辣椒GMS育性相关miRNA的鉴定和表达分析-《中国瓜菜》(2015年4期).docx

龙源版权所有 辣椒GMS育性相关miRNA的鉴定和表达分析作者：刘思月等来源：《中国瓜菜》2015年第04期 摘 要： 研究辣椒花药中sRNA分布，筛选育性相关miRNA，通过miRNA及其靶基因的表达分析探讨miRNA对雄性育性的调控。利用高通量测序技术对辣椒细胞核雄性不育两用系处于小孢子单核靠边期的花药进行sRNA测序，同时分析两材料间miRNA的表达差异。通过qRT-PCR技术验证差异分析结果，并对miRNA及其靶基因在小孢子发育不同时期的表达模式进行分析。在构建的不育株和可育株两个文库中共发掘出857个可信度高的miRNA；筛选得到42个表达差异显著的miRNA；通过靶基因预测与注释，预测出的差异表达miRNA的靶基因中与繁殖有关的基因有152 个，与生殖过程有关的基因有151个。通过qRT-PCR验证了选出的9条miRNA的存在，其表达差异与测序分析结果基本一致；单核靠边期多数miRNA负调控其靶基因，不同发育时期其调控模式会发生变化。本研究为揭示辣椒miRNA与雄性育性的关系提供了重要信息。 关键词： 辣椒； 细胞核雄性不育； miRNA； 高通量测序； qRT-PCR Identification and expression analysis of fertility-related miRNAs for the genic male sterile line in pepper （Capsicum annuum L.） LIU Siyue， LIU Chen， GUO Jinju， DA Fengjiao， SHEN Huolin （Beijing Key Laboratory of Growth and Developmental Regulation for Protected Vegetable Crops·College of Agriculture and Biotechnology，China Agricultural University，Beijing 100193，China） Abstract： The objective of this study is to investigate the distribution of sRNAs from anthers of pepper，and to screen miRNAs related to genic male sterility. By expression analysis of miRNAs and their target genes，we discussed the regulation mechanisam of male sterility by miRNAs. Using high-throughput sequencing technology，sRNA populations from anthers in late-uninucleate stage for GMS line in pepper were sequenced. qRT-PCR were performed to validate the results of sRNA sequencing and to analyze the expression profile in different developmental stages of microspore. Of the millions of high-quality sRNAs from 2 libraries，857 miRNAs of high credibility were identified，42 significant differently expressed miRNAs were screened. Prediction and annotation of target genes indicated that there are 152 targets involved in reproduction and 151 involved in reproductive process. qRT-PCR results confirmed the existence of the 9 selected miRNAs，and the expression files of most of the miRNAs is consistent with sequencing results. In late-uninucleate stage，most of miRNAs negatively regulate their targets，but the regu