The Breeding Of The Planthopper Resistant And Glyphosate Tolerate Transgfenic Rice

Rice is one of the most important crop in the world,it makes a big contribute to the food security.Insect pest is a very important reason that causes huge rice yield losses.Currently,rice planthoppers,especially brown planthopper(Nilaparvata lugens Stal;BPH),small brown planthopper(Laodelphax striatellus Fallen;SBPH)and white back planthopper(Sogatella furcifera Horvath;WBPH),have become more and more serious threats to the sustainability of intensive rice production system in China.To control the rice planthoppers damage,one could relay on using chemical pesticides,or try to breed planthoppers resistant rice varieties.However,the overuse and misuse of chemical pesticides have led to destroy the environment and become a hidden danger to food safety and food supply.One better alternative strategy of planthoppers control would be development resistant rice varieties.Three new Bt(Bacillus thuringiensis)genes ST1,ST2 and ST3 have been identified via in intro assay,which showed ST1 and ST2 genes together can resistant to small brown planthopper and ST3 gene confers brown planthopper resistant.In this study,those three Bt genes ST1,ST2 and ST3 sequence were optimized based on perferred rice codons and constructed into the new binary vector pTGH-1 in which there is a glyphosate tolerate marker gene called EPSPS.Two final transformation vectors,pTGH-1(ST1+ST2)and pTGH-1(ST3)were constructed and transformed into MH86 and ZH11 by the agrobacterium-mediated transformation.We used the PCR and Southern blot methods to analyze the T0 generation of the transgenic lines.RT-PCR was performed to detect the foreign genes' expression of the transgenic positive lines,then the transgenic lines showed higher expression level were chosen for further analysis via Western blot.The BPH and SBPH resistance level of Ti generation transgenic positive line was investigated through the standard seedbox screening technique.In addition,the Roundup(in which contains 41%glyphosate-isopropylammonium)diluted 150 times was treated with the transgenic positive lines to identify their glyphosate resistance capacity.The main results in the study are summarized as follows:1.Three Bt genes ST1,ST2 and ST3 sequence were optimized and synthesized based on perferred codons in rice.Two plants transformation vectors pTGH-1(ST1+ST2)and pTGH-1(ST3)were constructed.2.ST1 and ST2 genes are introduced into two rice varieties,MH86 and ZH11 by agrobacterium-mediated transformation.Positive transgenic lines in which the foreign genes are stably constructed in the rice genome were identified by molecular analysis.3.ST3 gene is also transformed into MH86 and ZH11 by the agrobacterium-mediated transformation,and currently,all the transgenic materials are at rooting stage.4.Although,the Ti generation positive transgenic plants that contain ST1 and ST2 genes showed no difference response to BPH feeding experiment compare to wild type,a improved resistance to SBPH was shown comparing to wild type.5.The transgenic positive lines that contain ST1 and ST2 genes also conferred a high resistance to the Roundup,which indicated that ST1,ST2 and EPSPS genes are closely linked and transformed into rice genome,and transgenic materials with both SBPH resistance and Roundup resistance have been development in this study.