Identification Of Target Genes Of HbMYCs In Hevea Brasiliensis

Hevea brasiliensis is main source of natural rubber. Rubber tree is the most important economic crops in Hainan. The latex is biosynthesized and accumulated in laticifer of the rubber tree. The number of laticifer rings affects the latex yield.Jasmonic acid (JA) and ethylene are the main phytohormones that control latex biosynthesis and drainage of rubber tree. JA is a key signal for regulating the laticifer differentiation. JAZ protein is important repressor in JA signaling pathway. JAZ protein inhibits the expression of downstream gene by interacting with JA related transcription factors. In previous work, three MYC transcription factors interacting with HbJAZ1 protein successfully identified by yeast-two-hybrid screening, termed as HbMYC2, HbMYC3 and HbMYC4, respectively. In this work, in order to identify the target genes regulated by the HbMYCs transcription factors, the following studies were carried out:(1) The interaction between HbJAZ1 protein and HbMYCs transcription factors was verified by bimolecular fluorescence complementary (BIFC) technique. The following vectors pairs were first constructed, including pSPYCE-HbJAZ1 and pSPYNE-HbMYC3,pSPYNE-HbJAZ1 and pSPYCE-HbMYC3 vectors. The vectors were prepared and transformed into protoplast of Arabidopsis and observed by laser confocal microscopy. The interaction between HbJAZ1 and HbMYC3 elicited yellow fluorescence regardless of the C-terminal or N-terminal fusioned with HbMYC3.The results of both BIFC expression vectors indicated that HbMYC3 interacts with HbJAZ1 in cell nucleus.(2) Screening of cis-elements interacted with HbMYCs transcription factors by yeast-one-hybrid technique. First, JRE, GCC, ABRE, ERE, G-Box, CACG and DRE cis-elements were inserted into pAbAi vector for yeast-one-hybrid. Then prey vectors e.g. pGADT7-HbMYC2, pGADT7-HbMYC3 and pGADT7-HbMYC4,were constructed. Two types of vectors were co-transformed into YIHGold yeast strain.Yeast-one-hybrid result showed that HbMYC2, HbMYC3 and HbMYC4 were found to interact with G-box.(3) Using the bioinformatics tool, gene promoter sequence containing the G-box(CACGTG) cis-element was scanned in genome-wide to search for the potential genes involved in latex biosynthesis or drainage. The promoter sequences were futher analyzed by the PlantCARE and PLACE online software to confirm that the promoter of aquaporin gene HbPIP2;1 contains G-box. Yeast-one-hybrid experiment showed that HbMYC2, HbMYC3,and HbMYC4 interacted with HbPIP2; 1 promoter.(4) The interaction and regulation relationship between HbMYCs transcription factors and HbPIP2 ; 1 promoter were verified in plants. The plant expression vector pSP-luc + NF-HbPIP2; 1-P, containing the HbPIP2; 1 promoter mediated luciferase reporter gene, was first constructed. Then the overexpression vector for HbMYCs transcription factors,e.g. pCAMBIA1300-HbMYC2, pCAMBIA1300-HbMYC3 and pCAMBIA1300-HbMYC4, were constructed. Two types of vectors were co-transformed into protoplast of Arabidopsis and observed by laser confocal microscopy. The interaction and regulation relationship between the HbMYCs transcription factors and the HbPIP2; 1 promoter was reflected by the activity level of luciferase. The results showed that HbMYC2, HbMYC3, and HbMYC4 interact with the HbPIP2 ; 1 promoter and up regulated the expression of the luciferase reporter gene.(5) The effects of ethylene on the expression of HbMYC2, HbMYC3, HbMYC4 and HbPIP2 ; 1 genes were analyzed by quantitative realtime-PCR (qRT-PCR). The cDNA of rubber tree leaves treated with ethylene for Oh, 3h, 6h,12h and 24h were analyzed by qRT-PCR. qRT-PCR results showed that ethylene treatment inhibited the expression of HbMYC2, HbMYC3 and HbMYC4 genes in rubber tree, but induced the expression of HbPIP2: 1. These results suggest that ethylene can affect the expression of HbMYC2, HbMYC3, HbMYC4 and HbPIP2; 1 genes in rubber trees.The results of this study show that the JA-related HbMYCs transcription factors HbMYC2, HbMYC3,and HbMYC4 can interact with G-box cis- elements to regulate their target genes. HbPIP2; 1 is one of the target genes regulating latex drainage. The results of this study have important theoretical value for revealing the molecular mechanism of JA regulation of rubber biosynthesis and drainage.