Development And Resistance Analysis Of Transgenic Plants With Cry2Aa~# Gene In Miscanthus Lutarioriparius And MlNAC2 Gene In M. Sinensis

Miscanthus lutarioriparius and M.sinensis belong to perennial giant grassy Miscanthus as renewable energy plant which has huge biomass. For ecological effects and economic benefits, they were the prime candidates to be planted on the vast marginal lands in China. Biotic and abiotic stress is the main restriction of Miscanthus growing on marginal lands, therefore development of the varieties and increasing breeding area of Miscanthus must focus on enhancing resistance characteristics of it. The Bt gene Cry2Aa was transferred into M.lutarioriparius and the transcription factor MlNAC2 was transferred into M.sinensis respectively in this study, which aimed at obtaining the new germplasm and offering germchits for the large-scale cultivation on marginal lands. Meanwhile, we intend to demonstrate the function of the target genes and to provide theoretical support for revealing the mechanism of genes. The main results were as follows:1 Optimization of callus receptor systemFor browning characteristics of callus during the induction process of Miscanthus, polyvinylpyrrolidone(PVP), vitamin C(VC) and citric acid(CA) were used as the browning inhibitors in our study. The results showed that treating with the 3 antioxidants could reduce the browning of explants and improve callus induction rate in a certain degree. CA is the best anti-browning agent of immature inflorescences culture of M. lutarioriparius, and 0.2 g/L CA could significantly increase the callus induction rate (P<0.01). The optimized medium for callus inducing in this study is MS+2 mg/L 2,4-D+0.5 mg/L 6-BA+0.2 g/L CA+8 g/L agar+30 g/L sucrose.2 Obtaining transgenic plants and functional analysis of Cry2Aa# gene in M. lutarioripariusThe Cry2Aa# gene has been transferred into M. lutarioriparius callus by agrobacterium-mediated transformation method. Regenerated plants were selected with Basta for Bar was the herbicide-resistant marker gene. Finally,6 transgenic plants were identified by PCR amplification.The test of smearring Basta confirmed that transgenic plants were resistant to Basta. Quantitative determination of Cry2Aa protein with ELISA showed that the expression level of protein was relatively low and different in transgenic plants.3 Obtaining transgenic plants and functional analysis of MNAC2 gene in M.sinensisThe MlNAC2 gene has been transferred into the callus of Msinensis with agrobacterium-mediated method and we obtained 4 positive plant lines via PCR appraisal.The semi-letha concentration of NaCl to transgenic plants of M.sinensis is 0.6%, which were analyzed via relative growth level, plant height, root number and root length of tissue culture seedling under different concentrations of NaCl stress.The transgenic lines were treated with 0.6%NaCl solution 0,1,3,9,12,24 or 48hours. And the relative expression level of MINAC2 has been measured by RT-qPCR. The results indicated that the expression level of MlNAC2 has significant differences within 0 to 48 hours of NaCl stress in different transgenic lines, and the relative expression level of MlNAC2 was significantly different in each transgenic lines in the same time of NaCl stress.