橡胶树PR107和CATAS8-79中胶乳蛋白的差异分析及磷酸化蛋白的鉴定 作者：王丹徐兵强孙勇彭存智常丽丽仝征
基利冈萨雷斯来源：《热带作物学报》2022年第05期丙硫咪唑
摘要：橡膠树是合成天然橡胶的重要植物。PR107和CATAS8-79是具有不同产排胶性状的2个无性系。在以往的研究中，胶乳中蛋白表达差异被认为是影响天然橡胶合成的关键因子之一。然而，这2个无性系之间与胶乳产量相关的蛋白质图谱尚未明确。本研究采用蛋白质组学方法对这些蛋白质进行鉴定，有助于探讨巴西橡胶树胶乳合成机理。经双向凝胶电泳和质谱鉴定分析，共获得65个差异表达蛋白信息。通过磷酸化蛋白质组分析，在PR107胶乳中鉴定出31个磷酸化蛋白质，含有74个磷酸化氨基酸残基，在CATAS8-79胶乳中鉴定出80个磷酸化蛋白质，含有166个磷酸化氨基酸残基。橡胶延伸因子/小橡胶粒子蛋白（REF/SRPP）家族成员被鉴定为差异表达蛋白和磷酸化修饰蛋白，这些蛋白在调节天然橡胶合成中起着重要作用。pro-hevein和hevamine也表现出不同的磷酸化修饰水平，它们主要在天然橡胶排胶过程中起作用。一种丝氨酸-苏氨酸蛋白磷酸酶激酶的磷酸化和去磷酸化修饰可能在天然橡胶合成中起调节作用。这些结果为天然橡胶生物合成调控机制的研究提供了新的理论依据。 丁惟宁关键词：巴西橡胶树；天然橡胶生物合成；磷酸蛋白质组；双向凝胶电泳（2-DE）中图分类号：S794.1文献标识码：A
Comparative Proteomics Analysis and Identification of Phosphorylated Protein in Latex of Rubber Tree Clones PR107 and CATAS8-79
奥密克戎BA.5.2变异株
Abstract：Hevea brasiliensis is an important plant for producing natural rubber. RP107 and CATAS8-
79 are two clones of H. brasiliensis with different properties of rubber production and expulsion. The study of protein function in the latex may help understand the regulatory mechanism related to the properties of rubber production and expulsion. This study aimed to compare and analyze the difference in latex protein between RP107 and CATAS8-79 at the level of protein accumulation and post-translational modification. Through the two-dimensional gel electrophoresis （2-DE）analysis， 65proteins derived from 88spots were found to be accumulated differently in the latex between the 2 clones. Among the proteins， 44 proteins had high accumulation in the latex of PR107 and 21 had high accumulation in the latex of CATAS8-79. The high-accumulation proteins （HAPs）in the latex of CATAS8-79were involved in intracellular organelles， external encapsulating
structure， and membrane-bound organelles in terms of cellular component， and most of them had drug-binding activity and hydrolase activity. Different from CATAS8-79， the HAPs in the latex of PR107 participated in a catalytic complex， nonmembrane-bound organelles， and apoplasts. Most of them had protein-binding and transferase activities.Furthermore， some proteins related to natural rubber synthesis and latex agglutination were found in DAPs. The rubber elongation factors（REFs）and small rubber particleproteins （SRPPs）were identified. The two classes of proteins played an important role in natural rubber biosynthesis in rubber trees. Some proteins mediating rub连轧
ber particle aggregation （RPA） and participating in response to tappingwere found in DAPs too. To determinate the phosphorylated proteins and amino acids in the latex of the two clones， the phosphopeptides were enriched using a Fe-NTA Phosphopeptide Enrichment Kit and shotgun analysis was performed through the high-throughput Tandem Mass Spectrometer （MS/MS）. 74 phosphorylated amino acid residues derived from 31 phosphorylated proteins， as well as 166 phosphorylated amino acid residues derived from 80 phosphorylated proteins， were identified in
PR107 and CATAS8-79， respectively. Among the phosphorylated proteins， 25 proteins of PR107 and 74 proteins of CATAS8-79 were specific in terms of the phosphorylation amino acids. Between the two clones， the members of REF/SRPP protein family， which regulate the synthesis of nature rubber （NR） in the latex， have high differentiation capacity at both protein accumulation and phosphorylation modification levels. The pro-hevein and hevamine proteins， which influence the process of rubber expulsion， also showed diversity at the phosphorylation modification level. The phosphorylation and dephosphorylation of an serine/threonine protein phosphatase kinase might play a regulatory role in NR synthesis. The results couldprovide a new theoretical basis for the study of the regulatory mechanism of NR biosynthesis.
Keywords：Hevea brasiliensis; natural rubber biosynthesis;phosphoproteome;two-dimensional gel e
季诺维也夫lectrophoresis （2-DE）
DOI： 10.3969/j.issn.1000-2561.2022.05.004
巴西橡胶树（Hevea brasiliensis）是目前最具商业价值的天然橡胶来源[1]。天然橡胶是在
橡胶树树皮下韧皮部乳管系统的乳管细胞中合成的，适当的割破树皮会获得胶乳[2]。自首次证明胶乳中含有蛋白质以来[3]，采用蛋白质组学技术研究发现，胶乳蛋白参与诸如天然橡胶的生物合成、对病原体的防御、伤口愈合和抗应激能力等代谢途径的调控[4]。基于双向凝胶电泳技术（2-DE）结合质谱的鉴定技术首先应用于胶乳及其亚细胞组织中蛋白质的检测，确定胶乳 中含有乳胶过敏原類蛋白质[5]、几丁质酶类蛋白[6]以及包括橡胶延伸因子（REF）/小橡胶粒子蛋白（SRPP）在内的天然橡胶合成关键蛋白[7]。随着质谱技术的发展，高通量非凝胶液质连用质谱检测技术应用于胶乳蛋白质研究，大量胶乳蛋白质及其翻译后修饰位点被成功鉴定[8-10]，稳定同位素标记相对和绝对定量（iTRAQ）技术的应用部分揭示了橡胶树胶乳中天然橡胶生物合成调控机制[11-12]。目前，虽然巴西橡胶树胶乳中蛋白质功能的研究取得一定突破，但这些蛋
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