Identification And Analysis Of Transcription Factor For Rubber Transferase HRT2 Gene

Laticifer is a specialized secretory tissue which synthesized the natural rubber,it consists of laticifer cells.The rubber particles in laticifer cells are the organelle of natural rubber synthesis.Natural rubber synthesis is a typical isoprenoid metabolic pathway in which one of the main reactions is rubber hydrocarbon chain’s extending.Until now,we don’t well know the biochemical mechanism of the rubber hydrocarbon chain’s extending,but it is consder that the isopentenyl transferase is indispensablein natural rubber synthesis.It is prove that the rubber transferase HRT2 plays an important role in the synthesis of rubber,but transcriptional regulation mechanism of this enzyme was rarely report.This study intend to identify the HRT2 rubber transferase gene transcription factor,it laysthe foundation for the study of natural rubber biosynthesis regulation mechanism.There sults were follows:(1) The expression of genes encoding for the rubber biosynthetic key enzyme HbFDPS, HbSRPP and HbHRT2 were significantly different between rubber tree clone Reyan 8-79 and PR107 with differential potential of rubber synthetic efficiency, the most difference of which is for the HbHRT2 expression.(2) A differential gene fragment was screened from comparative transcriptome database of Reyan8-79 and PR107 latex. Gene expression analysis showed that its transcript level in Reyan8-79 was about 1.7 times of that in PR107.(3) The differential gene fragment’s fulllength cDNA sequence was cloned. It is 1105bp in length, containing an open reading frame of 714bp which encodes 237 amino acids. Thel to 60 amino acids was the MADS domain, and named HbMADS3-2. Phylogenetic analysis showed the HbMADS3-2 shared 80% and 82% indentify with the MADS protein from Ricinus communis and Jatropha curcas, respectively.(4) The expression of HbMADS3-2 was tissue-specific. Its expression was high in the latex and weak in the flower and leaf while not detected in roots, fruit and young stems. The methyl jasmonate up-regulated the expression of HbMADS3-2 with in 8 hours. The ethrel up-regulated its expression at 2 h while down-regulated its expression from 1 d to 3 d.(5) The MADS-box binded CArG-box element was present in the promoter of HbHRT2 and HbSRPP,but not identified in the promoter of HbFDPS. Yeast one hybrid vector pGADT7-HbMAD3-2 was constructed. It was identified that the HbMADS3-2 combined with the promoter of rubber transferase gene HRT2 by piont-to-point yeast one hybrid, suggesting that HbMADS3-2 was the transcription factor of rubber transferase gene HRT2.(6) The interaction of HblMYC3 with HblMYC2 was identified by yeast two-hybrid screening. Alignment showed the HblMYC2 was as same as HbMYC which was reported to be the transcription factor of the rubber transferase gene HRT2. However, HblMYC3 could not bind the HbHRT2 promoter as detected by point-to-point yeast one hybrid.The conclusions as follows:the HRT2 gene’expression in the high efficiency rubber synthesis reyan 8-79 was significantly higher than the lower efficiency rubber synthesis PR107.The HRT2 gene was regulated by several transcription factors, except for the gene HbEIN3, there also have HblMYC2、HblMYC3 and HbMADS3-2..The transcription factors may form a complex to integrate the ethylene and JA signaling pathway on the transcriptional regulation of the HRT2 gene.