Comparative Analysis Of Structure And Function Of Three Seed-specific Promoters From Brassica Napus

With the development of biotechnology, directional improvement of seed quality through genetic engineering has become an important means of agriculture science and technology innovation, seed specific promoters can finely control gene expression as well as its spatial and temporal expression pattern in the seed. This will be of great significance in improving seed quality though genetic engineering. However, research reports on seed specific promoters in B.napus are few. In this study, three seed-specific genes were confirmed from B.napus after expression analysis and bioinformatics analysis. Three seed- specific promoters were cloned and used to drive the expressions of the GUS reporter gene and the constructs were introduced into wild-type Arabidopsis thaliana for functional verification. Then three promoters structural and sequences were analyzed. The major results are as follows:The results of gene chip analysis of the pod wall and seed of the B.napus in 21 days after pollination showed that the F7 K, F28 K and F33 K genes were expressed specifically in seeds. For validation of microarray results accuracy and the three genes in the pod wall and expression in seed during the silique development, we did the expression analysis of these three genes in B.napus after flowering at different developmental stages of silique. The results showed that the three genes were only expressed in the seeds during the seed development period.According to the B.napus genomic sequence, we designed primers and cloned three seed-specific promoters by inserting the promoter fragment to vector p BI121, the constructs with respective promoter-GUS reporter fusion were introduced into wild-type Arabidopsis via Agrobacterium-mediated transformation. Transgenic plants were obtained and the tissues from positive T2 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 in different tissue in Transgenic plants.The GUS gene driven by these three seed specific promoters expressed after seed germination for 2 days, There were some difference of expression pattern in 5d- and 10d-old day seedlings. F7 K promoter can drive the GUS gene to express in the vascular tissue of the root while F28 K and F33 K promoter can drive the GUS gene to express in cotyledon, the joint point of hypocotyl and root in 5d-old seedlings, but not in any organ of 10d-old seedlings.All the three promoters were not expressed at the apices, and showed some differences in their expression pattern in floral organs and pods. F7 K promoter can drive the GUS gene to express in anthers, stigma, filaments and petals as well as in funicle and abdominal suture in the early stage of pod development but only in funicle later. On the other hand, the F28 K and the F33 K promoter are not able to drive the GUS gene expressing in any organs of the flower and pod in different growth stages.Structural and sequences analysis showed that three promoters contain the TATA-box, CAAT-box, TC repeats and 5 UTR Py-rich stretch.At the same time the three promoters also contained the seed-specific cis-acting elements such as the ABRE motif,GCN4 motif or AACA motif.