大豆种子特异性启动子的克隆及序列分析

Isolation and Sequencing Analysis of the Seed-specific Promoter from Soybean

通过PCR技术从大豆品种吉林 4 3基因组DNA中分离到大豆β 伴球蛋白α 亚基基因启动子片段 (BCSP4 89) ,对此片段采用TAILPCR技术进一步延伸 ,获得了启动子片段BCSP6 6 6。序列分析表明 ,在启动子片段BCSP6 6 6中含有多种种子特异性启动子所特有的序列元件 ,如A T富含序列元件、RY重复序列元件、AGCCCA序列元件、TACACAT序列元件、ACGT序列元件、E盒等。据此推测该启动子片段具有种子特异性启动子活性。以该片段构建种子特异性表达载体pBI12 1 6 6 6 ,以FloralDip方法转化拟南芥 ,转基因拟南芥中的GUS酶荧光光度分析和组织化学检测均表明 ,GUS基因在启动子片段BCSP6 6 6调控下获得了种子特异性表达。

The low level of foreign genes' expression in transgenic plants is a key factor that limits plant genetic engineering.Because of the critical regulatory activity of the promoters on gene transcription, they are studied extensively to improve the efficiency of plant transgenic system.The constitutive promoters, such as the CaMV 35S promoter, were usually used in plant genetic engineering.But those constitutive promoters continuously expressed their downstream genes during the whole life span in all the tissues of the host plants.This was not only wasteful to host plants' energy, but also harmful to host plants and usually affecting their agronomic characters.In contrast, the seed-specific promoter only expresses its downstream genes from mid to late stage of seed maturation, and there is no expression or much lower expression in other tissues.So the seed-specific promoters are distinguished for the improvement having brought to plant quality engineering.It was the aim to characterize a new seed-specific promoter and improve the grain quality.The promoter region of β-conglycinin α-subunit gene was isolated from the genomic DNA of soybean Jilin 43 by PCR method, and successfully extended this fragment by TAIL PCR method and obtained the promoter fragment BCSP666(Fig.1-3).Sequencing analysis showed that the cloned fragment BCSP666 contained all of the motifs, such as RY repeat element, AG/CCCCA motif, TACACAT motif, ACGT motif, A/T rich motif, and E-box etc., that constituted the seed-specific promoter activity(Fig.4).Based on this sequencing analysis, the seed-specific promoter activity of the promoter fragment BCSP666 was predicted.And then the seed-specific expression vector pBI121-666, which contained GUS reporter gene, was constructed with the fragment BCSP666(Fig.5,6).Transformation of Arabidopsis thaliana plants by Agrobacterium-mediated floral-dip method with the recombined vector pBI121-666 was conducted.The transgenic plants were selected on the kanamicin-resistant MS medium, and confirmed by southern blot analysis(Fig.7).Fluorometric and histochemical analysis of GUS enzyme activity of the transgenic Arabidopsis thaliana plants (Fig.8,9) approved our original suggestion.So, the seed-specific promoter BCSP666 is obtained and characterized.