野生蕉miR395a前体克隆与进化特性及启动子分析

Cloning and evolution characteristics of pre-miR395a and promoter analysis in wild banana(Musa itinerans)

植物miR395参与硫代谢调控,并在应答逆境胁迫等方面起重要调控作用.为了解香蕉miR395的分子特性和进化规律,在参考香蕉基因组信息的基础上,结合RT-PCR技术,从三明野生蕉（Musa itinerans）叶片中获得81 nt miR395a前体序列,包含21 nt miR395a成熟体序列.进化特性分析表明,植物miR395a前体存在于27个物种中,野生蕉pre-miR395a序列与双子叶耧斗菜（Aquilegia viridiflora Pall.）最为接近,与单子叶的水稻、玉米、高粱最远,表明野生蕉pre-miR395a的起源比较特殊;前体序列长度差异较大（57-226 nt）;miR395a成熟体序列分析发现,来自5p臂上的miR395a序列特异性较大.野生蕉miR395a前体能形成典型的发卡结构,miR395a成熟体位于前体的3p臂上.转录起始位点分析表明,野生蕉pri-miR395a转录起始区域位于-87 bp--38 bp,A为转录起始位点.启动子顺式作用元件预测分析表明,野生蕉miR395a启动子包含多个激素、胁迫、生物钟及光响应元件,可能与响应胁迫密切相关.qPCR检测结果也发现miR395a的表达量在低温胁迫下极显著下调,表明响应低温胁迫.综上表明三明野生蕉在响应低温胁迫过程中miR395a起着重要调控作用,这对于进一步研究野生蕉miR395a在低温胁迫中的作用机制具有参考价值.

Mi R395 participates in sulfur metabolism control and plays an important regulatory role in stress response. To explore its structure, molecular characteristics, evolution, and promoter sequence characteristics in wild banana（Musa itinerans） from Sanming in Fujian Province, we referred to the data of the banana genome and performed RT-PCR to sequence pre-miR395 a, which contained 84 nt, including a 21-nt mature sequence. The evolution analysis showed that miR395 a was distributed in 27 plant species, among which the length of pre-miR395 a varied remarkably（between 57 and 226 nt）, and miR395 a-5 p was considerably specific; miR395 a was close to aqc-miR395 a, but very far from osa-miR395 a, zma-miR395 a, and abi-miR395 a, suggesting the possible specific origin of miR395 a in wild banana. Mfold predicted that pre-miR395 a spontaneously formed a typical, stable hairpin secondary structure, and the mature miR395 a sequence was located in the 3 p arm. Transcriptional start site（TSS） predicted that the TSS region of pri-miR395 a was likely between-87 bp and-38 bp with TSS A. The promoter sequence analysis indicated that the miR395 a promoter included many cis-elements such as hormone, light, stress responses, and circadian control. The q PCR assay further confirmed that miR395 a played a crucial role in cold stress response in wild banana. The 84 nt sequence of pre-miR395 a was obtained in wild banana, which was close to the dicot plant aqc-MIR395 a as per evolution analysis. Thus, pri-miR395 a might be associated with stress responses in plants. The q PCR assay indicated that the expression levels of miR395 a decreased very significantly during low temperature stress in wild banana. Therefore, miR395 a was inferred to play essential regulating roles in response to low temperature stress in wild banana, which was a valuable reference for further exploring the functional mechanism of miR395 a during low temperature stress in wild banana.