顺式-异戊烯基转移酶与橡胶粒子膜蛋白在杠柳天然橡胶生物合成中的作用机制研究

Natural rubber is an important industrial raw material and almost exclusively produced from Hevea brasiliensis latex. Because H. brasiliensis strict growing conditions limit its cultivation to specific tropical environments, the problem of insufficient capacity of natural rubber become more and more urgent that lead to the search for alternative sources in China. Periploca sepium Bunge is widely distributed throughout the country, especially in the arid and the semi-arid areas in the northern China. It has been confirmed to produce rubber latex containing rare cis-polyisoprene similar to that produced by H. brasiliensis, and is considered to be a suitable alternative source for the production of natural rubber in China. Base on our previous studies that most of the genes, which associated with rubber biosynthesis in the mevalonic acid (MVA), non-mevalonate (DXP/MEP) and polyisoprene pathways, have been isolated and characterized from P. sepium, we further expand our research to identify the rubber particle membrane proteins (RPMPs) in the laticifers of P. sepium by proteome analysis; Their relationship between cis-prenyltransferase (CPT) and the related RPMPs will be determined in vitro by enzymatic activity assay; Their regulations of expression will be investigated in situ during the laticifer and rubber particle growth by immunohistochemical localization; Their comprehensive functions and interactions will be confirmed in vivo by transformation in overexpression and RNAi-interference processes. These analytical results will elucidate the roles of CPT and RPMPs on the rubber molecular polymerization and chair elongation in P. sepium. Our research may provide useful insights into understanding the mechanism involved in rubber biosynthesis in the rubber-producing plants, and play a promoting role on development of P. sepium as an alternative source for the production of natural rubber in most parts of China.

天然橡胶是重要的工业原料，主要产自橡胶树。由于受该树种热带生长特性限制，我国适合种植的区域有限，产能严重不足，开发新的天然橡胶资源已成为迫切需要解决的问题。杠柳（Periploca sepium）广泛分布于我国各地，是北方干旱地区主要的野生灌木，其乳管具有橡胶生物合成能力，非常适合于我国作为生产天然橡胶的替代植物。我们在克隆和鉴定了杠柳甲羟戊酸、非甲羟戊酸和聚异戊二烯等橡胶生物合成途径中大部分基因的基础上，通过本项目进一步对其乳管乳汁的蛋白质组分析，确定与杠柳橡胶分子合成有关的橡胶粒子膜蛋白；利用体外酶活性测定探明顺式-异戊烯基转移酶与这些膜蛋白的互作关系；利用免疫组化方法分析其在橡胶粒子发育过程中的表达规律；利用转基因技术验证其在植物体内的整体功能，从而揭示杠柳橡胶分子的生物合成机制。该研究将为今后利用基因工程把杠柳培育成为我国大部分地区能够栽培的、生产天然橡胶的替代植物提供理论依据。

天然橡胶是重要的工业原料，主要产自橡胶树。由于受该树种热带生长特性限制，我国适合种植的区域有限，产能严重不足，开发新的天然橡胶资源已成为迫切需要解决的问题。杠柳（Periploca sepium）广泛分布于我国各地，是北方干旱地区主要的野生灌木，其乳管具有橡胶生物合成能力，非常适合于我国作为生产天然橡胶的替代植物。本项目通过对杠柳枝条、乳汁进行非标记定量蛋白质组学（Label-free）分析，联合以前的杠柳枝叶转录组测序结果，筛选出了与杠柳天然橡胶生物合成相关的肽段和蛋白质数目分别为194条和37条，确定了顺式-异戊烯基转移酶（CPT）、小橡胶粒子蛋白（SRPP）和橡胶延长因子（REF）等3个关键基因；利用同位素示踪技术测定了其酶活性，分析了CPT单独或与SRPP、REF以及洗净后的橡胶粒子组合，催化橡胶分子合成启始物烯丙基二磷酸，包括二甲基烯丙基二磷酸（DMAPP）、牻牛儿基二磷酸（GPP）、法尼基二磷酸（FPP）、双牻牛儿基二磷酸（GGPP）与14C标记的前体基质异戊烯基二磷酸（IPP）结合情况；通过构建CPT、SRPP和REF基因过表达（overexpression）载体和基因敲除（CRISPR/Cas9）载体，利用转基因技术验证其在植物体内的功能，从而明确了这3个基因对杠柳橡胶分子聚合和分子链延伸的作用。该研究将为今后利用基因工程把杠柳培育成为我国大部分地区能够栽培的、生产天然橡胶的替代植物提供理论依据。