橡胶树HbREF3基因的克隆及功能初步分析

橡胶延伸因子（REFs）是橡胶粒子上的一种主要膜蛋白,在橡胶生物合成途径中具有延伸橡胶烃和稳定橡胶粒子等重要作用。对橡胶树不同组织的转录组测序发现,HbREF3在胶乳中具有特异性的高表达,且其在HbREFs基因家族中表达水平也相对较高,仅次于表达丰度最高的HbREF1。HbREF3的开放阅读框为528 bp,编码175个氨基酸,对应的蛋白相对分子质量为19.62 kDa,理论等电点（pI）为5.28。系统进化树分析表明,HbREF3与橡胶树其他HbREFs虽均属于分组Ⅰ,但与HbREF1明显属于不同分支。HbREFs均含有REF保守结构域,但在保守motif的分布上,不同HbREFs蛋白含有保守motif数量不等,HbREF3比HbREF1多了一个motif。生信预测分析表明,该蛋白属于亲水性蛋白,无跨膜结构域,具有14个磷酸化位点。亚细胞定位分析发现HbREF3蛋白定位在内质网上,推测其可能参与橡胶粒子的形成和胶乳的再生。本研究结果为进一步阐明HbREF3基因在橡胶树胶乳再生中的作用机制奠定了基础。

Cloning and Primary Functional Analysis of HbREF3 Gene from Hevea brasiliensis

Natural rubber (NR) is an important strategic material related to national economy, livelihood and national security. It is of great significance to increase the production of natural rubber by focusing on the biosynthesis process of natural rubber. Rubber elongation factors (REFs) are mainly membrane proteins on rubber particles, which play an important role in extending rubber hydrocarbon and stable rubber particles during natural rubber biosynthesis process. It is an important approach for high-yield molecular marker-assisted selection breeding of rubber trees by identifying the key REFs gene that affects natural rubber yield. We found HbREF3 was highly and specifically expressed in latex, and its expression level was just less than that of HbREF1, which is the highest expression in latex in HbREFs gene family. The open reading frame of HbREF3 was 528 bp, encoding 175 amino acids. The molecular weight and theoretical isoelectric point of HbREF3 was 19.62 kDa and 5.28, respectively. Phylogenetic tree analysis showed that HbREF3 and HbREF1 belonged to different branches. All HbREFs proteins contain REF domain, and HbREF3 had one more motif than HbREF1. In silico studies showed that HbREF3 protein was a hydrophilic protein, without transmembrane domain, had 14 phosphorylation sites. The results indicated that HbREF3 may not be attached to the surface of rubber particles in a mosaic form, but directly or indirectly interacted with other proteins which embedded in the lipid monolayer membrane to form a complex attached on the surface of the rubber particles. The predicted localization result is different with HbREF1 which is embedded in the rubber particle monolayer membrane. The surface of rubber particles is composed of a monolayer of phospholipid membrane, the formation and development of rubber particles may be related to lipid synthesis. Subcellular localization analysis revealed that HbREF3 was localized in endoplasmic reticulum, we speculate that HbREF3 might be directly involved in rubber particle formation and latex regeneration by participating in lipid synthesis. Moreover, overexpression of REF/SRPPs family genes in nonrubber-producing plants (Arabidopsis and Capsicum) can significantly improve the drought resistance and accumulate a large number of lipid droplets. Therefore, we speculate that HbREF3 have a similar function to REF/SRPPs family proteins of nonrubber-producing plants in responding to drought in nature rubber harvesting process. We clarified the HbREF3 expression pattern and protein characteristics which would build a foundation for elucidating the molecular mechanism of HbREF3 in latex regeneration in rubber trees.