《植物生理学报》 2016, 52(4): 505-513

盐穗木HcmiR172e和预测的靶基因HcTOE3的克隆及盐胁迫下的靶向关系分析

张玉芳, 杨瑞瑞, 曾幼玲^*

新疆大学生命科学与技术学院, 新疆生物资源基因工程重点实验室, 乌鲁木齐830046

通信作者：曾幼玲；E-mail: zeng_ylxju@126.com

摘　要：

盐穗木(Halostachys caspica)广泛分布于新疆干旱盐碱地区, 属于藜科极端耐盐的盐生灌木。从600 mmol·L^-1 NaCl处理48 h的盐穗木根的小RNA文库中筛选到差异极显著的HcmiR172e做为研究对象, 利用盐穗木转录组数据预测其靶基因为HcTOE3, 开展HcmiR172e-HcTOE3的分子克隆和胁迫表达相关性工作。通过同源克隆和RACE技术获得盐穗木HcmiR172e前体和HcTOE3全长序列; 使用生物信息软件分析前体和靶基因信息及相关性; 采用qRT-PCR技术检测了不同盐浓度(200、400和600 mmol·L^-1 NaCl)处理48 h的盐穗木HcmiR172e及预测的靶基因HcTOE3的表达变化。结果显示, 克隆得到的HcmiR172e前体序列可形成颈环结构, 各项指标符合前体要求。其靶基因HcTOE3 cDNA全长为1 744 bp, 开放阅读框1 329 bp, 在编码区有一个高度匹配HcmiR172e的结合位点(CTGCAGCATCATCAGGATTC); qRT-PCR分析结果表明HcmiR172e与其预测的靶基因HcTOE3均响应盐的处理, 二者呈现一定的负相关性。后续将通过实验逐步阐明盐穗木HcmiR172e对Hc-TOE3在逆境中的靶向调控作用。

关键词：盐穗木; 盐胁迫; HcmiR172e; HcTOE3; 克隆; qRT-PCR; 相关性

收稿：2016-02-26   修定：2016-03-28

资助：国家自然科学基金(31160186)、新疆维吾尔自治区自然科学基金(2015211C274)和国家“973”计划前期研究专项(2012CB722204)。

Cloning, expression and correlated analysis of HcmiR172e with predicted target gene HcTOE3 in Halostachys caspica under salt stress

ZHANG Yu-Fang, YANG Rui-Rui, ZENG You-Ling^*

Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumqi 830046, China

Corresponding author: ZENG You-Ling; E-mail: zeng_ylxju@126.com

Abstract:

Halostachys caspica as a salt-diluted short shrub belonging to the Chenopodiaceae is distributed widely in acrid and saline-alkali region of Xinjiang and shows extremely salt-tolerant. A miRNA mature sequence (HcmiR172e) differentially expressed was candidated from the two small RNA libraries of the H. caspica roots established by 600 mmol·L^-1 NaCl treatment for 48 h and the target gene of HcmiR172e was predicted as HcTOE3 using the H. caspica transcriptome data. The HcmiR172e precursor (pre-HcmiR172e) and the full length HcTOE3 gene were obtained by homologous cloning and RACE technology. pre-HcmiR172e and Hc-TOE3 were analyzed by using the bioinformatics software. The expression pattern of HcmiR172e and HcTOE3 of the H. caspica assimilatings under different concentration NaCl treatment was detected using qRT-PCR method. The results showed that the obtained pre-HcmiR172e sequence can form the stem-loop structure, and the indexes can meet the requirements of the precursor using the Mold biological software. The full length of HcTOE3 gene was 1 744 bp, the open reading frame was 1 329 bp, and the highly binding site (CTGCAGCATCATCAGGATTC) of HcmiR172e was matched within the HcTOE3 encoding region. qRT-PCR analysis showed that HcmiR172e and predicted target gene HcTOE3 were responding to salt treatment and took on a negative correlation at a certain extend between HcmiR172e and HcTOE3. The regulated mechanism for a pair of the miR172e-TOE3 in H. caspica under aibotic stress will be elucidated in the further experiments.

Key words: Halostachys caspica; salt stress; HcmiR172e; HcTOE3; cloning; qRT-PCR; correlation