杠柳顺式-1,4-聚异戊二烯生物合成关键基因PsIPPI和PsCPT的克隆及功能分析

Cloning and Functional Identification of the Key Genes PsIPPI and PsCPT for cis-1,4-Polyisoprene Biosynthesis in Periploca sepium

天然橡胶是事关国计民生和国家安全的重要战略物资,其主要成分为顺式-1,4-聚异戊二烯,主要产自巴西橡胶树(Hevea brasiliensis)。由于受到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)乳管分泌的乳汁含有顺式-1,4-聚异戊二烯,非常适用于我国作为天然橡胶生产的替代植物。通过对杠柳树枝(包括皮、枝、叶)进行转录组测序,成功克隆了两个对顺式-1,4-聚异戊二烯生物合成起关键作用的基因PsIPPI和PsCPT,即异戊烯基二磷酸异构酶(isopentenyl diphosphate isomerase, IPPI)和顺式-异戊二烯基转移酶(cis-prenyltransferase, CPT)基因;以杠柳茎段为外植体,通过农杆菌介导的转化,将这两个基因以单独或组合形式在杠柳植物体内过表达,结果发现其基因相对表达量最高可增加200倍以上,但单独过表达PsIPPI的转基因植株,其聚异戊二烯含量和分子量分布没有明显变化;单独过表达PsCPT的转基因植株,其聚异戊二烯含量增加了约5倍,且高分子部分的比例提升了约1.6倍;同时过表达PsIPPI和PsCPT的转基因植株,其聚异戊二烯含量增加了5~6倍,高分子部分的比例提升了约2倍。本研究对杠柳顺式-1,4-聚异戊二烯生物合成关键基因的功能做了初步分析,证实利用部分关键基因的过表达可以提高杠柳的天然橡胶产量和高分子量的比例。随着对杠柳有关顺式-1,4-聚异戊二烯生物合成机制、编码各种酶系的关键基因的不断挖掘,可望将杠柳培育成为生产天然橡胶的替代植物,为解决我国天然橡胶贫乏的工业瓶颈和丰富橡胶资源提供技术支撑。

Natural rubber is a key strategic material that is vital to the people’s livelihood, the national economy and security. Its primary component is one kind of isoprenoids, known as cis-1,4-polyisoprene. The source of natural rubber is almost exclusive produced from Hevea brasiliensis whose strict growing conditions limit its cultivation to specific tropical environments. The problem of insufficient capacity of natural rubber production become more and more urgent that lead to the search for alternative sources in China. We found that the milk secreted by the laticifers in Periploca sepium contains cis-1,4-polyisoprene, and considered that it may be very suitable for China as a substitute plant for natural rubber production. Two genes encoding PsIPPI and PsCPT, namely isopentenyl diphosphate isomerase and cis-isopentenyl transferase, which play key roles in the biosynthesis of cis-1,4-polyisoprene, were successfully cloned from the branches(including barks, stems and leaves) of P. sepium according to its transcriptome sequencing. Using stem segments as explants, the two genes were overexpressed alone or in combination by Agrobacterium-mediated transformation, and their expression level were increased sharply up to 200-fold. However, the polyisoprene content and the molecular weight distribution had no significant change in the transgenic plants overexpressing PsIPPI alone, while the polyisoprene content and the proportion of high molecular weight increased about 5-fold and 1.6-fold in those of transgenic plants overexpressing PsCPT alone, as well as increased about 5-fold to 6-fold and 2-fold in those of PsIPPI and PsCPT co-overexpressing transgenic plantlets. In current research, the functions of the key genes associated with the biosynthesis of cis-1,4-polyisoprene in P. sepium were preliminarily identified, and the possibility was proved that the yield and high molecular weight of natural rubber could be improved by overexpression of some key genes. With the progress in the research on cis-1,4-polyisoprene biosynthesis mechanism, more and more importent enzymes and their encoded genes will be discovered and characterized, it is expected that P. sepium will be developed as an alternative source for the production of natural rubber, and completely solve the bottleneck of natural rubber existing in China.