Genetic Transformation Of Wheat With Resistance Genes Against Fusarium Head Blight And Identification Of Materials Transformed With Ethanol Genes

As an important staple food,wheat is cultivated worldwide.Infected by Fusarium Graminearum,Fusarium head bligh(FHB)is a global fungal disease especially in warm and humid regions.FHB not only causes severe yield reduction,but also secrets various toxins remaining in the food chain,which seriously threaten human and animal health.The best solution to solve the FHB is to cultivate the resistant varieties.However,the poor resistant germplasm is the key factor to limit the development of conventional breeding.With the rapid development of gene engineering and bioinformatics,people begin to search for FHB-resistant genes in a more extensive field.The FHB-resistant genes and RNAi fragments were construct as a minimal expression cassette,and then transformed into the Xiangmai 76 and Yangmai 158 through gene gun and Agrobacterium,respectively.The main purpose is to obtain FHB-resistant lines and provide the new material for the FHB-resistant breeding.Main results are as follows:1.Selection of positive wheat carrying RNAi fragments and identification of the resistance to FHB.1)Through gene gun,the two RNAi fragments of FHB chitin synthases gene-chs3bAS2-chs3bAS2,chs3bAS5-chs3bAS1,and marker gene-PMI,co-transformed into Xiangmai 76,and two transformed cultivars were obtained,naming ZC1 and ZC2.The PCR identification of T1 to T3 transgenic lines proved that transgenic genes can be stably inherited but two target genes separated.Single-floral inoculation of T2 and T3 transgenic lines indicated that FHB-resistance of ZC1 and ZC2 transgenic lines was significant increased compared with non-transgenic lines-Xiangmai 76;FHB-resistance of transgenic lines carrying both chs3bAS5-chs3bASl and chs3bAS2-chs3bAS2 is much better than carrying only chs3bAS5-chs3bAS1,and the significant difference was formed in T3 transgenic lines.HIGS technology,using the Chs3b gene's different RNAi fragments as target,can effectively improved wheat resistance to FHB and the resistance can be accumulated.2)RNAi fragments of FHB-related chitin synthase and glucan synthase genes glsAS3-glsAS6,chs3bAS2-chs3bAS2 and chs3bAS5-chs3bAS1,and marker gene-PMI were co-transformed into Xiangmai76,and a transgenic line A3-3 was obtained.PCR identification of T1 and T2 transgenic lines showed that the transgenic genes present linked inheritance;The transgenic lines of A3-3 were hybridized with Xiangmai 76,and the PCR identification indicated that transgenes through sexual hybridization transferred into non-transgenic Xiangmai 76 and were linked inheritance.3)Herbicide resistant gene-Bar and Fusarium RNAi fragments-CYP51chs3bAS2-chs3bAS2 and chs3bAS5-chs3bAS1 co-transformed into Xiangmai 76 by gene gun.6 transgenic lines were obtained through PCR identification and carried 3 different genotype:BC1 lines carrying both marker gene Bar and 3 target genes-CYP51?chs3bAS2-chs3bAS2 and chs3bAS5-chs3bAS1;BC4 lines only having Bar and chs3bAS2-chs3bAS2;while BA1,BA2,BC2,and BC3 lines only carry target gene chs3bAS2-chs3bAS2.4)The RNAi fragments of Fusarium Graminearum-glsAS3-glsAS6 and 6×chs3b5-2 having different regulatory elements and marker gene PMI was co-transformed into Xiangmai 76,with the result of obtaining 1 transgenic line DC.PCR identification of TO transgenic lines proved that both marker gene PMI and target genes glsAS3-glsAS6,6×chs3b5-2 were transformed.5)Through agrobacterium-mediated method,the Fusarium graminearum gene TamiR(4)and marker PMI were both transformed into Y4-2.2.Selection and analysis of transgenic wheat carrying FHB-resistant genes1)The anti-bacterial Fengyuan gene and marker gene PMI were transformed into Xiangmai 76 by gene gun.And then two transgenic lines FBI and FB2 carrying FneB and PMI were obtained.PCR identification of transgenic lines from T1 to T4 lines showed that target gene fneB and marker gene tend to be homozygous and linkage inheritance.3.Identification and agronomic analysis of transgenic wheat with the Glyoxylate pathway genes.1)T5 and T6 generation lines of ZQ1 and ZQ2(Xiangmai 76 as receptor cultivar),carrying 3 key enzyme genes(AtGDH,GcL,TsR)of glycolate pathway,were identified by PCR and RT-PCR.The results show that transgenes in both of two transgenic lines having been completely homozygous and highly expressed in RNA level.Agronomic analysis of ZQ's T6 generation lines:the plant type of ZQ1 and ZQ2 lines is compact,which is different from the wild type plant.2)The 3 transgenic lines labelling UD,CD and ZX,carrying glycolate dehydrogenase fusion gene-DEF were PCR identified.The promoter of UD and ZX lines transgene is Ubi,while CD line is CMPS.PCR identification showed that the transgene of T5 and T6 generation lines of UD is great segregated and the transgene of T3 and T4 generation lines-CD and ZX can be stabley inherited and tend to be homozygous.