Isolation Of Tissue-Specific Promoters And Specific Expression Of BnLAS Driven By The Promoters

In the present study, three leaf specific-expreesed promoters were cloned from Arabidopsis and Pharbitis after bioinformatics analysis. The cloned promoters were used to driven the expressions of the GUS reporter gene and the BnLAS gene in different constructs, respectively. These constructs were introduced into wild-type Arabidopsis thaliana for functional verification. The major results were as follows:1. Cloning and functional verification of leaf specific-expressed promotersPrimers were designed according to the genome sequence of Arabidopsis and Pharbitis and then used for cloning the three leaf specific promoters. Promoter and expression vector (pBI121) were connected, and transformed into wild-type Arabidopsis thaliana via Agrobacterium-mediated genetic transformation. Transgenic plants were produced and the tissues from Positive T1 plants were sampled for GUS staining. Results form GUS staining showed that all these promoters were able to drive the expression of the reporter gene.GUS gene driven by these three leaf specific promoters expressed highly in leaves. There is espression in 50d-old roots but not in 30d-and 70d-old roots. Seventy-day shoots also had no staining. There were some diiferrences of expression pattern in hypocotyl, floral organs and pod. Compared with the GUS staining in hypocotyl we found that PRbcS-1A expression in the two ends of hypocotyl, but the other two promoters expression are concentrated on the lower end of the hypocotyl. The differences of expression in flower were that PSTP3 can drive the GUS gene expressing in anthers and stigma, but not petals, sepals and style; PRbcS-1A can drive the GUS gene expressing in Sepals, stigma and stamen, but not style; PPNZIP can drive the GUS gene expressing in sepals, anthers, stigma and style.In pods, a small amount of expression were found in pod skin of the transgenic plants which transformed by PSTP3::GUS, but no expression in seeds. PRbcS-1A can highly express in pod skin, but in seeds. PPNZIP expressed little in pod skin, but highly in a part of seeds. 2. Effects of BnLAS gene driven by leaf specific promoters on plant growth and developmentIn order to understand the impact of BnLAS gene driven by leaf specific promoters on plant growth, three expression vectors of BnLAS gene driven by leaf specific promoter were constructed and transformed into wild-type Arabidopsis. Through the observation of To plants we found that expression of BnLAS gene driven by promoter PSTP3 and PPNZIP had significant effects on plant growth and development. The changes included, for example, reduced fertility, deepened leaf color, shrinkage of the edge of the leaves, delayed bolting or no bolting, branching reduction. Since only one positive plant transformed by PRbcS-1A::BnLAS vector was obtained, we can not determine the effects of positive plant transformed by PRbcS-1A::BnLAS.