沙冬青端粒酶逆转录酶基因(AmTERT)克隆及表达分析

Cloning and Expression Analysis of Telomerase Reverse Transcriptase(AmTERT) Gene from Ammopiptanthus mongolicus

利用分段克隆法从沙冬青中得到一条全长为3 459 bp的端粒酶逆转录酶基因(telomerase reverse transcriptase of Ammopiptanthus mongolicus,AmTERT),并对AmTERT进行了系统的生物信息学分析。结果表明,AmTERT编码1 153个氨基酸,蛋白相对分子质量为132.52 k Da,等电点为9.49,属于稳定性较差的亲水性蛋白。未发现跨膜结构存在,定位预测结果显示其在细胞质膜及线粒体上有定位,同时发现其有单分型(NLS-monopartite)和双分型(NLS-bipartite)细胞核定位信号,而在原生质体中的瞬时表达实验结果表明AmTERT定位在细胞核中。该氨基酸序列共有147个潜在磷酸化位点,氨基酸比对结果显示,AmTERT与大豆TERT相似性高达75%。另外,AmTERT蛋白具有端粒酶逆转录酶的保守功能结构域(TRBD及RT),并存在潜在的Akt激酶磷酸化位点,暗示其可能通过磷酸化作用发挥调节自身或其他基因功能的作用。荧光定量PCR结果显示,AmTERT在根中表达量较茎和叶组织都高,盐、干旱、热及低温胁迫均能导致沙冬青幼苗根和叶中AmTERT的表达量升高,说明植物细胞端粒酶可能和动物细胞一样具有应激保护功能。综上,研究结果表明,沙冬青端粒酶可能存在非端粒功能。

A segmented cloning method was used to clone a telomerase reverse transcriptase gene（AmTERT） of 3 459 bp in length from Ammopiptanthus mongolicus, and a systematic bioinformatic analysis of the telomerase reverse transcriptase gene（AmTERT） was carried out. The results showed that AmTERT encoded a protein of 1 153 amino acids with a relative molecular weight of 132.52 k Da and an isoelectric point of 9.49, which was a poorly stable hydrophilic protein. No transmembrane structure was found. The localization analysis results showed that it was probably located on the plasma membrane and in mitochondria, and also had NLS-monopartite and NLSbipartite nuclear localization signals. This amino acid sequence has a total of 147 potential phosphorylation sites and the result of amino acid alignment showed that the similarity between AmTERT and soybean TERT was as high as 75%. In addition, AmTERT protein has the conserved functional domains of telomerase reverse transcriptase（TRBD and RT）, and one putative Akt phosphorylation sites in AmTERT that conform to the consensus motif was predicted. Fluorescence quantitative PCR analysis showed that AmTERT expressed much more in roots than in stems and leaves. Furthermore, AmTERT expression increased under salt, drought, hot and cold stress, indicating that telomerase might be closely associated with genomic stability. Our research results implied that Ammopiptanthus mongolicus telomerase might have the non-telomere functions, and provided some new hints for further study to reveal the functions of plant telomerase.