巴西橡胶树HbMYB44转录调控天然橡胶生物合成机制研究

R2R3-MYB transcription factors regulate important physiological processes such as plant secondary metabolites and stress responses through combining with MBS motif in the promoters of target genes. How they function in natural rubber (NR) biosynthesis is still unclear. We identified a R2R3-MYB transcription factor HbMYB44 by screening the yeast one-hybrid library. Its expression was significantly upregulated by ethylene. Further study revealed that HbMYB44 interacted with MBS motif present in the promoters of key enzymes genes in the NR biosynthesis. We hypothesize that HbMYB44 plays an important role in regulating NR biosynthesis and ethylene signaling in rubber tree. In this study, we will focus on, 1) identification of interaction protein and target gene of HbMYB44 through yeast two-hybrid and chromatin immunoprecipitation technologies, respectively; 2) overexpression of HbMYB44 in Arabidopsis mutant atmyb44 for functional complementation analysis, and proving its function in regulating the NR biosynthesis and ethylene signaling; 3) systematically analyzing genes expressions of HbMYB44 and interaction protein under various stimuli (tapping, ethylene, etc.), and elucidation of the transcriptional regulation mechanism of HbMYB44 in NR biosynthesis. These results in this study will lay a foundation for further revealing the regulation networks of NR biosynthesis as well as developing new stimulants to increase NR yield in rubber tree.

R2R3-MYB转录因子通过结合靶标基因启动子的MBS元件调控植物次生代谢和逆境响应等重要生理过程，但其如何调控天然橡胶（聚异戊二烯）生物合成的机制尚不清楚。我们从酵母单杂交文库筛选出R2R3-MYB转录因子HbMYB44，该基因受乙烯诱导显著上调表达，进一步研究发现HbMYB44与橡胶生物合成关键酶基因启动子的MBS元件结合。我们推测，HbMYB44是橡胶树中调控橡胶生物合成和乙烯信号传导的重要环节。本项目拟采用酵母双杂交和染色质免疫沉淀技术筛选和鉴定HbMYB44互作蛋白及其调控的靶基因；通过转化拟南芥atmyb44突变体进行功能互补验证，证明HbMYB44调控橡胶生物合成关键酶及其在乙烯信号传导中的功能；系统分析HbMYB44及其互作蛋白基因在割胶、乙烯等刺激下的表达模式，阐明该基因转录调控天然橡胶生物合成的分子机制，为进一步阐明天然橡胶生物合成调控网络和新型产量刺激剂研发奠定基础。

R2R3-MYB转录因子通过结合靶标基因启动子的MBS元件调控植物次生代谢和逆境响应等重要生理过程，但其如何调控天然橡胶（聚异戊二烯）生物合成的机制尚不清楚。基于前期筛选出R2R3-MYB转录因子HbMYB44基因受乙烯诱导显著上调表达，并与橡胶生物合成关键酶基因启动子的MBS元件结合，我们提出HbMYB44是橡胶树中调控橡胶生物合成和乙烯信号传导的重要环节这一科学问题。本项目基于橡胶树基因组平台，鉴定了橡胶树31个MYB转录因子，并采用荧光定量PCR技术（qRT-PCR）证明HbMYB44显著受乙烯和白粉病等多因素诱导上调表达，说明HbMYB44是逆境信号和激素信号交互的核心；采用酵母双杂交（Y2H）和双分子荧光（BiFC）技术鉴定HbMYB44互作蛋白是橡胶树天然橡胶合成关键酶HbSRPP1；通过转化拟南芥atmyb44突变体进行功能互补验证，证明过表达HbMYB44基因拟南芥（OE）显著提高了对干旱、盐胁迫的抗性；OE中异戊二烯合成关键酶AtISPF1、AtDXR、AtIPP1和AtHGM1基因显著上调表达，证明HbMYB44上调拟南芥异戊二烯生物合成关键酶基因表达；系统分析HbMYB44在割胶、乙烯等刺激下的表达模式，阐明该基因转录调控天然橡胶生物合成的分子机制，为进一步阐明天然橡胶生物合成调控网络和新型产量刺激剂研发奠定基础。