The Studies About The Promoters Of Rubber Laticiferious Specific Expression Genes: Hevein And Ref

As a ideal place for plant bioreacter, rubber tree has more advantages such as easy getting and purifing of the foregion proteins expressing in it's laticiferious system. The key step for this is to isolate the laticiferious specific promoters. By using the method RT-PCR, a 680 bp fragment of hevein cDNA had been cloned. The sequence analysis indicated that the fragment had a complete open reading frame that encoded a polypeptide of 204 amino acids with the N-terminal region containing 17 amino acids signal peptide, the mature hevein containing 43 amino acids and the C-terminal region containing 144 amino acids polypeptide. Then, a 526 bp fragment of ref cDNA had been cloned. The sequence analysis indicated that the fragment had a coding region of 414bp encoding 138 amino acids and a 3' end non-coding region of 112bp.Furthermore, By using the method of genome walking, a 1306bp fragment of hevein 5' end sequences, a 763bp fragment of ref 5' end sequences and a 317bp fragment of hmgl 5' end sequences, had been cloned respectively on the baisis of cloning these genes. The analysis of hevein 5' end sequences with the software of promoter prediction indicated that the core promoter regions could be in 865-909 and 1147~1189. The analysis of this sequence with the software of plant core found some elements such as At 1-motif, HSE, I-box, ABRE, ERE and EIRE. In the same way, the analysis of ref 5' end sequences found a core promoter region in 219-268 and the elements of Atl-motif, HSE, I-box, WUN-motif, P-box and G-box. Similarly the analysis of hmgl 5' end sequences found a core promoter region in 155~205 and the elements of CGTAC-motif, HSE, I-box and G-box.The 1306bp fragment of hevein 5' end sequence had been deleted into 4 fragments by the method of PCR, which replaced the CaMV 35 S promoter of pBI121 and constructed the plant chimeric plasmids including: pBIHl(1241bp), pBIH2(805bp), pBIH3(348bp) and pBIH4(224bp). So did the 763bp fragment of ref 5' end sequence, and a chimeric plasmid pBIRl(378bp) had been constrcted. The results of these chimeric plasmids, pBI121 and CK-in vitro transient expression test (3 days later) indicated that the samples contained pBIHl(l~1241) or pBIH2(436~1241) showed bright blue respectively, but the samples contained pBIH3(893~1241), pBffl4(1016~1241), pBIRl, pBI121 and CK- didn't showblue. It made out that the 1241bp and the 805bp fragments of hevein promoters were strong promters especially in latex. ABA could improve the expression of some of these chimeric plasmids. The samples contained 0.1%ABA(1 day later) including pBIHl, pBIH2, pBIH3 and pBIRl had been detected strong GUS activites, but samples contained 0.1%ABA(1 day later) including pBI121, pBM4 and CK- had not been detected GUS activites. pBIH2 had been tansfered into leaves of rubber tree. The results of the transient expression in leaves indicated that the veins of the leaves bombarded by the microcarriers coated with pBIH2 showed bright blue; veins of the leaves bombarded by the microcarriers coated with ddHaO did not show blue; veins of the leaves bombarded by the microcarriers coated with pBI121 did not show blue but in parenchyma cells there were some blue spots.In the research of transfering pBIH2 into tobacco through agrobacterium, 28 plantlets of Kan resistance had been obtained. 25 of them were detected by PCR and made out following results: A 800 bp fragment could be amplified in pBIH2 transgenic plantlets. We totally got 15 transgenic plantlets and used X-gluc for detecting GUS activities. The results showed that none GUS activitie had been detected in transgenic tobacco's roots, leaves and veins.