中国农业科学 ›› 2017, Vol. 50 ›› Issue (22): 4266-4276.doi: 10.3864/j.issn.0578-1752.2017.22.003

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

谷子SiARGOS1的克隆、表达分析和功能标记开发

王智兰1,杜晓芬1,王军1,杨慧卿1,王兴春2,郭二虎1,王玉文1,袁峰1,田岗1,刘鑫1,王秋兰1,李会霞1,张林义1,彭书忠1   

  1. 1山西省农业科学院谷子研究所/杂粮种质资源发掘与遗传改良山西省重点实验室,山西长治 046011; 2山西农业大学生命科学学院,山西太谷030800
  • 收稿日期:2017-04-17 出版日期:2017-11-16 发布日期:2017-11-16
  • 通讯作者: 王军,Tel:13333550810;E-mail:128wan@163.com。郭二虎,Tel:13994673899;E-mail:guoerhu2003@163.com
  • 作者简介:王智兰,Tel:15234582699;E-mail:wangyan11111ai@163.com。杜晓芬,Tel:18636509393;E-mail:dxf6285210@126.com。王智兰和杜晓芬为同等贡献作者。
  • 基金资助:
    山西省重点科技创新团队项目(2015013001-09)、山西省农业科学院种业发展专项(2015ZYZX-04)、山西省农业科学院科技自主创新能力提升工程项目(2016ZZCX-09)、山西省青年基金(2015021143)、山西省农业科学院农业科技创新研究课题(ZDSYS1504)

Molecular Cloning, Expression Analysis and Development of Functional Markers for SiARGOS1 Gene in Foxtail Millet

WANG ZhiLan1, DU XiaoFen1,WANG Jun1, YANG HuiQing1, WANG XingChun2, GUO ErHu1, WANG YuWen1, YUAN Feng1, TIAN Gang1, LIU Xin1, WANG QiuLan1, LI HuiXia1, ZHANG LinYi1, PENG ShuZhong1   

  1. 1Millet Research Institute, Shanxi Academy of Agricultural Sciences/Shanxi Key Laboratory of Genetic Resources and Breeding in Minor Crops, Changzhi 046011, Shanxi; 2 College of Life Science, Shanxi Agricultural University, Taigu 030800, Shanxi
  • Received:2017-04-17 Online:2017-11-16 Published:2017-11-16

摘要: 【目的】从谷子中分离受激素诱导表达、参与器官大小控制的拟南芥ARGOS(Auxin-regulated gene involved in organ size)基因家族的同源基因,进行生物信息学分析,明确其在不同组织器官及其受植物激素诱导的表达模式,分析基因编码区及其启动子序列差异,开发功能标记,为谷子产量性状相关基因的改良提供依据。【方法】通过对已有ARGOS蛋白保守结构域进行BLAST,明确谷子ARGOS家族成员数目并进行蛋白序列分析,采用同源克隆方法获得谷子ARGOS家族成员之一——SiARGOS1编码区及其启动子序列,用生物信息学方法分析SiARGOS1启动子的顺式作用元件,通过实时荧光定量PCR分析该基因在谷子各器官中以及不同植物激素条件下的诱导表达模式,利用基因编码区及启动子序列的SNP和插入缺失序列开发分子标记,同时利用85份谷子品种的穗重(panicle weight,PW)、穗粒重(grain weight,GW)和千粒重(thousand-grain weight,TGW)等产量性状数据进行基因型间的差异显著性分析,挖掘用于检测该基因与谷子产量性状相关优异等位变异的功能标记。【结果】获得6个谷子ARGOS家族成员,均具有典型的保守OSR(organ size related)结构域,包含2个跨膜螺旋结构和1个高度保守富含亮氨酸区域,克隆了与拟南芥AtARGOS同源的家族成员之一——SiARGOS1编码区及其启动子序列,该基因位于谷子第8染色体上,开放阅读框为342 bp,无内含子,编码113个氨基酸,启动子区域为2 109 bp,含有与生长素、乙烯、茉莉酸和赤霉素等多种植物激素调控有关的元件。表达分析发现,SiARGOS1在谷子根、茎、叶和穗等器官中均有表达,在根中表达量最高,其次为茎和叶,穗中表达量最低。SiARGOS1对生长素吲哚乙酸(indole-3-acetanic acid,IAA)不敏感,但受乙烯利(ethephon,ETH)上调表达。不同基因型谷子SiARGOS1序列分析发现,SiARGOS1编码区151 bp(起始密码子83 bp)处存在1个SNP(C/G),导致该基因第28个氨基酸发生突变(Ala/Gly),据此设计一个CAPS- AccⅡ标记;另外,启动子区存在19个SNP和2个InDel,根据-1 652—-1 651处(TA)2/3和-1 165—-1 163处(TCA)1/2的序列差异分别设计SSR引物AP-1和AP-2。同时,用这些标记对85份谷子品种进行检测,CPAS-AccⅡ和AP-1检测到不同基因型的穗重、穗粒重和千粒重的差异均不显著,而AP-2检测的2种基因型间除千粒重差异不显著外,穗重和穗粒重在2015和2016两年间差异均达到显著水平。【结论】在谷子中发现6个ARGOS家族成员,均具有保守OSR结构域,其中,谷子SiARGOS1开放阅读框为342 bp,无内含子,与拟南芥AtARGOS同源,该基因对生长素不敏感,但受乙烯利上调表达。在该基因启动子区开发的SSR标记AP-2可作为功能标记,用于谷子穗重和穗粒重等产量性状相关优异等位变异的鉴定和筛选。

