橡胶树形成层组织的酵母双杂交cDNA文库构建及HbHDA6互作蛋白筛选

Construction of yeast two-hybrid cDNA library in cambium tissue of Hevea brasiliensis and screening of HbHDA6 interacting proteins

次生乳管是天然橡胶合成和贮存的场所,是由橡胶树树干树皮中维管形成层细胞分裂分化而来。次生乳管的数量与天然橡胶产量直接相关,而这些乳管的数量取决于形成层分化次生乳管的频率(乳管分化能力),是橡胶树产量育种的主要指标。前期研究中,我们发现组蛋白去乙酰化酶(HDA)抑制剂曲古抑菌素A(TSA)能诱导橡胶树乳管分化且组蛋白去乙酰化酶基因(HbHDA6)能够参与橡胶树乳管分化调控。由于组蛋白乙酰化修饰调控橡胶树次生乳管分化的分子机制尚未阐明,因此该文使用冠菌素(COR)诱导橡胶树形成层分化产生次生乳管的实验系统,以分离形成层组织为材料,构建酵母双杂交cDNA文库,以HbHDA6基因为诱饵来筛选酵母双杂交文库,确定与HbHDA6相互作用的蛋白。结果表明:(1)利用Gateway技术构建的均一化COR诱导橡胶树形成层组织的酵母双杂交cDNA文库,初级文库的容量为6.34×10^(6)CFU·mL^(-1),总单克隆数为1.27×10^(7),文库重组率为100%;次级文库的容量为7.72×10^(6)CFU·mL^(-1),总单克隆数为1.54×10^(7),文库重组率为100%。初级文库和次级文库的插入片段平均长度分别为1.1 kb和1.2 kb。(2)成功构建了筛选HbHDA6互作蛋白的pGBKT7-HbHDA6诱饵载体,并确认无自激活活性。(3)使用该诱饵载体对构建的酵母双杂交cDNA文库进行筛选,并通过NCBI_BLAST比对和去除重复以后,获得了22个与HbHDA6发生互作的蛋白,包括CLP1、ERF3、ERF4、HSP82、LARP6a、APT5、PP2A、FBA6等。该研究成果为解析组蛋白乙酰化修饰调控橡胶树次生乳管分化的分子机制提供了理论基础,为转基因改良橡胶树的产胶潜力提供了候选基因,为高性能天然橡胶遗传改良育种提供了新线索。

The secondary laticifer is the position for synthesis and storage of natural rubber(NR),which is differentiated from the vascular cambium cells of bark in stem of rubber trees(Hevea brasiliensis).The quantity of secondary laticifer is depended on the frequency of the secondary laticifer differentiation from cambia,which is the main index of yield breeding of rubber tree.In previous studies,we found trichostatin A(TSA),an inhibitor of histone deacetylase(HDA),can also induce laticifer differentiation,and the histone deacetylase gene(HbHDA6)is a participator in laticifer differentiation.Because of the molecular mechanism of secondary laticifer differentiation regulated by histone acetylation has not been clarified.Therefore,we construct a yeast two-hybrid cDNA library used the vascular cambium tissues treatment by coronatine(COR),and screening the yeast two-hybrid library by HbHDA6 gene as the bait,for determining the proteins interacting with HbHDA6.The results were as follows:(1)The homogenized yeast two-hybrid cDNA library of vascular cambium was constructed by the technology of Gateway.The capacity of the primary library was 6.34×10^(6)CFU·mL^(-1),the total number of clones was 1.27×10^(7),and the capacity of secondary library was 7.72×10^(6)CFU·mL^(-1),the total number of clones was 1.54×10^(7),and the recombination rates of two libraries were 100%.The average length of inserted fragments was 1.1 kb and 1.2 kb in primary and secondary library,respectively.(2)The bait vector of pGBKT7-HbHDA6 for screening the proteins interacting with HbHDA6 was successfully constructed and confirmed no self-activation activity.(3)The pGBKT7-HbHDA6 bait vector was used to screen the constructed yeast two-hybrid cDNA library,and 22 proteins interacting with HbHDA6 were obtained by NCBI_BLAST comparison and removing duplicates,including CLP1,ERF3,ERF4,HSP82,LARP6a,APT5,PP2A,APT5,FBA6,etc.The results provide a theoretical basis for analyzing the molecular regulatory network of the secondary laticifer differentiation of rubber tree,and provide candidate genes for the rubber production potential of genetically modified and a new clue for the genetic improvement and breeding of high-performance NR.