旱柳NAC基因家族的全基因组鉴定及其在淹水和盐胁迫下的表达分析

Whole genomic identification of NAC gene family and expression analysis under flooding stress and salt stress in Salix matsudana

  • 摘要: 【目的】 对旱柳(Salix matsudana)NAC基因家族成员进行鉴定,并分析其在淹水胁迫和盐胁迫下的表达差异,为研究SmNAC家族成员参与逆境胁迫调控机制及培育高耐非生物胁迫旱柳品种提供理论基础。【方法】 以敏淹旱柳品种Yanliu No. 1、耐淹旱柳品种Suliu 795和敏盐旱柳种质Yanjiang和耐盐旱柳品种9901等4份旱柳种质为试验材料,通过基因组数据挖掘鉴定出旱柳全基因组范围内的NAC基因家族成员,并对其蛋白理化性质、保守基序、系统发育关系、基因结构及其染色体位置、共线性关系进行预测分析。对SmNAC基因家族成员进行淹水胁迫和盐胁迫下的表达谱分析;选取对淹水胁迫和盐胁迫均具有明显响应的基因构建拟南芥过表达载体并获取转基因拟南芥,通过实时荧光定量PCR检测该基因在野生型和转基因拟南芥中的相对表达量,并分析转基因拟南芥和野生型拟南芥在淹水胁迫下的表型差异,测定其生理指标。【结果】 鉴定出290个SmNAC基因家族成员,命名为SmNAC001~SmNAC290;SmNAC家族基因编码的氨基酸数量范围为105~710个,分子量为12.7~77.8 kD,理论等电点(pI)为4.33~9.65,大多数蛋白成员定位于细胞核。根据SmNAC家族蛋白与拟南芥NAC蛋白的系统发育进化关系及其保守基序分析结果,将SmNAC家族蛋白分为16个亚家族。大部分SmNAC家族基因中含有3个外显子,同一亚家族内的SmNAC成员基序组成和排列模式相似,如NAP和OsNAC7亚家族。大部分SmNAC家族基因成员启动子含有茉莉酸甲酯以及缺氧胁迫等响应元件。SmNAC基因家族中有244个基因随机分布在38条染色体上,其中存在13对串联复制事件。转录组数据结 果 显 示 ,淹 水 胁 迫 和 盐 胁 迫 后 ,ATAF 亚 家 族(SmNAC007SmNAC008SmNAC009SmNAC010SmNAC011SmNAC012基因)表达量在不同抗性旱柳种质中均明显上调。实时荧光定量PCR结果显示,转基因拟南芥中SmNAC007基因的相对表达量明显高于野生型,为10~15倍;与野生型拟南芥相比,转基因拟南芥中总蛋白含量显著降低(P<0.05)。【结论】 鉴定出290个SmNAC基因家族成员,其中分别有102个和151个家族成员在淹水胁迫和盐胁迫中差异表达;初步证实SmNAC007基因在淹水胁迫响应中发挥正向调控功能。

     

    Abstract: 【Objective】 The purpose of the study was to identify members of the NAC gene family in Salix matsudana and analyze their expression differences under flooding stress and salt stress,so as to provide theoretical basis for studying the mechanism of SmNAC family members involved in stress regulation and cultivating S. matsudana varieties with high tolerance to abiotic stress. 【Method】 Using the 4 S. matsudana germplasms(flooding-sensitive S. matsudana variety Yanliu No. 1,the flooding-tolerant S. matsudana variety Suliu 795,the salt-sensitive S. matsudana germplasm Yanjiang, and the salt-tolerant S. matsudana variety 9901)as experimental materials,the NAC gene family members within the whole genome of S. matsudana were identified through genomic data mining,and their protein physicochemical properties,conserved motifs,phylogenetic relationships,gene structures,chromosome localization and collinearity relationships were predicted and analyzed. Expression profiles of SmNAC gene family members under flooding stress and salt stress was conducted;genes that showed obvious response to both flooding stress and salt stress were selected to construct Arabidopsis thaliana overexpression vectors and obtain transgenic A. thaliana. The relative expression levels of these genes in wild-type and transgenic A. thaliana were detected by real-time fluorescence quantitative PCR,and the phenotypic differences between transgenic A. thaliana and wild-type A. thaliana under water flooding stress were analyzed,and the physiological indicators were measured. 【Result】 A total of 290 SmNAC gene family members were identified,named SmNAC001 to SmNAC290;the number of amino acids encoded by SmNAC family genes ranged from 105 to 710,with molecular weights ranging from 12.7 to 77.8 kD and theoretical isoelectric points(pI)ranging from 4.33 to 9.65. Most of the protein members were localized in the nucleus. Based on the phylogenetic evolutionary relationship between SmNAC family proteins and A. thaliana NAC proteins and the results of conserved motifs,the SmNAC family proteins were divided into 16 subfamilies. Most SmNAC family genes contained 3 exons,and SmNAC members within the same subfamily had similar motif composition and arrangement patterns,such as the NAP and OsNAC7 subfamilies. Most SmNAC family gene members promoters contained elements responsive to methyl jasmonate and hypoxia stress. In the SmNAC gene family,244 genes were randomly distributed on 38 chromosomes,among which there were 13 pairs of tandem duplication events. Transcriptome data showed that after flooding stress and salt stress,the expression levels of the ATAF subfamily(SmNAC007SmNAC008SmNAC009SmNAC010SmNAC011,and SmNAC012 genes)was up-regulated in the S. matsudana genplasms with different resistance. Real-time fluorescence quantitative PCR results showed that the relative expression level of the SmNAC007 gene in transgenic A. thaliana was greatly higher than that in the wild-type by 10 to 15 times;compared to wild-type A. thaliana,the total protein content in transgenic A. thaliana was significantly reduced(P<0.05). 【Conclusion】 A total of 290 SmNAC gene family members are identified,among which 102 and 151 family members are differentially expressed under flooding stress and salt stress respectively;the SmNAC007 gene is preliminarily confirmed to play a positive regulatory role in the response to flooding stress.

     

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