pathogens. The Chiâ…¤transformants of T. harzianum results showed that it improve the inhibition activity to these pathogens compared with original strain of T. harzianum. The SOD transformants of T. harzianum results showed that there was no obvious difference in inhibition activity to these pathogens between original strain of T. harzianum and its transformants obtained by A. tumefaciens. These results indicated that our transgenic operation didn't influence its antifungal ability. Then stress experiment of wild T. harzianum and its transformant were tested in high temperature and salt conditions. The result indicated that transformant still suvived. Moreover, dual cultured with plant pathogens, the SOD transformant also keep the inhibition activity to these pathogens.however, wild T. harzianum could not live in high temperature and salt conditions.The Chiâ…¤gene expression in the T. harzianum obviously improved the ability to the inhibit the pathogens. The SOD gene in the T. harzianum improved the ability to the high temperature and salt.This established the foundation of the application for more fields.Based on the EST sequences of T. harzianum obtained by our laboratory, The cloned sod and hsp24 were inserted into prokaryotic expression vector, respectively, and the recombinant expression vectors were named pET-sod and pET-hsp24. Double enzymes digestion and sequencing were performed for confirming whether the expression vector constructed successfully. Genes of sod and hsp24 were expressed respectively in E. coli BL21 induced by IPTG, the protein bands of 15.7kD and 24kD were observed respectively by SDS-PAGE.The enzyme activity of SOD and survival to high temperature of hsp24 were tested. The result conformed to expectation, indicating that these genes have been expressed in E. coli, also suggesting that genes from T. harzianum could express well in E.coli. All these genes had registered in GenBank.Genes of sod and hsp24 from T. harzianum were respectively inserted into pYES2 vector, an expression vector of Saccharomyces cerevisiae with high efficiency promoter, and transformed them into the cells of S. cerevisiae INVSc1, generating transformants of sod and hsp24. Stress testing showed that S. cerevisiae cell transformed with sod and hsp24 both enhanced their tolerance to Na₂CO₃ and drought, indicating the two genes are involved in stress tolerance in T. harzianum.The cloning and functional studies of these genes has built a solid technical platform for cloning and study other genes from T. harzianum, and have a significant effect in further exploiting and application of important biocntrol genes in T. harzianum. In this study, genes involved in the functions of biocontrol and stress response have been cloned. The cloning of these genes will make an important role in investigating the biocontrol of T. harzianum under stress and exploiting its important enzymes genes, and is the beginning of studying the biocontrol mechanism of T. harzianum systemically and broadly. The chiâ…¤and sod gene was transformed into T. harzianum and well expressed in T. harzianum, which means that we have obtained a genetic engineering strain efficiently. This study enhanced the resistance of T. harzianum, ensured its excellent biocontriol function in various adverse conditions so as to widen the application area of T. harzianum.