中国农业科学 ›› 2017, Vol. 50 ›› Issue (17): 3259-3273.doi: 10.3864/j.issn.0578-1752.2017.17.002

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甜菜WRKY转录因子全基因组鉴定及其在非生物胁迫下的表达分析

孔维龙1,于坤2,但乃震1,杨绍宗1,包满珠1,黄向荣1,傅小鹏1

 
  

  1. 1华中农业大学园艺林学学院/园艺植物生物学教育部重点实验室,武汉 430070;2石河子大学农学院/新疆生产建设兵团绿洲生态农业重点实验室,新疆石河子 832003
  • 收稿日期:2017-02-23 出版日期:2017-09-01 发布日期:2017-09-01
  • 通讯作者: 傅小鹏,E-mail:fuxiaopeng@mail.hzau.edu.cn
  • 作者简介:孔维龙,E-mail:Asuraprince@126.com
  • 基金资助:
    国家自然科学基金(31000918)、中央高校基本科研业务费专项(2662015PY052,2662016PY041)

Genome-Wide Identification and Expression Analysis of WRKY Transcription Factor Under Abiotic Stress in Beta vulgaris

KONG WeiLong1, YU Kun2, DAN NaiZhen1, YANG ShaoZong1, Bao ManZhu1, HUANG XiangRong1, FU XiaoPeng1   

  1. 1College of Horticulture and Forestry Sciences, Huazhong Agricultural University/Key Laboratory of Horticultural Plant Biology, Ministry of Education, Wuhan 430070; 2College of Agronomy, Shihezi University/Key Laboratory of Oasis Ecological Agriculture, Xinjiang Production and Construction Group, Shihezi 832003, Xinjiang
  • Received:2017-02-23 Online:2017-09-01 Published:2017-09-01

摘要: 【目的】WRKY转录因子是一类植物响应生物、非生物胁迫,对生长发育都起重要调控作用的转录因子。在甜菜全基因组信息分析的基础上,鉴定WRKY家族基因(BvWRKYs),解析其组织特异性及盐、热胁迫下的表达情况,为该类基因的功能研究提供参考,为观赏甜菜和石竹目其他观赏植物的基因工程打下基础。【方法】以75条拟南芥WRKY蛋白为参考,根据WRKY保守蛋白序列(PF03106)利用hmm和BLAST同源性搜索对甜菜WRKY家族基因进行鉴定。利用MapInspect、GSDS2.0、MEGA5.0、DNAMAN5.0、WebLogo 3、MEME生物信息学工具对甜菜WRKY家族基因染色体定位、系统发生关系、基因结构、蛋白质保守结构域、保守元件进行预测和分析。利用RNA-seq和qRT-PCR分析甜菜WRKY组织表达特异性,盐胁迫、热胁迫条件下WRKY表达情况。【结果】甜菜WRKY家族基因包含40个成员,其中39条不均匀地分布在9条染色体上,另外1条定位到随机片段上。根据WRKY保守域特征并与拟南芥WRKY蛋白进化分析,可将40个成员分为Ⅰ、Ⅱ、Ⅲ 3类,Ⅰ类有9个成员,Ⅱ类有26个成员,Ⅲ类有5个成员。根据进化关系Ⅱ类可进一步分为Ⅱa(1个)、Ⅱb(4个)、Ⅱc(9个)、Ⅱd(5个)和Ⅱe(7个)5个亚类。基因结构分析发现,甜菜WRKY外显子和内含子数目具有高变异性(2—7个外显子),即使同一亚类内也都差异较大。保守元件分析显示同一类或亚类内成员具有相同的保守元件。WRKY保守域分析发现2个WRKY七肽域变型:WRKYGKK和WRKYGEK。每个WRKY至少在2个组织中表达,30个WRKY在叶中表达,40个WRKY在花序中均有表达,36个WRKY在幼叶中有表达,38个WRKY在直根中有表达,39个WRKY在幼苗中有表达,36个WRKY在种子中有表达。各WRKY表达量差异较大,可分为低表达、高表达基因两类,如BvWRKY23BvWRKY3BvWRKY11BvWRKY7BvWRKY6BvWRKY26BvWRKY4BvWRKY40BvWRKY24BvWRKY2BvWRKY28在各组织中均有较高表达,而BvWRKY38BvWRKY13BvWRKY36BvWRKY35BvWRKY5BvWRKY34在各组织中均表达较低。热胁迫条件下BvWRKY16BvWRKY21BvWRKY20BvWRKY22BvWRKY32BvWRKY33BvWRKY34上调表达;盐胁迫条件下BvWRKY1BvWRKY6BvWRKY19BvWRKY31BvWRKY33呈现不同程度上调表达;BvWRKY33对热、盐2种胁迫均有明显响应。【结论】甜菜WRKY蛋白结构高度保守,基因序列长度和内含子数量变化很大,在不同组织中呈现出多种表达模式,部分WRKY响应热或盐胁迫,对甜菜逆境生理调控起重要作用。

