Gene Expression Anaylysis Of ZjGA20ox In Zoysiagrass(Zoysiajaponica Steud.) And Methodological Improvement Of Agrobacterium-mediated Transformation Of RNA Interference ZjGA20ox Gene

GAs is an important class of plant hormones, participating in a variety of plant growth developmental stages and physical process. The gibberellin20-oxidase played an important role in the biosynthesis of gibberellic acid as one of key oxidase enzymes regulating directly the generation of biological GAs. Zoysiagrassis widely used in south area of China, howerver, the grass has some shortages, such as its short green period, poor resistance to some biotic and abiotic stress. The objective of this study was to investigate the possible function of GA20ox in responses to abiotic stresses by analyzing gene expression pattern of GA20ox in Zoysiagrass. In addition, RNAi expression vector was constructed and methods of agrobacterium-mediated RNAi vector transformation was improved and optimized. This study proposed by transformating the RNAi vector into Zoysiagrass, in order to obtain the transgenic plants of dwarf phenotype and good resistance.Mature seeds of Zoysia japonica Steud.were selectedasresearch materials. Using grinding, soaking and random vaccination methods to induce embryo callus and plants were regenerated, a lot of high quality original embryo callus material were obtained and provde solid base for later methodologica improvement of agrobacterium-mediated transformation of Zoysiagrass.Three important factors affecting agrobacterium mediated genetic transformation system were evaluated, and optimized parameters were listed:bacteria liquid concentration (OD600value of0.5-0.6), infection time (15min) and trained time (2-3d). Combined with the improved culture medium, the efficiency eventually reached over50%. Through optimizing the parameters of embryo callus cleaning, agrobacterium pollution ratio was reduced in the process of screening culture which greatly saved time, cost and avoided the waste of callus materials compared with the previous agrobacterium mediated method. Finally the improved agrobacterium-mediated system of zoysiagrass was established.On the basis of the establishment of regeneration system and the establishment of improved agrobacterium-mediated transformation system of Zoysiagrass, the283bp-fragment of ZjGA20ox was amplified by PCR from genomic DNA of zoysiagrass and used to construct the hairpin RNAi vector pC1301-ZjGA20ox-RNAi, and then this vector was transferred into zoysiagrass. After screening, differentiation and regeneration of resistant callus, semi-quantitative PCR was used to detect the presence of GA20ox-RNAi vector for resistant plants. Some transgenic plants were found a semi-dwarfing phenomenon. PCR and RT-PCR test results showed that ZjGA20ox gene of semi-dwarfing plants showed no gene expression which confirmed that ZjGA20ox-RNAi interference gene was integrated into genome of Zoysia japonica.Finally, the differential expression of ZjGA20ox in different growth stages and different tissues of the non-transgenic plants, as well as differential expression induced by abiotic stresses including low temperature (4℃), H2O2(8μM), salt stress (250mMNaCl),25%PEG6000, and high temperature (42℃)were investigated by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). Results showed that the ZjGA20ox gene was expressed in the leaves, but were not expressed in the roots. ZjGA20ox gene expression in leaves were showed decreasing from15day to5month, and to10month (Jan./2008-Oct./2008). Higher expression of ZjGA20ox was observed in young leaf and sheath, compared to senescent leaf, blade, and stolon. Therefore, The expression patterns of2jGA20ox gene was tissue specific and age dependent. Expression of2jGA20ox decreased under low temperature(4℃),25%PEG6000, and high temperature(42℃) compared to non-stressed plants. The highest expression of ZjGA20ox was observed when plants were treated with8μM H2O2for10h and with250mM NaCl for5h. Furthermore, the physiological and biochemical experiment results showed that application of exogenous GA3alleviated tissue damage from low temperature treatment, During recovery from cold stress, ZjGA20ox gene upgulated resulting in the increased synthesis of endogenous gibberellins.