Study On Genitic Transformation Of Lespedeza Bicolor Turcz. With BADH Gene & Antisense 4CL Gene

Lespedeza Michx.plants are excellent fodder shrubs and also trees which can be used to conserve soil and water.It's important on improving their tolerance to abiotic stress in agricultural and forestry production.Many transgenic results showed that the transgenic plants carring betaine aldhyde dehydrogenase(BADH) gene could improve their tolerance to drought or salt stress. 4-coumarate:CoA ligase(4CL) is the key enzyme in the process of lignin synthesis. Transgenic plants with antisense 4CL gene reduced the lignin content.In this paper,the BADH & antisense 4CL genes were selected as objective genes.The pBI121-xylA-BADH plant expression vector with xylose being the selective agent was built.Another plant expression vector named antisense 4CL-BADH was built too.We expect to use gene engineering to improve the characters of Lespedeza in the aspect of the lignin content and abiotic resistance.Lespedeza bicolor Turcz.from America which has abundant harvest was selected as the research object.The high frequency regeneration system of L.bicolor was optimized.L.bicolor genetic transformation system based on xylose as the selective agent was built,and the marker free transgenic system was built too.The xylA-BADH genes and antisense 4CL-BADH genes were introduced into L.bicolor.Some functions of transgenic plants were assayed.The research findings will have theoretic & real significance on promoting the development of fodder industry which Lespedeza Michx.plants are used as fodder.The main research work and results are as follows:(1) The xylose isomerase(xylA) gene was cloned from E.coli.The pBI121-xylA-BADH plant expression vector with xylose being the selective agent was built. Another new plant expression vector without marker gene named antisense 4CL-BADH was built too.The plasmid DNA of pBI121-xylA-BADH and antisense 4CL-BADH were transformed into LBA4404,EHA105 individually.(2) During the building and optimization of regeneration system of L.bicolor,the main medium named MS^* with having a half of the macronutrients in MS medium was used.The optimal medium for adventitious buds regeneration of L.bicolor cotyledonary node was MS^* + 6-BA 1.0 mg.L^-1 + NAA 0.2 mg.L^-1 + sucrose 3%+ agar 6 g.L^-1.The optimal medium for segment proliferation was MS^* + 6-BA 1.0 mg.L^-1 + NAA 0.1 mg.L^-1 + IBA 0.2 mg.L^-1 + sucrose 3%+ agar 6 g.L^-1.The optimal medium for rooting of L. bicolor plants in vitro was MS^* + NAA 0.1 mg.L"l + IBA 0.2 mg.Lt + sucrose 3%+ agar 6 g.L^-1.(3) The agrobaeterium-mediated xylose selective system and marker free selective system of L.bicolor were studied.200～300 mg.L^-1 Cefotaxine(Cef) could restrain the growing of agrobacterium during the adventitious buds regeneration of L.bicolor cotyledonary node.100 mg.L^-1 Cef was enough to restrain agrobacterium during proliferation or rooting.When pBI121-xylA-BADH plasmid DNA was introduced into L.bicolor,3%xylose could be the selective agent at adventitious buds regeneration of cotyledonary node and 0.5%sucrose & 2.5%xyiose could be the selective agent at proliferation or rooting.The methods of introducing antisense 4CL-BADH into L.bicolor were studied,1g.L^-1 betaine aldehyde chloride was the selective agent for adventitious buds regeneration of cotyledonary node.The mixture of 0.3 g.L^-1 betaine aldehyde chloride & 0.7 g.L^-1 NaCl was the selective agent for proliferation or rooting.(4) After PCR,PCR-Southern and Southern assays had been done,xylA-BADH and antisense 4CL-BADH were sure to be introduced into L.bicolor.The study on tolerance to salt stress showed that the xylA-BADH transgenic plants had the best tolerance and the antisense 4CL transgenic plants were the worst.The tolerance of antisense 4CL-BADH transgenic plants to salt stress was slight lower than wild-type plants. After being cultivated for some days at 1.0g.L^-1 NaCl stress,the activity of betaine aldehyde dehydrigenase in antisense 4CL-BADH transgeinic plants was 2.5 times than that of in the wild-type plants.The stem slices of L.bicolor were observed by the histochemical staining of M(a|¨)ule responses,Weisner responses,Rosoff responses and fluorescence of lignin responses.The preliminary result showed that the transgeinic L.bicolor with antisense 4CL-BADH maybe could reduce the lignin content.The reliability of the assay methods about the antisense 4CL transgenic plants was discussed.In addition,the number of idioblastic cells in the stem slices of transgeinic L.bicolor with antisense 4CL-BADH was more than those of wild-type L.bicolor.After discussion of the relationship among idioblastic cells,tolerance to abiotic stress and betaine,the author thought the reason of transgenic plants having an increased number of idioblastic cells was that BADH was introduced and much betaine might be synthesized.