谷子光敏色素基因光周期、非生物胁迫响应特性及关键自然变异位点鉴定

The responsive characteristics of phytochrome genes to photoperiod, abiotic stresses and identification of their key natural variation sites in foxtail millet (Setaria italica L.)

摘要:

比较分析光敏色素基因家族成员对光周期、非生物胁迫响应模式并鉴定其有利自然变异类型，为全面了解光敏色素基因家族在光周期调控谷子生长发育及应对非生物胁迫中的作用机制、开展关键性状的分子辅助选择奠定基础。文中利用RT-PCR技术从超晚熟谷子农家种‘毛粟’中克隆了3个光敏色素基因SiPHYA、SiPHYB和SiPHYC；在进行生物信息学分析后，利用荧光定量PCR技术研究了3个基因的光周期调控模式及对聚乙二醇（polyethylene glycol，PEG）模拟干旱、自然干旱、脱落酸（abscisic acid，ABA）、高温、NaCl五种非生物胁迫的响应特性；最后检测这3个基因在160份谷子材料的突变位点，通过单倍型分析确定基因的功能效应。结果表明，获得了基因SiPHYA、SiPHYB和SiPHYC包含完整编码区的cDNA序列，长度分别为3 981、3 953、3 764 bp，其中基因SiPHYB和SiPHYC具有较近的进化关系。基因SiPHYA、SiPHYB和SiPHYC均受光周期调控，但光周期对SiPHYB、SiPHYC昼夜表达模式的影响要强于SiPHYA；短日照条件临近抽穗SiPHYA、SiPHYB表达水平显著低于长日照，暗示其在谷子长日照抑制抽穗中发挥作用。SiPHYB和SiPHYC共同响应PEG模拟干旱、自然干旱、ABA、高温4种胁迫；SiPHYA和SiPHYB以不同方式响应盐胁迫，SiPHYC没有参与谷子盐胁迫响应过程。基于160份谷子材料重测序数据发现基因SiPHYB高度保守，基因SiPHYA含有两个错义突变：单核苷酸多态性（single nucleotide polymorphism，SNP）7 034 522^C→T，SNP7 036 657^G→C，导致延迟抽穗、增加株高。基因SiPHYC含有一个错义突变（SNP5 414 823^G→T），导致短日照缩短抽穗期，长日照延长抽穗期，对株高、穗长的增加作用不受光温环境影响。光周期对基因SiPHYA、SiPHYB和SiPHYC具有不同的调控作用，除了盐胁迫，SiPHYB和SiPHYC共同响应多种非生物胁迫，相比参考基因型，SiPHYA、SiPHYC突变延迟抽穗、增加株高和穗长。

关键词:谷子;光敏色素基因;非生物胁迫;表达分析;单倍型

Abstract:

The responsive patterns of phytochrome gene family members to photoperiod and abiotic stresses were comparatively analyzed and the favorable natural variation sites of these genes were identified. This would help understand the mechanism of phytochrome gene family in photoperiod-regulated growth and development and abiotic stress response. In addition, it may facilitate the molecular marker assisted selection of key traits in foxtail millet. In this study, we used RT-PCR to clone three phytochrome genes SiPHYA, SiPHYB and SiPHYC from ultra-late maturity millet landrace variety ‘Maosu’. After primary bioinformatics analysis, we studied the photoperiod control mode and the characteristics of these genes in responding to five abiotic stresses including polyethylene glycol (PEG)-simulated drought, natural drought, abscisic acid (ABA), high temperature and NaCl by fluorescence quantitative PCR. Finally, we detected the mutation sites of the three genes among 160 foxtail millet materials and performed haplotype analysis to determine the genes' functional effect. We found that the cloned cDNA sequences of gene SiPHYA, SiPHYB and SiPHYC were 3 981, 3 953 and 3 764 bp respectively, which contained complete coding regions. Gene SiPHYB and SiPHYC showed closer evolutionary relationship. Photoperiod regulated all of the three genes, but showed more profound effects on diurnal expression pattern of SiPHYB, SiPHYC than that of SiPHYA. Under short-day, when near heading, the expression levels of SiPHYA and SiPHYB were significantly lower than that under long-day, indicating their roles in suppressing heading of foxtail millet under long-day. SiPHYB and SiPHYC were responsive to PEG-simulated drought, natural drought, ABA and high temperature stresses together. SiPHYA and SiPHYB responded differently to salt stress, whereas SiPHYC did not respond to salt stress. Re-sequencing of 160 foxtail millet materials revealed that SiPHYB was highly conservative. Two missense mutations of SiPHYA, such as single nucleotide polymorphism (SNP) 7 034 522^C→T and SNP7 036 657^G→C, led to delaying heading and increasing plant height. One missense mutation of SiPHYC, such as SNP5 414 823^G→T, led to shortening heading under short-day and delaying heading under long-day, as well as increasing plant height and panicle length regardless of photo-thermal conditions. Photoperiod showed different regulatory effects on SiPHYA, SiPHYB and SiPHYC. SiPHYB and SiPHYC jointly responded to various abiotic stresses except for the salt stress. Compared with the reference genotype, mutation genotypes of SiPHYA and SiPHYC delayed heading and increased plant height and panicle length.

Key words:foxtail millet;phytochrome genes;abiotic stress;expression analysis;haplotype