Cloned And Functional Analysis Of Cotton Seed-specific Promoter

With the development of biotechnology, plant seeds become not only the carrier of traditional agricultural products but also the carrier of high-value chemicals and pharmaceutical products. Orientational modification of the ingredients in plant seeds through genetic engineering has become an important part of technology innovation in agricultural sciences in this century. Constitutive Promoter is the most generally used promoter in plant genetic engineering. This kind of promoter can drive the non-specifie, sustained, stable expression of target gene, but not regulate the gene expression with the temporal and spatial control, which may result in the waste of plant nutrition, and even damage to the transgenie plant. So the tissue-specificity, the strength, time and space location of the gene expression of the seed-specific promoter show a significant advantage. Therefore, it is urgent to study expression control mechanism of seed-specific promoter for theoretical and practical significance.Late embryogenesis abundant protein (LEA protein) is a protein family accumulated in late embryonic development of seed. Their sequence can provide a good source to obtain the seed-specific promoter. In order to provide the experimental proof and theoretical basis of seed-specific promoter, this study has cloned two seed-specific promoters (D113 and D34) from Gossypium arboreum L. and analyzed their seed-specific sequence components and functions.In this study, the 1262 bp 5′flanking sequence of D113 gene and the 1450 bp 5′flanking sequence of D34 gene were cloned by PCR from Gossypium arboreum L. The similarity of the promoter from D113 and D34 promoter were 97.43% and 94.27% respectively. Bioinformatic analysis showed that both of them contain the seed-specific cis-acting elements such as the ABREs, G-box, E-box and the A\T rich sequence and in the D113 sequence also found RY box. Further analysis suggests that the interaction between promoter elements can enhance the seed-specific of promoter.Two new plant expression vectors named pBI121-D113 and pBI121-D34 were successfully constructed by fitting method, in which the reporter gene GUS is drived by D113 and D34 promoter.These two vectors were used to transferred into Arabidopsis thaliana by mediation of Agrobacterium, and transgenetic plants were obtained from T0. Histochemical analysis of both of the two kind of T1 transgenic plants showed that the leaves, roots, stems were dyed, except for seeds, which indicated that both promoters were seed-specific and nomally can not induce gene expression in other organisms.The genetic transfer systeme for Arabidopsis thaliana used in this research was efficient. Tested of concentration on kanamycin and Agar A by gradient concentreation. The results showed that the most optimal of kanamycin was 50 mg / L and the Agar A was 58 g / L.