橡胶树膜系统酵母双杂交cDNA文库构建及HbSRPP7互作蛋白筛选  

Construction of a Normalized Hevea brasiliensis Latex MYTH cDNA Library and Screening of HbSRPP7 Interacting Proteins

作　　者：聂智毅[1] 康桂娟[1] 覃怀德[1] 曾日中[1] NIE Zhiyi;KANG Guijuan;QIN Huaide;ZENG Rizhong(Rubber Research Institute,Chinese Academy of Tropical Agriculture Sciences/Key Laboratory of Biology and Genetic Resources of Rubber Tree,Ministry of Agriculture and Rural Affairs,Haikou,Hainan 571101,China)

机构地区：[1]中国热带农业科学院橡胶研究所/农业农村部橡胶树生物学与遗传资源利用重点实验室,海南海口571101

出　　处：《热带作物学报》2023年第9期1735-1744,共10页Chinese Journal of Tropical Crops

基　　金：海南省基础与应用基础研究计划(自然科学领域)高层次人才项目(No.2019RC328);国家自然科学基金项目(No.31770642)。

摘　　要：橡胶树橡胶粒子上的蛋白质在橡胶生物合成一系列反应过程中起着关键作用,它们直接决定着橡胶烃分子的数量和大小,影响天然橡胶的产量和质量。小橡胶粒子蛋白(small rubber particle protein,SRPP)是丰度仅次于橡胶延伸因子(rubber elongation factor,REF)的橡胶粒子蛋白组分,与橡胶粒子发育和橡胶生物合成密切相关。目前已知橡胶粒子上存在多种SRPP家族蛋白,但大部分成员的功能尚不清楚。SRPP可能通过与其他橡胶粒子蛋白相互作用发挥功能。为了筛选SRPP蛋白家族成员之一Hb SRPP7的互作蛋白,本研究利用位点特异性重组技术构建了原始库容为1.5×10^(7)CFU的均一化橡胶树胶乳膜蛋白酵母双杂交系统(membrane yeast two-hybrid system,MYTH)cDNA文库,该文库插入片段平均长度大于1500 bp,重组率约为100%。构建了用于MYTH系统筛选HbSRPP7互作蛋白的pBT3STE-SRPP7和pBT3SUC-SRPP7诱饵载体并确认其能在NMY32酵母菌株中正确表达,无自激活活性。使用pBT3STE-SRPP7诱饵质粒对胶乳MYTHcDNA文库进行筛选,获得21个HbSRPP7候选互作的蛋白,包括3个REF家族蛋白(HbREF1、HbREF3和HbREF8)、2个SRPP家族蛋白(HbSRPP1、HbSRPP2)、2个活性氧清除相关蛋白(thioredoxin H-type-like、L-ascorbate peroxidase 2)以及5个胁迫相关蛋白(high mobility group Bprotein 2-like、RPM1-interacting protein 4-like、stress-related protein-like、salt stress-induced hydrophobic peptide ESI3-like和F-box/kelch-repeat protein)。结果显示HbSRPP7除了可能通过与橡胶生物合成相关的橡胶粒子蛋白互作参与橡胶生物合成,也可能通过与生物和非生物逆境胁迫相关蛋白互作,响应橡胶树乳管生物和非生物逆境胁迫系统信号,参与橡胶粒子上的橡胶生物合成调控。该研究结果有助于了解SRPP家族蛋白的功能,为揭示橡胶粒子上参与橡胶生物合成的蛋白复合体组成,阐明橡胶生物合成及其调控的分子机制奠定基础。The proteins on rubber particles of Hevea brasiliensis play key roles in a series of reactions of rubber bio-synthesis.They directly determine the number and size of rubber molecules,thus affecting the yield and quality of natural rubber.Small rubber particle protein(SRPP)is the second highest abundance protein in rubber particles follow-ing after the rubber elongation factor(REF),which is closely related to rubber particle development and rubber biosyn-thesis.At present,it is known that there are many SRPP family proteins on rubber particles,but the functions of most SRPP family proteins have not been identified.SRPP may function by interacting with other rubber particle proteins.To screen the interaction protein of HbSRPP7,a normalized H.brasiliensis latex cDNA library was constructed based on the membrane yeast two-hybrid(MYTH)system.The capacity of the cDNA library was 1.5×10^(7) CFU,the average li-brary recombination rate was about 100%,and the average amplification sizes of insert fragments in the cDNA library were above 1500 bp.The pBT3STE-SRPP7 and pBT3SUC-SRPP7 bait vectors were constructed and confirmed that they could be correctly expressed in the NMY32 yeast strain without self-activating activity.The latex MYTH cDNA library was screened using the bait plasmid pBT3STE-SRPP7,and 21 candidate proteins of HbSRPP7 were obtained,including three REF family proteins(HbREF1,HbREF3,and HbREF8),two SRPP family proteins(HbSRPP1 and HbSRPP2),two reactive oxygen species scavenging-related proteins(thioredoxin H-type-like and L-ascorbate peroxi-dase 2),and five stress-related proteins(high mobility group B protein 2-like,RPM1-interacting protein 4-like,stress-related protein-like,salt stress-induced hydrophobic peptide ESI3-like and F-box/kelch-repeat protein).It is shown that HbSRPP7 may participate in rubber biosynthesis through interaction with the rubber particle proteins related to rubber biosynthesis.In addition,HbSRPP7 may also participate in the regulation of rubber biosynthesis on rubber particles by inte

关 键 词：橡胶树 HbSRPP7 膜蛋白酵母双杂交系统 CDNA文库 互作蛋白 

分 类 号：S435.661[农业科学—农业昆虫与害虫防治]