Comparative Study Of The Seed Specific Promoters FAE1 And NapB Isolated From Brassica Napus On Their Structure And Function

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.A seed-specific promoter is an indispensable component of gene regulation in seed. It decides the tissue-specificity, the strength, time and space location of the gene expression. This study was aimed to clone two seed-specific promoters pNapB and pFAE1 from rapeseed and to elucidate their sequences, and the functions as regard to their strength, time and space location of the gene expression.The upstream regulatory region of the storage protein gene NapinB and the fatty acid elongase gene FAE1 were separated from Brassica napus c.v.Zhongyou 821 genomic DNA using homology-based method. Bioinformatic analysis showed that pNapB with 1136bp and pFAE1 with 1420 bp both contain the seed-specific cis-acting elements such as the RY box, G-box ,E-box though in different distribution. The NapB promoter also contained the ABA response distB and proxB (or B-box ABA response complex) and GCN4 motif, but FAE1 promoter did not.Plant expression vectors were constructed by ligating pNapB and pFAE1 with GUS gene and transferred into tobacco and rapeseed by mediation of Agrobacterium tumefaciens, respectively. A large number of transgenic tobacco plants were obtained. Through the histochemical analysis of T0 transgenic plants, it was shown that both pNapB and pFAE1 were seed-specific, but with leakage expression in other tissues. In transgenic tobacco, transformants with pNapB-GUS showed earlier and stronger GUS activity in the embryo development stage than those with pFAE1-GUS. However, two promoters showed a little difference in the final color of the matured embryoes after histochemically stained. The real-time quantitative PCR assay was made on the transgenic tobaccos plants expressing pNapB-GUS and pFAE1-GUS, respectively. Results indicated that the NapB-GUS mRNA started accumulation at about 12DAF (day after flowering) and soon reached the maximum level and kept plateau till the seeds mature. The pFAE1-GUS mRNA started accumulation at about 17DAF and reached the peak value at 24DAF and then decreased to a lower level.The same plant expression vectors pNapB and pFAE1 were transferred into rapeseed (B. napus), respectively by A. tumefaciens. Only pFAE1-GUS transgenic plants were obtained. Histochemical analysis of T1 transgenic plants showed that the seed–specific pFAE1-GUS was expressed not only in seeds but also in leaves and roots to a lesser extent. In pFAE1-GUS transformants blue color developed in the ovules since 25 DAF. Blue pigment was found in cotyledon but not in the seed coats. To determine the temporal developmental pattern of the pFAE1 and pNapB activity, we analyzed different torpedo stage embryos of rapeseed through the real-time quantitative PCR. pNapB-GUS mRNA began to accumulate since the 25DAF, earlier than PFAE1-GUS mRNA. On the other hand pFAE1-GUS mRNA began to accumulate since the 30DAF and increased rapidly to reach their maximum at the 35 DAF, followed by a decline as the seed approached maturity. The level of the pNapB-GUS mRNA increased linearly while pFAE1-GUS mRNA changed with the seed maturation in a way like a bell shape.This study compared two promoters pNapB and pFAE1 on their tissue specificity, activity, time and space patern to provide a theoretical basis of the application in genetic modification of seed components.