Genetic Transformation And Characterization Of Stress-related Genes In Rice

Abiotic stresses such as high salinity, drought, and low temperature are majoradverse environmental factors that affect the growth of plants and accout for the yieldreductions of crops, and even lead to death of plants. Increasing evidence suggest thatgenetic engineering has been one of the most important strategies to improve stressresistance of crops in modern crop breeding programs. Under abiotic stress conditions,some critical genes involved in signal transduction regulate the expression of functionalgenes. A number of studies suggest that overexpressing some of these importantregulatory or functional genes in the transgenic plants can significantly improve stressresistance.In this study, a few stress-related genes in rice were studied for their expressionlevels under various stress conditions and in different tissues. These genes weretransformed into rice cultivar to test their effectivenees on improving stress resistance.Transgenic plants were identified for the copy number and expression level of transgenesand positive transgenic families were tested for drought resistance and salt tolerance. Inaddition, a stress-inducible promoter, potentially useful for gene engineering, wasidentified for one of the stress-related genes. Results obtained in this study providedevidence on improving stress resistance of crops by genetic engineering of thesestress-related genes as well as references for understanding the molecular basis of stresstolerance. The main results are as following:1. Twelve constructs were made for five stress-responsive genes and transformed intojaponica rice Zhonghua 11.2. Northern blot analysis revealed that three genes, OsCK1 (encoding a protein kinaseof CIPK family), OsDhnI (encoding a dehydrin-like protein), and OCPI1 (encoding aputative chymotrypsin-like proteinase inhibitor), were induced strongly by variousstresses including drought, high salinity and abscisic acid (ABA). These three geneswere focused in this study for testing the stress resistance of transgenic plants.3. PCR analysis was performed for all T₀ transgenic plants and 90ï¼…of them were transgenic positive. A total of 139 positive transgenic plants from sevenoverexpression constructs were analyzed for the overexpression (81 plants withoverexpression) and copy number (more than 30ï¼…plants had single copy of T-DNA)of the transgenes.4. The overexpression and single copy families of OsCK1, OsDhnI and OCPI1 wereplanted in the field for drought stress at anthesis stage. The results showed that bothgrain yield and seed setting rate of OsDhnI- and OCPI1-transgenic families weresignificantly higher than that in the wild type rice Zhonghua 11. The transgenicplants of the two genes also showed stronger salt tolerance than the wild typegrowing on MS medium containing 100 mM NaCl. Theses results suggested thatoverexpression OsDhnI and OCPI1 could significantly improve stress resistance. Theassay of proteinase inhibitor activity suggested that the endogenouschymotrypsin-like activity in the OCPI1-overexpressed transgenic plants in rice wasinhibited.5. The OCPI1 promoter, potentially useful in making stress-inducble constructs, wasisolated and identified. By histochemical assay, expression level of GUS wasdetected in callus, leaf, root, stem, sheath, ligule, auricle, glume, pistil and stamen.By Northern blot and GUS activity assay, the OCPI1 promoter was induced stronglyby dehydration and salt stresses. Interestingly, similar results were obtained by usingthe antisense sequence of OCPI1 promoter perform GUS assay, suggesting thatOCPI1 promoter has a bidirectional stress-inducible activity.6. Site-directed mutation of OsCK1 was generated for expressing protein withconstitutive kinase activity. The mutated OsCK1 constructes for overexpression wastransformed into rice and single copy transgenic plants were identified for furtheranalyses such as stress tolerance testing and kinase activity assay.