落叶松体胚发育中12个针叶树特异microRNAs表达分析  

Expression Analysis of 12 miRNA Families Specific to Conifers during Somatic Embryogenesis of Larch

作　　者：张俊红[1] 吴涛[1] 韩素英[2] 齐力旺[1] 张守攻[1] 

机构地区：[1]中国林业科学研究院林业研究所,北京100091 [2]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4期411-418,共8页Forest Research

基　　金：国家"973"计划项目(2009CB119100);国家自然基金(30830086)

摘　　要：以落叶松体细胞胚胎发育8个阶段的材料构建小RNA文库,进行了Solexa测序、miRNA鉴定、靶基因预测、qRT-PCR定量分析等研究。结果表明:发现的87个miRNAs中,29个为针叶树特异的,来自12个家族,分别为miR946、miR947、miR950、miR951、miR1311、miR1312、miR1313、miR1314、miR1316、miR3701、miR3702和miR3704,其中25个长度为22nt,且在小RNA文库中的最低拷贝数为18(miR1311),最高拷贝数为33 009(miR950)。发现12个miRNA家族的34个靶基因可能参与生长发育、抗病、AGO反馈调节等多方面遗传调控;qRT-PCR分析表明:72%miRNAs表达次高峰在后期单胚或早期子叶胚,在中期子叶胚最低,最高峰在后期子叶胚,分别与子叶形成、胚胎后熟、胚胎休眠的生理活动相吻合,而miRNA前体的表达模式不同于miRNA,前体变化幅度小于成熟体。In order to identify miRNA and investigate it＇ s expression profile during somatic embryogenesis （SE） of larch, a small RNA library was constructed and sequenced by Solexa technique, and expression analysis of miRNAs and four precursors during eight developmental stages of SE was evaluated by qRT-PCR. The results demonstrated that a total of 87 miRNAs was identified, of which 29 miRNAs from 12 families were specific to conifers, including miR946, miR947, miR950, miR951, miR1311, miR1312, miR1313, miR1314, miR1316, miR3701, miR3702 and miR3704, which could be used for further analysis and next experiment. Of these, 25 miRNAs were 22nt in length, while 4 miRNAs were 2Int. The abundance of miRNAs varied largely in the small RNA library, such as miR1311 was detected by 18 copies, while miR950 with 33 009 copies. 34 potential target genes were predicted for 12 miRNA families, which were linked to many respects of regulation, including plant growth and development, dis- ease resistance, AGO protein feedback regulation, qRT-PCR analysis showed that, for 72% miRNAs, the minor ex- pression peak was at late single embryo or early cotyledonary embryo, the lowest level was at middle cotyledonary embryo, while tile major peak was at late cotyledonary embryo, which was consistent with the physiology events of cotyledon formation, embryo pre-mature, and embryo dormancy. However, the expression model of miRNA precursor was differed from that of miRNA, and precursor expression with much smaller extent than that of miRNA. The discovery of miRNAs specific to conifers and their targets using embryogenic tissue could provide references for the study on the growth and development of gymnosperms.

关 键 词：日本落叶松 体细胞胚胎 microRNA 针叶树 表达分析 

分 类 号：S791.22[农业科学—林木遗传育种]