Cloning And Characterization Of The MYB Gene Family Of Hevea Brasiliensis

Rubber biosynthesis and drainage process in Hevea brasiliensis is a complicated wounding response that is regulated by various environmental factors and phytohormones such as jasmonic acid(JA)and ethylene(ET).JA and ET regulate the physiological responses by regulating downstream transcription factors,such as regulating nicotine biosynthesis and cold stress response by regulating MYB gene expression.In order to study the molecular mechanism of rubber biosynthesis and abiotic stress regulated by the JA-and ET-related MYB genes,the MYB gene family of Hevea brasiliensis was cloned and its function was identified.The following results were obtained:(1)In silico analysis of transcriptome data and cDNA sequences deposited in NCBI GenBank,141 MYB genes of Hevea brasiliensis(HbMYBs)were obtained through comparison of highly conserved DNA domain of the MYB gene.(2)The reversed Northern blotting(Macroarray)was used to analyze the differential expression patterns of HbMYBs genes after wounding,ET and JA treatments.Ten differentially expressed HbMYBs genes were verified by qRT-PCR and then subjected to bioinformatics analysis.(3)The pCAMBA1300-GFP-HbMYBs vector and the pGBKT7-/HbMKYBs vector were constructed,and the subcellular localization and yeast transcriptional activation of HbMYBs were analyzed.The results showed that most HbMYBs genes were located in the nucleus and showed transcriptional activity,suggesting that these proteins have characteristics of transcription factor.(4)The pGADT7-HbMYBs vector was constructed,and it was co-transformed with the existing pGBKT7-JAZs,pGADT7-HbICEs and other vectors into Y2H Gold yeast strains,and then screened for protein interaction.The result showed that HbMYB15,HbMYB75 can interacted with HbICE1;HbMYB15,HbMYB75 and HbMYB21 interacted with HbICE2,suggesting that these HbMYBs may be involved in cold stress response of rubber tree.The qRT-PCR results also revealed that these three genes were regulated by cold treatment.(5)The pGADT7-HbMYBs vector was co-transfected with pAbAi vector containing multiple cis-acting elements into Y1H Gold yeast strains,and the cis-acting elements that interacted with the rubber MYB transcription factor were screened.The results showed that HbMYB15 HbMYB21,HbMYB35,HbMYB75 can interact with G-box,JRE;HbMYB76 can interact with ABARE,GCC,DRE;HbMYB17,HbMYB77 can interact with GCC.The results of this study indicate that HbMYBs transcription factors were induced by multiple hormones in Hevea brasiliensis.The HbMYB15,HbMYB75 and HbMYB21 genes may be involved in the cold stress response.HbMYBs can bind to a variety of cis-acting elements,which lay a foundation for screening HbMYBs gene target genes in the future.