Genetic Transformation Of Atritficially Modified Endochitinase And Glucanase Genes In Maize

Maize is one of the most important crops in China.But each year as a result of the disease caused by the loss of up to about10%of Maize.In Southwest China, maize sheath blight has become the one of the major diseases due to high tempera-ture and high humid. In recent years, although in order to reduce the sheath blight on southwestern corn damage and loss,a series of measures has been implemented, but due to the introduction of implementation of free(less)cultivation, increasing inf-ertilizer input, Promotion of the Planting density and the lack of resistantvarieties, the sheath blight has a growing trend.So breeding the maize which is resistant to sheath blight is imminent.Numerous studies reveal that sheath blight is cased by the infection of DeuteromycotinaRhizoctonia solani. While endochitinase and glucanase kill the pathogenic bacteria by degradation of Rhizoctonia solani cell wall,At the same time,the two has a synergistic effection to make it more effective suppression of the effect of the sheath blight.So molecular biology techniques will be able to inhibit exogenous gene into Maize,and it is the most effective method of the cultiv-ating resistant maize to sheath blight and controling completely the sheath blight.In this study, the target genes of Trichoderma endochitinase and soybean gluca-nase were modified by the codon preference.Two recombinant vectors were constru-cted with constitutive promoter CaMV35S and Ubiquitin,named pCAMBIA3301-glu-chi, we transformed the exogenous source gene,followed by Agrobacterium-mediated transformation into maize embryogenic callus of inbred line18-599(red).Resistant plants were obtained by resistance screening and regenerative differentiation.The main results are as follows:1.Acquisition of target gene Designed Trichoderma endochitinase and soybean glu-canase genes nucleotide sequences were send to Beijing AudioC-odes peak Biotech CoLtd for the synthesis of the whole sequence.The synthetic genes were sequenced, the sequencing results were consistent with the design of gene sequences.After mo-dified,by the genes with the wild-type Trichoderma endochitinase and soybean glucanase CDS sequences comparison analysis,we found the total alteration of endo chilinase base numbers were218, the numbers of codons were192, the content of G+C was from53%to64%; but the total alteration of glucanase base numbers were263, the numbers of codons were239, the contents of G+C were from44%to65%.2.Construction of expression vector After modified, the genes were cloned into the vector pCAMBIA3301.And the maize ubiquitin promoter and CaMV35S promo-ter-driven plant expression vector(pCAMBIA-3301-glu-chi)was constructed with bar geneas a selectable marker.3. Callus induction and genetic transformation Embryogenic callus were obtained froml8-599(red) immature embryo of maize inbred lines.By agrobacterium-mediated and gene gun-mediated transgenic resistant plants were screened with Basta.At last,10were positive TO transgenic material by PCR,and T1generation of transgenic materials were for PCR to verify the stability of hereditary.