杠柳橡胶合成关键基因CPT、SRPP和REF启动子克隆与表达特性分析

Cloning and characterization of the promoters of the key genes CPT, SRPP and REF involved in Periploca sepium rubber biosynthesis

摘要:

巴西橡胶树（Hevea brasiliensis）是天然橡胶的主要来源。受热带气候条件限制，我国巴西橡胶树种植面积有限，天然橡胶自给率低。杠柳（Periploca sepium）可以合成顺式-1，4-聚异戊二烯（天然橡胶的主要成分），是一种天然橡胶的新型替代植物，但其合成调控机理尚不明确。本研究利用染色体步移方法，克隆获得了杠柳顺式-1，4-聚异戊二烯合成关键酶顺式-异戊烯基转移酶（cis-prenyltransferase，CPT）、小橡胶粒子蛋白（small rubber particle protein，SRPP）和橡胶延长因子（rubber elongation factor，REF）基因的启动子序列。以β-葡糖醛酸酶（β-glucuronidase，GUS）基因为报告基因，利用农杆菌（Agrobacterium）介导稳定遗传转化法解析了这3个启动子的组织表达特性。结果显示，这三者在杠柳的叶片和茎中均有表达，特别是在叶脉和维管束位置共同表达，提示在这些部位REF和SRPP与CPT可能相互作用，共同完成天然橡胶的主要成分顺式-1，4-聚异戊二烯的聚合。本研究为解析杠柳中天然橡胶的生物合成机制以及培育高产天然橡胶新品种提供了理论基础。

Abstract:

Hevea brasiliensis is the main source of natural rubber. Restricted by its tropical climate conditions, the planting area in China is limited, resulted in a low self-sufficiency. Periploca sepium which can produce natural rubber is a potential substitute plant. cis-prenyltransferase (CPT), small rubber particle protein (SRPP) and rubber elongation factor (REF) are key enzymes involved in the biosynthesis of cis-1,4-polyisoprene, the main component of natural rubber. In this study, we cloned the promoter sequences of CPT, SRPP and REF through chromosome walking strategy. The spatial expression patterns of the three promoters were analyzed using GUS (β-glucuronidase) as a reporter gene driven by the promoters through Agrobacterium-mediated genetic transformation. The results showed that GUS driven by CPT, SRPP or REF promoter was expressed in leaves and stems, especially in the leaf vein and vascular bundle. The GUS activity in stems was higher than that in leaf. This study provided a basis for analyzing the biosynthesis mechanism of natural rubber and breeding new varieties of high yield natural rubber.