Cloning and function analysis of starch synthase SSⅡa promoter in maize

Objective To clone maize (Zea mays) starch synthase SSⅡa promoter, analyze its function, and provide a basis for its future research and application.

Method The SSⅡa 5′ flanking sequence was found on Maize GDB by BLASTing the maize genome sequence published on NCBI, and the SSⅡa promoter was cloned from maize B73 using PCR. We analyzed the cis elements of the promoter using PlantCare. Fragments of 1 407, 867, 633, 483, and 365 bp were cloned with specific primers, and were inserted into the plant expression vector pCAMBIA3301, respectively. Five plant expression vectors with different 5′ deletions of the SSⅡa promoter were constructed and named P1, P2, P3, P4 and P5.The transgenic Arabidopsis thaliana plants were obtained through Agrobacterium-mediated transformation.

Result A DNA fragment of 2 526 bp was obtained by PCR amplification with maize B73 genome DNA as template and SSⅡaF/SSⅡaR as specific primers. The positive A.thaliana plants, which were screened by herbicide, had gus gene by PCR detection. The histochemical analysis of GUS showed that the expression vectors of five promoters were blue in leaves and pods at maturity. The quantitative analysis of gus gene showed that among five transgenic A.thaliana at maturity, the expression level of P1 in leaves was the highest and the others were basically the same, and the expression levels of P1 and P2 in seeds were similar, both being higher than those of P3, P4 and P5.

Conclusion The maize SSⅡa promoter has been successfully cloned. The five constructed expression vectors with different 5′ deletions of the SSⅡa promoter all have activities in transgenic A.thaliana, and the promoters with the length of 1 407 bp (P1) and 867 bp (P2) have endosperm specificity.