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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5 d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 results of sequencing analysis of the transcriptome of the cold acclimation system in the early stage of the research group, six EST sequences highly similar to the neutral / alkaline invertase genes were selected and verified by electronic splicing and RT-PCR to obtain a full length of 2101 bp nucleic acid sequence. The gene contains a 1923 bp ORF encoding 640 amino acids with a molecular weight of 71.8 kD and a theoretical isoelectric point of 5.69. According to BlastX homology comparison, this gene has the highest similarity (80%) with litchi LcNI. It belongs to the G100 family and belongs to the neutral / alkaline invertase gene and named it CsINV10 (GenBank accession number KT359348). Phylogenetic tree analysis of the CsINV10 amino acid sequence showed that it had the closest relationship with cassava MeNINV8. Further analysis showed that the amino acid sequence of CsINV10 has no N-terminal signal peptide, no transmembrane domain, is a hydrophilic protein and is located on the chloroplast. Quantitative real-time PCR analysis showed that CSINV10 possessed tissue-specific expression, highest in tea leaves and flowers, and lowest in roots. The results showed that the expression of CsINV10 in mature leaves was gradually increased after treated with low temperature (4 ℃), drought and salt stress for 1 d, respectively. However, under the condition of ABA treatment, the expression of CsINV10 increased firstly and then decreased. After 5 days, the gene was not expressed, indicating that the gene may be involved in the response of tea plants to various stresses, which laid the foundation for the further study on the role of invertase gene in stress-tolerance such as cold tolerance of tea plants.