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  

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

作　　者：张玉芳[1] 杨瑞瑞[1] 曾幼玲[1] 

机构地区：[1]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

出　　处：《植物生理学报》2016年第4期505-513,共9页Plant Physiology Journal

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

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 Na Cl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q RT-PCR技术检测了不同盐浓度(200、400和600 mmol?L-1 Na Cl)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 c DNA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);q RT-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HcTOE3在逆境中的靶向调控作用。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 mi RNA mature sequence（HcmiR172e） differentially expressed was candidated from the two small RNA libraries of the H. caspica roots established by 600 mmol？L-1 Na Cl treatment for 48 h and the target gene of HcmiR172 e was predicted as HcTOE3 using the H. caspica transcriptome data. The HcmiR172 e precursor（pre-HcmiR172e） and the full length HcTOE3 gene were obtained by homologous cloning and RACE technology. pre-HcmiR172 e and HcTOE3 were analyzed by using the bioinformatics software. The expression pattern of HcmiR172 e and HcTOE3 of the H. caspica assimilatings under different concentration Na Cl treatment was detected using q RT-PCR method. The results showed that the obtained pre-HcmiR172 e 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 HcmiR172 e was matched within the HcTOE3 encoding region. q RT-PCR analysis showed that HcmiR172 e and predicted target gene HcTOE3 were responding to salt treatment and took on a negative correlation at a certain extend between HcmiR172 e and HcTOE3. The regulated mechanism for a pair of the mi R172e-TOE3 in H. caspica under aibotic stress will be elucidated in the further experiments.

关 键 词：盐穗木 盐胁迫 HcmiR172e HcTOE3 克隆 QRT-PCR 相关性 

分 类 号：Q943.2[生物学—植物学]