Construction Of Tetravalent-target Expression Vector Carrying Cry1Ac,Bar And RNAi Fragments Resistance To Virus Diseases And Genetic Transformation In Sugarcane

Sugarcane is an important crop for sugar and biofuel.Sugarcane production is badly affected by plant diseases and insects especially by sugarcane mosaic virus(ScMV),yellow leaf virus(ScYLV)and borers.Varieties resistant to herbicide are the need to modern mechanized production to reduce input.Sugarcane is a highly heterozygous allopolyploid plant with a complex genetic background and short of germplasm resources resistant to diseases,insects and herbicide.Therefore,it is very difficult to get novel varieties with integrated traits through crossbreeding.Genetic engineering and RNAi technology offer new ways for integrated breeding.Consensus sequences of CP and HC-pro genes of ScMV and CP gene of ScYLV were selected out as RNAi fragments to target genes respectively.The RNAi fragments against to ScMV and ScYLV were linked together to form a fusion fragment.According to the restriction enzyme sites,the fusion fragment was inserted into the RNAi plant expression vector forwardly and reversely to form a bivalent RNAi structure.Introduction of gene Cry 1 AC into pHANNIBAL-Z,after introduction of the bivalent RNAi structure into pHANNIBAL-Cry1AC,and Npt ? by Bar replacement into pART27,Finally the Cry1AC and the bivalent RNAi structure were introduced into pART27-Z to form tetravalent expression vectors with genes resistant to insects and herbicide and ScMV and ScYLV.The tetravalent vectors were transformed into sugarcane calli of FN3 9?ROC22 and FN15 mediated by Agrobaterium strains and gene gun respectively.The calli were subjected to PPT stress and transgenic sugarcane lines were identified by PCR and expression of the genes were analyzed by quantitative PCR and protein strips.The main achievements of this paper were as follows:1,The intermediate vector pHANNIBAL was modified with the restriction enzyme site Sma ? inserted in between Spe ? and Sph ? and the expression vector pART27 was modified with bar to replace existing NPT ?;(2)The tetravalent expression vectors carrying Cry1AC,bar and the bivalent RNAi structures were constructed successfully as pART27-MC1YCP-Cry1AC,pART27-MC2YCP-Cry1AC and pART27-MYCP-Cry1AC;(3)The concentrations of PPT(phosphinothricin)for screening PPT-resistant calli and plants of ROC22?FN39 and FN15 were determined as 0.8 mg/L for all three varieties in callus stage,and 0.9 mg/L,0.8 mg/L and 0.9 mg/L in seedling stage for ROC22?FN39 and FN15 respectively;.(4)Populations of transgenic sugarcane plants were created via agrobacterium mediated transformation and ballistic bombardment and PPT screening;(5)33 tetravalent transgenic sugarcane plants were identified and expression of the target genes was also detected by PCR,quantitative PCR and protein strips,of which 5 were mediated by Agrobacterium and 28 were from ballistic bombardment;...