橡胶树中碱性转化酶基因HbNIN8的克隆与表达分析

Molecular Cloning and Expression Analysis of a Neutral/Alkaline Invertase Gene(HbNIN8) from Hevea brasiliensis

蔗糖是高等植物光合产物的重要存储方式,而转化酶和蔗糖合酶是水解蔗糖的主要酶。研究基于橡胶树的基因组和转录组数据,采用RT-PCR技术克隆到一个中碱性转化酶基因HbNIN8的全长cDNA。该基因预测编码630个氨基酸。同源和亚细胞定位分析表明,HbNIN8属于α类中碱性转化酶,叶绿体定位。应用毕赤酵母真核表达获得其重组蛋白,分析了其酶活性特点,HbNIN8在毕赤酵母中重组蛋白的最适pH为7.5,最适温度为45℃。实时荧光定量PCR分析表明,HbNIN8主要在叶片中表达,其表达量随着叶片的成熟而逐渐增加,在稳定期叶片中达到最大,在成熟叶片中,HbNIN8的表达呈现明显的昼夜差异,晚上的表达水平相对高于白天。因此,HbNIN8很可能是负责将叶绿体中的蔗糖水解为单糖,从而调控橡胶树叶绿体内蔗糖和淀粉的分配。

Sucrose is an important storage form of photosynthetic products in higher plants, whereas invertases and sucrose synthases are the main enzymes to hydrolyze sucrose. Based on available genome and transcriptome data, the full-length cDNA of HbNIN8, a neutral/alkaline invertase gene was cloned from Hevea brasiliensis by RT-PCR, which was predicted to encode 630 amino acids. According to homologous and subcellular analysis, HbNIN8 is more likely located in chloroplast that belongs to α group of neutral/alkaline invertase. By using the Pichia pastoris expression system, the enzymatic properties of the recombinant protein were analyzed: the optimum pH of HbNIN8 is 7.5, and the optimum temperature is 45 ℃. qRT-PCR results showed that HbNIN8 is mainly expressed in leaves, gradually increasing with leaves development and peaking at the mature stage. HbNIN8 also exhibits an obvious diurnal fluctuation in mature leaves, where the expression level is relatively higher in night than in day. Thereby, HbNIN8 may hydrolyze sucrose into monosaccharides in chloroplast, participating in the distribution of sucrose and starch in chloroplast of Hevea brasiliensis.