茶树中性/碱性转化酶基因CsINV10的克隆与表达分析

摘要

基于课题组前期对茶树冷驯化系统的转录组测序分析结果,从中挑选出6条与中性/碱性转化酶基因高度相似的EST序列,电子拼接和RT-PCR验证后获得一条全长为2101 bp的核酸序列。该基因包含1923 bp的ORF,编码640个氨基酸,蛋白分子量为71.8 kD,理论等电点为5.69。根据BlastX同源性比对显示,该基因与荔枝LcNI相似性最高(80%),为G100家族成员,属于中性/碱性转化酶基因,将其命名为CsINV10(GenBank登录号为KT359348)。对CsINV10氨基酸序列的系统进化树分析显示,其与木薯MeNINV8亲缘关系最近。进一步分析显示, CsINV10的氨基酸序列无N端信号肽,无跨膜结构域,属于亲水性蛋白,并定位在叶绿体上。荧光定量PCR分析表明, CsINV10具有组织表达特异性,在茶树叶和花中的表达量最高,根系中最低。分析发现,低温(4℃)、干旱和盐胁迫分别处理茶树1 d 后,成熟叶片中 CsINV10的表达呈逐渐上升趋势；而在 ABA 条件处理下,该基因呈先升高后降低趋势,在处理5d 后基本不表达,表明该基因可能参与茶树对多种逆境胁迫的响应,这为后续进一步研究转化酶基因在茶树抗寒等逆境胁迫中的作用奠定基础。%Based on the comprehensive RNA-Seq analysis of tea plant during cold acclimation stage, we picked out six ESTs sequences with a high similarity to neutral/alkaline invertase gene to get a splicing. As a result, a full-length of 2101 bp nucleotide sequence was obtained from tea plant after validated by using RT-PCR technique. Bioinformatics analysis showed that the se-quence containing 1923 bp ORF (Open Reading Fram) and encoding 640 amino acid residues with a putative molecular mass of 71.8 kD and theoretical isoelectric point of 5.69, was named as CsINV10 (GenBank accession number:KT359348). BlastX and phylogenetic analysis indicated that the protein encoded by CsINV10 shared the highest identity (80%) with LcNI in Litchi, and had a closest genetic relationship with MeNINV8 in Manihot esculenta Crantz. Also, CsINV10 as a neutral/alkaline invertase gene could be classified into G100 family. The protein had no N-terminal signal peptide and transmembrane domain, and was predi-cated to be a hydrophilic protein localized in chloroplast. The expression analysis of CsINV10 under different abiotic stress condi-tions showed that cold, PEG and salt stresses could gradually promote the expression of CsINV10 when treated for one day, how-ever, it had a transient increase when treated by ABA for three hours. Consequently, we speculated that CsINV10 might be in-volved in the tea plant response to abiotic stresses. Moreover, we found that CsINV10 had a tissue-specific expression pattern in leaf and flower. These results provide a theoretical basis for the functional analysis of invertase genes of tea plant in response to various stresses.