茶树CsAAPs亚家族基因的克隆与表达分析

doi:10.13305/j.cnki.jts.20200612.002

摘要/Abstract

摘要： 采用RT-PCR技术,从龙井43中成功克隆了5个茶树AAPs（Amino acid permeases,氨基酸通透酶）基因。氨基酸序列比对结果表明,5个CsAAPs亚家族蛋白的序列同源性较高,为70.27%。根据氨基酸序列同源性构建系统发育树,结果显示5个CsAAPs分属3组。生物信息学分析表明,CsAAPs均含有9~10个跨膜区域和16~18个AAP保守基序。为了研究该亚家族对氮素的响应情况,选用3个茶树品种的扦插苗为试验材料,氮饥饿两周后,分别供应不同浓度的NH₄NO₃,然后利用qRT-PCR对CsAAPs在不同组织、氮素水平及茶树品种中的表达情况进行分析。研究发现CsAAPs在营养组织中均有表达,但是存在一定的组织表达差异,其中CsAAP3在茎中表达量最高,CsAAP8的主要表达部位为根和茎。在给氮素饥饿处理的茶苗从新供氮之后,CsAAP3的基因表达在3个氮素利用效率不同的品种间差异较大;在氮高效品种中茶302茎中表达的CsAAP3和CsAAP8,可以快速地对低氮条件做出响应,在低氮处理3 h后,基因表达水平明显增加。此研究预示着CsAAPs亚家族在茶树体内可能通过复杂的氨基酸转运参与氮代谢调控。

关键词: 茶树, 氨基酸通透酶, 基因克隆与表达, 氮素

Abstract: In this study, five sequences of the amino acid permease (AAP) genes were successfully cloned from tea cultivar Longjing 43 by RT-PCR. The amino acid sequence alignment analysis revealed the high homology among the five CsAAP proteins (70.27%). Phylogenetic tree based on amino acid sequences showed that five CsAAPs could be classified into three groups. Further bioinformatics analysis demonstrated that these CsAAPs contained 9-10 transmembrane structures and 16-18 conserved AAP motifs. To investigate the response of this subfamily to nitrogen, cutting seedlings of three tea cultivars were fed with NH₄NO₃of different concentrations after two weeks of nitrogen starvation and then used for qRT-PCR analysis. The results show that these five CsAAPs were expressed in all vegetative tissues of tea cutting seedlings, but there were certain differences in tissue expressions. Among them, CsAAP3 showed the highest gene expression levels in stems and the main expression sites of CsAAP8 were roots and stems. The expression levels of CsAAPs varied temporally and spatially with nitrogen treatments. The gene expression of CsAAP3 was significantly changed among the tea cultivars with different nitrogen use efficiency. The expressions of CsAAP3 and CsAAP8 in the stem of the high nitrogen efficient cultivar Zhongcha 302 could quickly respond to low nitrogen levels. Their gene expression levels were increased significantly after 3 h of the low nitrogen treatment. These results implies that CsAAPs subfamily might play important roles in the regulation of nitrogen metabolism through the intricate transports of amino acids in tea plants.

Key words: Camellia sinensis, amino acid permeases (AAPs), gene cloning and expression, nitrogen

中图分类号:

郭玲玲, 张芬, 成浩, 韦康, 阮丽, 吴立赟, 王丽鸳. 茶树CsAAPs亚家族基因的克隆与表达分析[J]. 茶叶科学, 2020, 40(4): 454-464. doi: 10.13305/j.cnki.jts.20200612.002.

GUO Lingling, ZHANG Fen, CHENG Hao, WEI Kang, RUAN Li, WU Liyun, WANG Liyuan. Molecular Cloning and Expression Analysis of CsAAPs Gene Subfamily in Camellia Sinensis[J]. Journal of Tea Science, 2020, 40(4): 454-464. doi: 10.13305/j.cnki.jts.20200612.002.

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