The Study On Construction Of Two-Component Male Sterile Line By Split-Intein Mediated-Assembly Of Functional Protein

At present, plant nucleus infertility have been achieved by means of genetic engineering, but only 50% of sterile plants can be obtained when nucleus sterile line is crossed maintainer line, and the remaining 50% fertile plants need to be removed. How to get 100% of sterile plants is the focus of plant genic male sterile male sterility research. In order to solve the problem, Barnase toxic protein gene and glyphosate-resistant G2-EPSPS gene were split into inactive N-terminal and C-termial fragments, inactive fragments were fused in-frame to Ssp DnaE intein and Npu DnaE intein N-terminal and C-termial respectively. Then, pCABarESN, pCABarESC, pCABarESP, pCABarSNZESN, pCABarSCZESC plant expression vectors and pCABarESP, pCABarABn control vectors were constructed. Above vectors were transformed into mustard and cabbage plants to study the feasibility of 100% male sterility line by split-Intein mediated protein structure and function reconstruction.The main results were as follows: 1、The function reconstruction in mustard of split-EPSPS gene mediated by Npu DnaE mtempCABarESN, pCABarESC, pCABarESP control vectors were transformed into mustard By Agrobacterium-mediated method. The PCR and RT-PCR analysis results showed ESN、ESC、G2-EPSPS gene have been transformed into mustard and transcription. The further PPT resistance screening results showed that ESN, ESC gene insert mustard genome at multiple sites in some pCABarESN, pCABarESC transgenic plants. Glyphosate resistance screening results showed that the expression only N-terminal and C-terminal fragments of G2-EPSPS gene in pCABarESN and pCABarESC transgenic mustard plants were sensitive to glyphosate, but intact G2-EPSPS expression in pCABarESP transgenic mustard plants were glyphosate resistant. The hybrid between pCABarESN and pCABarESC transgenic mustard plants showed strong glyphosate resistance. The indicated a functional G2-EPSPS protein was assembled in the plants mediated by Npu DnaE intein.2、Generation the parental plants of two-component male sterility of mustard The pCABarSNZESN, pCABarSCZESC plant expression vectors were transformed into the mustard, the PCR results indicated the parental plants of two-component male sterility of mustard had been obtained.3、Generation of transgenic male sterile cabbageRibonuclease Barnase gene driven by BcA9 promoter in plant expression vector pCABarABn transformed into Brassica oleracea ’TF’ variety by Agrobacterium-mediated transformation of hypocotyls, and transgenic cabbage plants were generated with herbicide Bastaresistance. Cytological microscopy results showed the pollen mother cells degeneration as a result of premature tapetum cells degradation, and no normal pollens development in mature anthers. Compared with wild-type plants, smaller flower organ, more severe wrinkles petals, bend pistil, thin stamen were observed. In addition, there were a large number ofbuds dead when the temperatures is below 22℃, but the temperature is higher than 25℃, dead bud phenomenon would relieved. Field survey results showed that transgenic Brassica oleracea plants can not seed by self-pollination, well seedbut shorter.pods was observed when pollinated with pollens of wild-type plants.4、Generation the parental plants of two-component male sterility of cabbageThe pCABarSNZESN, pCABarSCZESC plant expression vectors were transformed into the cabbage, The PCR results indicated the parental plants of two-component male sterility of mustard with herbicide PPT resistance had been obtained. RT-PCR analysis results showed that ESN, ESC genes have transcripted respectively in the leaves of pCABarSNZESN, pCABarSCZESC transgenic cabbage.Moreover, SN, SC genes in the buds of pCABarSNZESN, pCABarSCZESC transgenic cabbage also have been transcripted.