橡胶树β-淀粉酶基因HbBAM1的克隆与表达分析

Molecular Cloning and Expression Analysis of HbBAM1 from Hevea brasiliensis

叶绿体内的淀粉是植物光合作用产物的重要临时储存形式,这些淀粉的降解需要β-淀粉酶(BAM)。β-淀粉酶基因是一个多基因家族,分别在不同组织中起着不同的作用,叶绿体β-淀粉酶在叶绿体淀粉降解过程中起着关键作用。利用橡胶树的转录组和基因组数据库,通过RT-PCR的方法获得一个橡胶树BAM基因,命名为HbBAM1。利用实时荧光定量PCR分析其在叶片中的表达模式,通过原核表达获得其重组蛋白,并分析其酶活性特点。同源性分析和亚细胞预测分析结果表明,HbBAM1定位于叶绿体中;HbBAM1原核表达产物的最适酶活性温度是35℃,其酶活性受到氧化型谷胱甘肽和双氧水等氧化剂的抑制。HbBAM1基因主要在橡胶树的花和叶片中表达;HbBAM1基因随着叶片的发育进程表达量逐渐增加,在淡绿期和稳定期叶片中表达量最大;HbBAM1基因在成熟叶片中的表达呈现明显的昼夜差异,在光合作用最强的上午10:00和下午16:00的表达量最大,晚上的表达量最低。上述结果表明,HbBAM1可能参与橡胶树叶片叶绿体内淀粉的降解,控制叶片气孔的开放与关闭,从而调控橡胶树叶片的光合作用。

In chloroplast,starch is an important temporary storage form of photosynthetic products, and the degradation of the starch requires β-amylase（BAM）activity. β-amylase is a multi-gene family, which has different functions in different tissues. Plastic β-amylase plays a key role in starch degradation in chloroplast. Based on the transcriptome and genome data,an β-amylase gene, HbBAM1 was cloned by RT-PCR from Hevea brasiliensis. Real time RT-PCR（q RT-PCR）was used to characterize the expression pattern of HbBAM1 in the leaves. The recombinant protein of HbBAM1 was obtained by prokaryotic expression, and the characteristics of the enzyme activity was analyzed. HbBAM1 located in the chloroplast based on homology analysis and subcellular prediction. The optimum enzyme temperature of HbBAM1 was35 ℃, the enzyme was suppressed by oxidant, oxidized glutathione and hydrogen peroxide. HbBAM1 was mainly expressed in the flowers and leaves. The expression of HbBAM1 was gradually increased in the leaf development process, peaked at the light green and stable stages. HbBAM1 displayed obvious circadian variations in the stable leaves,which had the highest expression level during the strongest photosynthesis time-10 o＇clock and 16 o＇clock,the lowest at night. It was presumed that HbBAM1 maight participate in the starch degradation in the chloroplast of H. brasiliensis,control the open and close of the stomas,regulates the photosynthesis of H. brasiliensis.