关键词: 甜菜, WRKY转录因子, 生物信息学, 胁迫, 表达分析

Abstract: 【Objective】 WRKY transcription factor is a kind of transcription factor which plays an important role in plant response to biotic, abiotic stress, plant growth and development. Based on the sugar beet genome information, the WRKY family genes (BvWRKYs) were identified, tissue-specific expression profiles and expression pattern under salt and heat stresses were analyzed. The aim of the study is to provide reference for the WRKY gene function research, and lay a foundation for ornamental beet (B. vulgaris) and other Dianthus ornamental plant genetic engineering research. 【Method】 A total of 75 Arabidopsis WRKY proteins were used as references, according to WRKY conserved protein sequence (PF03106), the WRKY genes of B. vulgaris were identified by hmm and BLAST homology searches, and the chromosome location, phylogeny, gene structure, conserved domain, conserved element were also analyzed by MapInspect, GSDS2.0, MEGA5.0, DNAMAN5.0, WebLogo 3 and MEME bioinformatics tools. The specificity of WRKY genes expression and expression pattern under salt stress and heat stress of B. vulgaris were analyzed by RNA-seq and qRT-PCR analysis. 【Result】 B. vulgaris WRKY gene family contained 40 members, 39 were unevenly distributed on 9 chromosomes, 1 on the random fragment. According to the WRKY conserved domain features and the evolution analysis with Arabidopsis thaliana, 40 members were divided into three classes: Ⅰ, Ⅱ, and Ⅲ. ClassⅠhad 9 members, class Ⅱ had 26 members and class Ⅲ had 5 members. According to the evolutionary relationship, class Ⅱ further divided into five subclass: Ⅱa (1), Ⅱb (4), Ⅱc (9), Ⅱd (5) and Ⅱe (7). Genetic structure analysis showed that exon and intron number of B. vulgaris WRKY genes had high variability (2-7 exons), even within the same subgroup. Conserved element analysis showed that within the same class or subclass had the same conserved elements. WRKY conserved domain analysis revealed two mutations in the WRKY domain: WRKYGKK and WRKYGEK. Each WRKY was expressed in at least two tissues, 30 WRKYs were expressed in leaf, 40 WRKYs were expressed in inflorescence, 36 WRKYs were expressed in young leaf, 38 WRKYs were expressed in root, 39 WRKYs were expressed in seedling, and 36 WRKYs were expressed in seed. The expression levels of WRKY genes were also different. All WRKY genes were divided into two types: low expression genes and high expression genes. Such as: BvWRKY23, BvWRKY3, BvWRKY11, BvWRKY7, BvWRKY6, BvWRKY26, BvWRKY4, BvWRKY40, BvWRKY24, BvWRKY2 and BvWRKY28 were highly expressed in all tissues; BvWRKY38, BvWRKY13, BvWRKY36, BvWRKY35, BvWRKY5 and BvWRKY34 were low expressed in all tissues. BvWRKY21, BvWRKY20, BvWRKY22, BvWRKY32, BvWRKY33 and BvWRKY34 genes were up-regulated under heat stress, and BvWRKY1, BvWRKY6, BvWRKY19, BvWRKY31 and BvWRKY33 were up-regulated under salt stress. In addition, BvWRKY33 had a significant response to both heat and salt stress. 【Conclusion】 B. vulgaris WRKY proteins are highly conserved, the length of the gene sequence and the number of introns varied widely, all WRKY genes showed a variety of expression patterns in different tissues, some WRKY genes responded to heat or salt stress, which play an important role in stress physiological regulation.

Key words: Beta vulgaris, WRKY transcription factor, bioinformatics, stress, expression analysis