盐穗木miR166a前体和预测靶基因ATHB8like的克隆及二者靶向关系的鉴定
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新疆自治区自然科学基金(2015211C274)


Clone and Correlation Identification of Halostachys caspica miR166a Precursor and Its Predicted Target ATHB8like
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    摘要:

    以盐生植物盐穗木为实验材料,从前期构建的高盐胁迫下盐穗木根的小RNA文库中候选了差异表达的miR166a,利用生物信息学从盐穗木转录组数据中预测其靶基因;采用5′ RLMRACE技术鉴定盐穗木miR166a对预测靶基因ATHB8like的靶向性;通过PCR和RACE技术克隆盐穗木miR166a前体和预测靶基因ATHB8like全长基因序列,并进行相应的生物信息学分析。结果显示:盐穗木miR166a预测的靶基因为ATHB8like;通过实验鉴定确实存在靶向切割,具体的切割位点位于miR166a成熟体的14~15碱基之间;miR166a成熟体序列在不同植物中高度保守;克隆获得的盐穗木miR166a前体可折叠成完整的颈环结构,符合miRNA的前体特征,候选植物miR166a前体在进化上没有表现出保守性;预测的靶基因ATHB8like cDNA全长为2 786 bp,开放阅读框为2 526 bp,编码841个氨基酸,ATHB8like具有一个HDZIP Ⅲ结构域,在进化上具有保守性。该研究结果为进一步开展盐穗木miR166a和ATHB8like的生物学功能奠定了基础。

    Abstract:

    In this study, miR166a with differential expression was candidated from the small RNA libraries of the halophyte Halostachys caspica roots established under high salinity (600 mmol·L-1 NaCl), some work was carried out as follows. miR166atargeted gene was predicted from this species transcriptome by bioinformatic analysis; the miR166atargeting was identified with the technology of 5′ RLMRACE; miR166a precursor and full length of ATHB8like were obtained by PCR and RACE methods and fully analyzed by bioinformatic softwares. The results showed that predicted target was ATHB8like; 5′ RLMRACE validated the accurate cleavage site of the target by H. caspica miR166a between 14th to 15th base of its mature miRNA; H. caspica mature miR166a was highly conserved compared to different plant species; the miR166a precursor could be folded into complete hairpin structure and was met with various indexes of miRNA precursor and did not reflect the conservation in evolution by aligning with plant species available; the full length cDNA of ATHB8like is 2 786 bp that open reading frame is 2 526 bp with putative 841 amino acids containing a HDZIP III domain and H. caspica ATHB8like was conserved compared to those ATHB8 of different plants. This work will lay the foundation for further studying biological functions of H. caspica miR166a and its target.

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张素伟,易小娅,曾幼玲.盐穗木miR166a前体和预测靶基因ATHB8like的克隆及二者靶向关系的鉴定[J].西北植物学报,2017,37(6):1073-1079

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  • 在线发布日期: 2017-07-17
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