金鱼草WAK/WAKL基因家族鉴定及抗核盘菌候选基因挖掘

Genome-wide identification and mining of candidate genes against Sclerotinia sclerotiorum of WAK/WAKL gene family in snapdragon(Antirrhinum majus L.)

  • 摘要: 【目的】鉴定金鱼草细胞壁相关受体激酶(Wall-associated kinase,WAK)及类WAK蛋白(WAK-like kinases,WAKL)基因家族成员,并挖掘抗核盘菌的候选基因,为深入解析金鱼草抗核盘菌的WAK/WAKL分子调控机制提供理论参考。【方法】以拟南芥WAK/WAKL家族蛋白为参考序列,利用生物信息学方法从金鱼草基因组数据库中鉴定出WAK/WAKL基因家族成员,并对其理化性质、系统发育、基因结构、保守基序、顺式作用元件及共线性关系等进行预测分析,并通过转录组测序和实时荧光定量PCR对该家族基因在金鱼草抗感核盘菌材料中的表达模式进行分析。【结果】从金鱼草基因组中共鉴定出27个WAK/WAKL基因家族成员,其中WAK家族基因16个(AmWAK1~AmWAK16),WAKL家族基因11个(AmWAKL1~AmWAKL11),编码的氨基酸数量为615~1085个;理论等电点(pI)为4.94~8.69,绝大多数蛋白呈酸性;所有蛋白的总平均亲水性指数(GRAVY)值小于0,均为亲水性蛋白;大多数蛋白亚细胞定位于细胞质膜区域,少部分定位于细胞外、液泡、高尔基体和细胞核。27个WAK/WAKL基因家族成员不均匀地分布在金鱼草的8条染色体上,且与拟南芥WAK/WAKL家族基因存在7对共线性同源基因;基因启动子区域含有与防御和胁迫响应、茉莉酸甲酯响应及水杨酸响应等顺式作用元件。通过叶片离体接种核盘菌试验,筛选出抗性差异明显的易感病品种Am1和抗病品种Am6。有12个WAK/WAKL家族基因在金鱼草抗感材料中显著差异表达(P<0.05),有9个基因表达模式与转录组测序结果基本一致。这9个基因中,有5个基因(AmWAK7AmWAKL2AmWAKL8AmWAK6AmWAK13)在金鱼草的根、茎和叶组织中高表达,而剩余4个基因(AmWAK2、AmWAK8、AmWAK9和AmWAK12)在根、茎和叶中基本不表达。【结论】筛选出的27个金鱼草WAK/WAKL基因家族成员,具有相似的基因结构和蛋白功能结构域,其中AmWAK6AmWAK7AmWAK13AmWAKL2AmWAKL8为金鱼草抗核盘菌的关键候选基因,具有介导抗病的潜在功能。

     

    Abstract: 【Objective】To identify the gene family members of the cell wall-associated kinase(WAK) and WAK-like kinases(WAKL) of Antirrhinum majus L.,and explore the candidate genes against Sclerotinia sclerotiorum. This study provided a theoretical reference for further analysis of WAK/WAKL molecular regulatory mechanism of A. majus against S. sclerotiorum. 【Method】Using Arabidopsis thaliana L. WAK/WAKL family proteins as reference sequences,the WAK/WAKL gene family members were identified from the A. majus genome database by bioinformatics method,and their physicochemical properties,phylogeny,gene structure,conserved mods,cis-acting elements and collinear relationships were predicted. Transcriptome sequencing and real-time fluorescence quantitative PCR were used to analyze the expression patterns of the family genes involved in S. sclerotiorum resistance. 【Result】A total of 27 WAK/WAKL gene family members were identified from the A. majus genome,including 16 WAK genes(AmWAK1-AmWAK16) and 11 WAKL genes(AmWAKL1-AmWAKL11),encoding 615-1085 amino acids. The theoretical isoelectric point(p I) ranged from 4.94to 8.69,and the most proteins were acidic. The total mean hydrophilic index(GRAVY) of all proteins was less than 0,indicating that all proteins are hydrophilic proteins. Most of the subcellular proteins were located in the plasma membrane region of the cell,and a few were located in the extracellular,vacuole,Golgi apparatus and nucleus. There were 27 members of WAK/WAKL gene family distributed unevenly on 8 chromosomes of A. majus,and 7 pairs of collinear homologous genes with A. thaliana WAK/WAKL gene family. The gene promoter region contained cis-acting elements involved in defense and stress response,methyl jasmonate response and salicylic acid response. The susceptible variety Am1 and resistant variety Am6 were screened by inoculating the leaves of sclallodiscus in vitro. There were 12 WAK/WAKL family genes significantly differentially expressed(P<0.05) among the resistant materials,and the expression patterns of 9 genes were basically consistent with the transcriptome sequencing results. Of the nine genes,five(AmWAK7,AmWAKL2,AmWAKL8,AmWAK6 and AmWAK13) were highly expressed in the root,stem,and leaf tissues of A. majus,while the remaining four(AmWAK2,AmWAK8,AmWAK9 and AmWAK12) were largely unexpressed in the root,stem and leaf tissues.【Conclusion】Twenty-seven members of the WAK/WAKL gene family are screened,which have similar gene structure and protein functional domains. Among them,AmWAK6,AmWAK7,AmWAK13,AmWAKL2 and AmWAKL8 are key candidate genes for resistance to S. sclerotiorum,and have potential functions of mediating disease resistance.

     

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