Cloning And Functional Analysis Of The Gene Promoter Of HbSERK1in Hevea Brasiliensis

SERK is a protein to make a plant cell obtained embryonic ability which belongs to the LRR-RLK super family. SERK gene was first isolated in hypocotyls embryonic cell culture of carrot, and studies have demonstrated that SERK gene is a molecular marker which has embryonic ability. It was shown that the key to the tissue culture of the rubber tree is to obtain the somatic embryogenesis. Based on HbSERKl gene, we isolated and analyzed the function of HbSERK1promoter. These researches provide a theoretical basis for expressional regulation mechanism of HbSERK1in somatic embryogenesis in Hevea brasiliensis.In this thesis, the promoter region of HbSERK1from Hevea brasiliensis was cloned. The result of the comparison with the genome sequence of Hevea brasiliensis and the sequence of HbSERK1can prove the HbSERK1promoter is a1395bp sequence in the upstream of the translation initiation site. A/T content in this sequence is up to66.2%, which meet the characteristic of eukaryotic promoter sequence. We also found many elements such as TATA-box, CAAT-box, CAT-box,02-site, ERE, ARE, TCA-element, GCN4-motif, ACA-motif in this promoter region of HbSERK1.In order to verify the HbSERK1promoter function, we constructed the corresponding deletion plant expression vectors after cloning the five5’end of the deletion promoters. We make deletions plant expression vector into tobacco leaves, by Agrobacterium-mediated vacuum infiltration method. The leaves cultured in dark for2days used to test GUS activity of situ analysis and quantitative analysis. The results showed that the four deletion expression vectors can drive the expression of GUS except for SP5. It proved that-1395bp-252bp area in HbSERK1promoter can drive expression of GUS, which suggests HbSERK1promoter is active.The plant expression vectors were transformed to the tobacco by Agrobacterium-mediated. Our preliminary gain transgenic tobacco containing HbSERK1promoter deletion expression vectors after Hygromycin B screening and PCR test. It also showed the conclusion that the four deletion expression vectors can drive the expression of GUS except for SP5. It proved that-1395bp-252bp area in HbSERK1promoter can drive expression of GUS, which suggests HbSERK1promoter is active. The results of stable expression and transient expression have the consistency in GUS activity test, which proved HbSERK1promoter has bioactivity.