关键词: 谷子, SiARGOS1, 启动子, 表达分析, 功能标记

Abstract: 【Objective】The homologous genes of Arabidopsis ARGOS (auxin-regulated gene involved in organ size) family were isolated from foxtail millet, which were induced expression by hormone and participate in the regulation of plant organ size. Bioinformatics, including alignment of amino acid sequences and promoter cis acting elements, and the expression pattern in different tissues and plant hormones were analyzed. Functional markers were developed from sequences analysis of gene encoding region and promoter, which will play an important role in genetic improvement of yield traits, accelerate breeding process and increase yield of foxtail millet.【Method】The number of ARGOS family members and the sequence of the ARGOS family in foxtail millet were analyzed through BLAST of conserved domain with the existing ARGOS in other plants. The encoding region and promoter sequences of SiARGOS1 gene, one of the family members of ARGOSin foxtail millet, were obtained with homologous cloning method. The promoter cis acting elements of SiARGOS1 was analyzed by the bioinformatics analysis. Expression patterns in different tissues and plant hormone of SiARGOS1 were analyzed using real-time PCR. Functional markers were developed from sequence analysis of SNP and insertion/deletion in gene encoding region and promoter. Functional markers of elite alleles of yield related traits were tested using data analysis of significant differences between genotypes associated with the yield traits such as panicle weight, grain weight and thousand-grain weight in 85 cultivars. 【Result】A total of six ARGOS family members were found to have a conserved OSR (Organ Size Related) domain consisting of two transmembrane helical structures and a highly conserved proline-rich motif in foxtail millet. the open reading frame (ORF) and the promoter of SiARGOS1, one of the family members homologous to Arabidopsis AtARGOS were cloned. Sequence analysis showed that the ORF of SiARGOS1 on chromosome 8 encoding a putative protein composed of 113 amino acids was 342 bp in length, without intron. The promoter contains a variety of regulation related components including auxin, ethylene and other plant hormone with 2 109 bp in length. SiARGOS1 is expressed in all organs of foxtail millet, at high levels in root, whereas at lowest level in panicle. The gene was insensitive to indole-3-acetic acid (IAA), but was up-regulated by ethephon (ETH). On the basis of the alignment of gene SiARGOS1 genomic DNA sequence, CAPS-AccⅡmarker was developed on the missense polymorphism site (C/G→Ala/Gly) at the 150 bp of the encoding region, two SSR markers, AP-1 and AP-2, were developed at -1652--1651(TA)2/3 and -1165--1163(TCA)1/2 of promoter, respectively. No significant association was found among the yield associated traits such as panicle weight, grain weight and thousand-grain weight with CAPS-AccⅡ and AP-1 in 85 cultivars. However, AP-2 proved to be highly correlated with panicle weight and grain weight in 85 cultivars, in addition to thousand-grain weight. The average values of panicle weight for two genotypes were 15.52 g and 20.61 g, respectively, while the ones of grain weight for two genotypes were 12.24 g and 16.74 g, respectively. 【Conclusion】Six ARGOS family members were found in foxtail millet , all of which had conserved OSR domain. The open reading frame (ORF) of SiARGOS1, homologous to Arabidopsis thaliana AtARGOS, is 342 bp in length without intron. The SSR marker AP-2, which was developed in the promoter region of thegene, could be used as functional marker for the selection of superior allelic variation in yield traits such as panicle weight and panicle weight of foxtail millet varieties.

Key words: foxtail millet, SiARGOS1;promoter, expression analysis, functional marker