Cloning And Expression Of Transcription Factor For Cis-isopentenyl Transferase Gene From Hevea Brasiliensis

Natural rubber is white latex by Hevea brasiliensis synthesis and secretion, the main component is polyisoprene, which has high temperature resistance, wear resistance and high flexibility and other advantages. Hevea brasiliensis production function mainly depends on distributed latex duct system implementation in the tree body. Natural rubber is a large number of cis-isoprene polymerization of high molecular weight polymer. Latex is the isoprenoid precursor, most of all isoprenoid precursor is C5 compound of isopentenyl pyrophosphate (IPP) and its dimethylallyl diphosphate (DMAPP), the present study shows that, IPP and DMAPP can be synthesized by MVA and MEP ways. From the analysis of synthetic rubber way, the key enzyme of participate in rubber biosynthesis, including a series of IPP isomerase and prenyl transferase, such as FPP synthase, GGPP synthase, rubber transferase enzyme (RT) and so on. This shows that the study of rubber transferase which dentification and regulation is the key to clarify the regulation mechanism of rubber biosynthesis in plants.The 5’regulatory sequence of HRT1 was isolated by the PCR-based DNA method based on the HRT1 sequences. And HbRT 5’regulatory was subcloned into the yeast bait vector. By yeast one-hybrid technology, the selection of relevant transcription factors, its 5 ’flanking regulatory functions carried out a preliminary study. Finally three transcription factors were identified, designated HbRZ, HbEIN and HbMYC.Bioinformatics analysis shows that the deduced HbRZ protein consisted of 156 amino acid residues with a calculated molecular weight of 17.5 kDa and isoelectric point (pI) of 5.92, HbRZ shared 66%、62%、62% and 62% identity with the HbRZ proteins from Ricinus communis, Vitis vinifera, Glycine max and Populus trichocarpa, respectively. A conserved domain search revealed that HbRZ contained a typical Zinc finger domain which has been found in ZIP protein. The deduced HbEIN protein consisted of 620 amino acid residues with a calculated molecular weight of 70.40 kDa and isoelectric point (pI) of 5.70 HbEIN shared 88%、82%、76%、71% and 70% identity with the HbEIN proteins from Ricinus communis, Populus trichocarpa, Cucumis melo, Nicotiana tabacum and Vigna radiata, respectively. A conserved domain search revealed that HbEIN contained a typical EIN3 superfamily domain which has been found in EIN3 protein.The deduced HbMYC protein consisted of 476 amino acid residues with a calculated molecular weight of 53.76 kDa and isoelectric point (pI) of 5.73 .HbMYC shared 67%、64%、60%、43% and 41% identity with the HbMYC proteins from Ricinus communis, Vitis vinifera, Populus trichocarpa, Brassica oleracea and Arabidopsis thaliana, respectively. A conserved domain search revealed that HbMYC contained an typical E-box/N-box specificity site, centromere-binding protein 1, cbp-1, HLH superfamily domain which has been found in HLH protein.Transcription pattern analysis revealed that HbRZ had high transcription in laticifer, low in leaf and flowers. Semi-quantitative RT-PCR results showed that HbEIN had high transcription in barks, low in leaf and bud, spent almost no expression. HbMYC had high transcription in laticifer, low in bud and barks, leaf and flowers also nearly no expression.Transcriptions of HbRZ, HbEIN, HbMYC in latex were not induced by ethylene. But transcription of HbRZ in latex was reduced by jasmonate, HbEIN in latex has no effect on the expression, the expression of HbMYC was first to reach the peak on the latex samples was treated of 2 hours by jasmonate and then showing a gradual downward.