谷子SiARGOS1的克隆、表达分析和功能标记开发  

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

作　　者：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 

机构地区：[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

出　　处：《中国农业科学》2017年第22期4266-4276,共11页Scientia Agricultura Sinica

基　　金：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);山西省农业科学院农业科技创新研究课题(ZDSYS1504);山西省青年基金(2015021143)

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-acetic 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),�[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 ARGOS in 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 amin

关 键 词：谷子 SI ARGOS1 启动子 表达分析 功能标记 

分 类 号：Q943.2[生物学—植物学] S515[农业科学—作物学]