日本落叶松小RNA文库构建及其microRNA鉴定  

Construction of Small RNA Library and Characterization of MiRNAs in Larix kaempferi

作　　者：吴涛[1,2] 张俊红[1] 韩素英[3] 杨文华[1] 李万峰[1] 齐力旺[1] 

机构地区：[1]中国林业科学研究院林业研究所,细胞生物学实验室,北京100091 [2]云南省林业科学院,云南省森林植物培育与开发利用重点实验室,云南昆明650201 [3]中国林业科学研究院森林生态环境与保护研究所,北京100091

出　　处：《林业科学研究》2012年第6期677-684,共8页Forest Research

基　　金：国家自然科学基金重点项目(30830086);国家“973”项目(2009CB119106)资助;国家“863”计划重点项目(2011AA100203)

摘　　要：以2年生日本落叶松实生苗为材料构建其小RNA文库,对283个克隆进行PCR验证后测序,通过生物信息学分析鉴定文库中的microRNA并进行qRT-PCR验证。结果表明:(1)小RNA文库的体积为50 mL,总库容量为5.25×106cfu,重组率为92.6%,插入片段长度约65 bp,与小RNA连接片段长度吻合。(2)去除rRNA(86个)、tRNA(14个)等非目的序列后,获得日本落叶松28个保守miRNAs,属于17个miRNA家族;其中,15个miRNAs(miR159c、miR160a、miR162a、miR164b、miR165a、miR166a、miR166a*、miR166b*、miR169a、miR169b、miR171a、miR171b、miR172a、miR319b、miR396a)的序列与其它植物完全一致,其余13个miRNAs(miR156a、miR159a、miR159b、miR164a、miR166b、miR168a、miR169b*、miR319a、miR396b、miR408、miR482a、miR2111、miR3701)则高度相似。(3)测序结果与本室落叶松转录组数据进行序列比对,新发现4个miRNAs(lle-miR1、lle-miR2、lle-miR3、lle-miR4)及其前体。(4)qRT-PCR验证表明,miR159a、miR159b等16个保守的和lle-miR1 lle-miR4等共20个miRNAs存在于日本落叶松中。(5)通过靶基因预测及UniProt数据库分析发现,32个miRNAs中有24个对应69个靶基因,其功能主要为转录因子、信号转导、胁迫抗性和代谢相关酶及未知功能蛋白。In this study, a small RNA library mixed equally sRNA of needle leaves, stem arid root tissues was con- structed for 2-year-old seedling of Japanese larch （ Larix kaempferi）. Two hundred and eighty three recombinants from library were partially selected randomly and sequenced. These sequenced sequences were searched and com- pared in Rfam, Repbase, miRBase and PMRD databases by BLASTN to remove rRNA, tRNA, snRNA suoRNa and other non-candidate small RNA. The results indicated that 28 miRNAs were acquired and these sequences were smne （miR159c, miR160a, miR162a, miR164b, miR165a, miR166a, miR166a＊, miR166b＊, miR169a, miR169b, miR171a, miR171b, miR172a, miR319b, and miR396a） as or highly homologous （miR156a, miR159a, miR159b, miR164a, miR166b, miR168a, miR169b＊, miR319a, miR396b, miR408, miR482a, miR2111, and miR3701 ） to other known plant miRNAs, which belonging to 17 miRNA families. Additional 4sequences could be matched to stem-arm sequences of stem-loop structure M-folded with our lab＇s transcriptome data （contigs/singlets）. The 4 sequences could be characterized novel miRNAs of Japanese larch. Quantitative real-time polymerase chain reaction （qPT-PCR） analysis demonstrated that 20 miRNAs were expressed in larch. The putative target genes of these miRNAs were predicted using psRNATarget online service. There were 69 targets obtained by the prediction for 24 miRNAs among 28 conserved and 4 novel larch miRNAs, which are highly enriched in tran- scription factor （TF）, signal transduction protein, stress-induced-associate protein and other uncharacterized pro- tein.

关 键 词：日本落叶松 小RNA 文库 MIRNA 转录组 

分 类 号：S791.223[农业科学—林木遗传育种]