毛竹miR397和miR1432的克隆及其逆境胁迫响应表达分析

摘要/Abstract

摘要： [目的] miRNA作为一种非编码基因在植物生长发育以及抗逆等过程中起着重要的调控作用。通过分析毛竹miR397和miR1432前体序列的结构特点,研究其组织表达特异性,分析其经光照、温度、干旱、NaCl等非生物胁迫以及激素处理后的表达变化,以期为进一步揭示其功能以及未来竹子抗逆育种的分子设计提供参考。[方法] 通过茎环引物法和RT-PCR技术,以毛竹为材料分离miR397和miR1432的前体序列,利用在线平台Web LOGO分析miRNA成熟序列的碱基保守性,用MEGA 6.0软件构建基于miRNA前体的系统进化树。借助在线平台RNAfold WebServer预测二者前体二级结构。直接从BambooGDB下载phe-miR397和phe-miR1432前体上游的1 500 bp序列,利用在线平台Plant CARE分析其所含作用元件。采用实时定量PCR方法分析phe-miR397和phe-miR1432在毛竹根、茎、叶和鞘中的表达情况,以及检测经黑暗、强光(1 500 μmol·m^-2s^-1)、高温(42 ℃)、低温(4 ℃)、NaCl(250 mmol·L^-1)、GA₃溶液(100 μmol·L^-1)、ABA溶液(100 μmol·L^-1)处理2 h后毛竹叶片中phe-miR397和phe-miR1432的表达变化。[结果] 从毛竹中分别克隆出miR397和miR1432的前体序列,均为88 bp,且分别包含其成熟序列,均为21 bp。前体序列均能形成稳定的茎环结构,且成熟序列均产生于前体茎环结构5'端臂上。miR1432家族成熟序列的碱基保守性整体高于miR397家族。毛竹二者前体上游调控区均含有启动子基本作用元件,如TATA-box,CAAT-box; 同时存在很多逆境(光、干旱、温度等)胁迫相关响应元件以及激素响应元件等,意味着phe-miR397和phe-miR1432可能受到逆境胁迫和激素的调节。phe-miR397和phe-miR1432均在叶鞘中表达丰度最高,最低丰度phe-miR397出现在幼茎中,而phe-miR1432则在叶片中。经强光、黑暗、高温、低温、NaCl等胁迫处理后,叶片中phe-miR397和phe-miR1432的表达均下调; 干旱和ABA处理后,phe-miR397的表达下调,而phe-miR1432则上调; GA₃处理后,phe-miR397 的表达上调,phe-miR1432则下调。[结论] phe-miR397和phe-miR1432作为毛竹中2个保守型miRNA,在受光照、温度、干旱和NaCl的胁迫以及ABA和GA₃的处理时,其表达均呈现了不同程度的下调或上调,这暗示着它们可能在毛竹应对非生物胁迫的抗逆过程中起重要的调控作用,且与内源激素的调节相关联。

关键词: 毛竹, miRNA, 非生物胁迫, 激素

Abstract: [Objective] Stress is one of the main factors affecting growth, development and formation of biological production and quality of plant. As non-coding genes, miRNAs play important roles in regulating plant growth and the processes of stress resistance. To reveal the function of miR397 and miR1432 in bamboo and provide a basis for molecular design of bamboo resilience breeding in future, the structural features of miR397 and miR1432 precursor sequences from Phyllostachys edulis were analyzed, the tissue-specific expression analysis as well as the expression changes of miR397 and miR1432 under abiotic stresses of drought, temperature, light, NaCl and hormone treatment were carried out.[Method] The precursor sequences of miR397 and miR1432 of P. edulis were isolated using stem-loop primer method and RT-PCR technology, online platform Web LOGO was used for the conservation analysis of mature miRNA. MEGA 6.0 software was used for the construction of phylogenetic tree with miRNA precursor sequences. The secondary structure of the two precursors was predicted with the online platform RNAfold WebServer. The sequences of 1500 bp upstream of the precursor of phe-miR397 and phe-miR1432 were direct-downloaded respectively from BambooGDB and acting elements analysis was performed using online platform Plant CARE. Real-time quantitative PCR method was used for the tissue-specific expression analysis of phe-miR397 and phe-miR1432 in bamboo roots, stems, leaves and sheaths, and the expression changes in bamboo leaf after 2 h treatments of darkness, high light intensity (1 500 μmol·m^-2s^-1), high temperature (42 ℃), low temperature (4 ℃), NaCl (250 mmol·L^-1), GA₃ solution (100 μmol·L^-1) and ABA solution (100 μmol·L^-1) respectively.[Result] The isolated precursor sequences of phe-miR397 and phe-miR1432 were all 88 bp, containing 21 bp mature sequences correspondingly. The precursors of phe-miR397 and phe-miR1432 could fold into stable stem-loop structure, and the mature sequences were generated at 5' end of the arm in the stem-loop structure respectively. Overall, the nucleotide conservative in mature sequence of miR1432 family was higher than that of miR397 family. Both are containing the essential elements of promoter, such as TATA-box and CAAT-box, in the regulatory region of the upstream precursors of phe-miR397 and phe-miR1432. At the same time, many stress (light, drought, temperature, etc.) related response elements and hormone-responsive elements were found, indicating phe-miR397 and phe-miR1432 may be regulated by stress and hormone. Both phe-miR397 and phe-miR1432 expressed in leaf sheath with the highest level, while the lowest in young stem for phe-miR397 and in leaf for phe-miR1432. The expression of phe-miR397 and phe-miR1432 in leaf were all down-regulated after 2 h with the treatments of high light intensity , darkness, 42 ℃, 4 ℃ and NaCl. The expression of phe-miR397 was down-regulated under drought treatment as well as ABA, while that of phe-miR1432 was up-regulated. The expression of phe-miR397 was up-regulated and phe-miR1432 was down-regulated for GA₃ treatment.[Conclusion] As two conservative miRNAs in bamboo, the expression of phe-miR397 and phe-miR1432 were either up- or down-regulated under the stress treatments of light, temperature, drought and NaCl, as well as ABA and GA₃ treatments, indicating that they might play important regulatory roles in respond to abiotic stress resilience in bamboo, which were also associated with the regulation of endogenous hormones.

Key words: Phyllostachys edulis, miRNA, abiotic stress, hormones

中图分类号:

S718.46

王丽丽, 赵韩生, 孙化雨, 董丽莉, 娄永峰, 高志民. 毛竹miR397和miR1432的克隆及其逆境胁迫响应表达分析[J]. 林业科学, 2015, 51(6): 63-70.

Wang Lili, Zhao Hansheng, Sun Huayu, Dong Lili, Lou Yongfeng, Gao Zhimin. Cloning and Expression Analysis of miR397 and miR1432 in Phyllostachys edulis under Stresses[J]. Scientia Silvae Sinicae, 2015, 51(6): 63-70.

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