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园艺学报 ›› 2022, Vol. 49 ›› Issue (5): 958-972.doi: 10.16420/j.issn.0513-353x.2021-0103

• 研究论文 • 上一篇    下一篇

miR168家族进化特性及其在砂梨休眠期的表达模式分析

梁沁1,2, 张延晖3, 康开权4, 刘瑾航1, 李亮1, 冯宇1, 王超1, 杨超1,*(), 李永裕1,*()   

  1. 1福建农林大学园艺学院,福州 350002
    2广安市广安区现代农业园区服务中心,四川广安 638500
    3福建省永泰县农业农村局经济作物站,福州 350700
    4福建省连江市国有林场,福州 350500
  • 收稿日期:2022-01-11 修回日期:2022-04-01 出版日期:2022-05-25 发布日期:2022-05-25
  • 通讯作者: 杨超,李永裕 E-mail:yycc32@126.com;lilin3182@163.com
  • 基金资助:
    国家自然科学基金项目(31501694);国家自然科学基金项目(31902067);高等学校博士学科点专项科研基金项目(2011351511011)

Molecular Evolution of MiR168 Family and Their Expression Profiling During Dormancy of Pyrus pyrifolia

LIANG Qin1,2, ZHANG Yanhui3, KANG Kaiquan4, LIU Jinhang1, LI Liang1, FENG Yu1, WANG Chao1, YANG Chao1,*(), LI Yongyu1,*()   

  1. 1College of Horticulture,Fujian Agriculture and Forestry University,Fuzhou 350002,China
    2Guang’an Modern Agricultural Park Service Center,Guang’an,Sichuan 638500,China
    3Economic Crop Station,Agricultural and Rural Bureau of Yongtai County,Fuzhou 350700,China
    4Lianjiang State-Owned Forest Farm in Fujian Province,Fuzhou 350500,China
  • Received:2022-01-11 Revised:2022-04-01 Online:2022-05-25 Published:2022-05-25
  • Contact: YANG Chao,LI Yongyu E-mail:yycc32@126.com;lilin3182@163.com

摘要:

为了探究miR168家族的进化特性及其在梨休眠进程中的表达模式,通过检索实验室前期构建的砂梨(‘黄花’梨)miRNA文库、转录组数据库和miRBase数据库,得到植物miR168家族成员的成熟体和前体序列。从‘黄花’梨花芽中克隆了两条miR168前体,发现梨miR168家族包含2条前体序列和4条成熟体序列。采用生物信息分析和实时荧光定量PCR(qRT-PCR),对梨miR168家族的成熟体序列及前体(pre-miR168)序列进行分析,与已知植物miR168构建系统发育进化树,预测其靶基因,分析其在梨休眠发生进程中的表达情况。结果表明,梨pre-miR168序列均能形成稳定的发夹结构;植物pre-miR168两端序列较保守,而中间序列不保守;mi168成熟体保守性较高,进化树分析表明梨pre-miR168与苹果的亲缘关系最近,miR168成熟体主要分为两大类。梨miR168调控的潜在靶基因主要包含Argonaute1蛋白基因(protein argonaute 1,AGO1)和胼胝质合成酶3基因(callose synthase 3,CalS3)共21条mRNA,都是以裂解靶标的方式进行调控。qRT-PCR分析结果表明,梨miR168a相对表达量在整个休眠期间呈先上升后下降的单峰动态变化;靶基因AGO1的表达呈上升趋势,靶基因CalS3的表达呈下降趋势;AGO1的表达趋势与梨花芽的萌芽率呈正相关关系,随着萌芽率的升高而升高,而CalS3表达趋势与梨花芽的萌芽率呈负相关关系,随萌芽率升高而降低。梨miR168与其他植物miR168的序列具有高度保守性,miR168与AGO1可能通过ABA来调控梨花芽的冬季休眠,通过上调表达梨miR168a而抑制CalS3表达的方式促进梨花芽冬季休眠的解除。

关键词: 梨, miR168, 进化特性, 休眠, 表达模式

Abstract:

To explore the evolutionary characteristics of miR168 gene family and its expression pattern in dormancy process of pear,the mature and precursor sequences of all miR168 family members in plants were obtained from miRBase 22.0 and miRNA library of pear(‘Huanghua’)created by our laboratory. We cloned two precursors of miR168 from the pear flower buds,which contained two mature sequences. Bioinformatics analysis and real-time quantitative PCR(qRT-PCR)were used to analyze the mature sequences and the pre-miR168 sequences of the miR168 family of pear. The phylogenetic tree was constructed with known plant miR168,and based on target genes prediction,the expression of miR168 and target genes during dormancy development in pear was analyzed. RNAfold prediction analysis showed that the pre-miR168 sequence of pear could form stable hairpin structure. Further analysis showed that the sequences of both ends of plant pre-miR168 were conservative,while the intermediate sequences were not. The mature miR168 was highly conserved,and the evolutionary tree analysis was mainly divided into two categories. Phylogenetic analysis found that the affinity of pre-miR168 of pear was closely related to apple,which showed that species affinity is the main factor for the evolutionary characteristics of the precursor of miR168 family. PsRNA target prediction showed that a total of 21 potential mRNAs of target genes were regulated by miR168 by means of cleavage,which mainly contained protein argonaute 1AGO1)and callose synthase 3CalS3). qRT-PCR analysis showed that a dynamic change of “rise-decline expression pattern of miR168a was observed during the dormancy process of pear bud. In addition,the expression of target gene AGO1 showed an increase trend,while the expression of target gene CalS3 showed a decrease trend. Furthermore,the expression of CalS3 showed a negative correlation with germination rate of pear bud,declined with the increase of germination rate. Taken together,the work also showed that the sequence of plant miR168s was highly conserved,and may regulate flower bud dormancy together with AGO1 through ABA. The up-regulated expression of miR168a in pear may inhibit the expression of CalS3 and thus promote the release of dormancy in pear during dormancy in winter.

Key words: pear, microRNA168, evolutionary characteristics, dormancy, expression pattern

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