大豆NAC转录因子生物信息学分析及GmNAC-1克隆和亚细胞定位  

Bioinformatics Analysis of Soybean NAC Transcription Factor and Cloning and Subcellular Localization of GmNAC-1

作　　者：曲硕 刘芳 孙浩文 宋庚辰 胡世豪 姜海鹏 赵雪[1] 韩英鹏[1] QU Shuo;LIU Fang;SUN Haowen;SONG Gengchen;HU Shihao;JIANG Haipeng;ZHAO Xue;HAN Yingpeng(College of Agronomy Key Laboratory of Soybean Biology and Genetic Breeding,Ministry of Agriculture and Rural Affairs,Northeast Agricultural University,Harbin 150030,China)

机构地区：[1]东北农业大学农学院/农业农村部东北大豆生物学与遗传育种重点实验室,黑龙江哈尔滨150030

出　　处：《大豆科学》2024年第5期523-538,共16页Soybean Science

基　　金：黑龙江省重点基金(ZD2022C002);黑龙江省重点研发项目(JD22A015);国家自然科学基金(U22A20473);国家现代农业岗位体系项目(CARS-04-PS07);东北农业大学科研项目(NEAU2023QNLJ-003)。

摘　　要：为研究抗大豆胞囊线虫NAC基因,对大豆东农L10(抗SCN3)和黑农37(感SCN3)进行SCN3逆境胁迫处理,通过RNA-seq筛选差异表达GmNAC基因,采用生物信息学方法分析大豆NAC蛋白理化性质、磷酸化、亲水性和蛋白结构,并进行亚细胞定位、基因共线性分析和启动子区元件分析。克隆GmNAC-1,构建过表达GmNAC-1的pCAMBIA3300质粒,通过农杆菌注射法转化至烟草中,进行亚细胞定位。对SCN3胁迫后抗、感大豆的根、茎、叶进行荧光定量PCR。结果表明:NAC家族蛋白总体呈现电中性;二、三级结构无规则卷曲比例最高;亚细胞定位表明NAC家族蛋白存在于细胞核;蛋白总体呈现亲水性,不存在染色体偏好性;丝氨酸对维持蛋白功能发挥主要作用;蛋白总体上为可溶性蛋白;存在多个响应逆境胁迫应答元件LTR、MRE、TGACG-motif等;共线性分析表明不同物种中的NAC基因存在一定同源性。荧光定量PCR结果表明抗、感大豆在受到SCN3胁迫后,GmNAC-1基因均存在上调表达,但抗病比感病大豆根系GmNAC-1基因上调表达更明显。亚细胞定位结果表明GmNAC-1定位于细胞核中,与生物信息学预测结果一致。本研究通过RNA-seq筛选差异表达基因GmNAC-1,该基因参与SCN逆境胁迫,结果为进一步研究抗大豆抗胞囊线虫基因奠定基础。To explore the soybean cyst nematode-resistant NAC gene,soybean Dongnong L10(SCN3-resistant) and Heinong 37(SCN3-sensitive) were subjected to SCN3 adversity stress treatment,and the differentially expressed GmNAC genes were screened by RNA-seq,and the physicochemical properties,phosphorylation,hydrophilicity,and protein structure of the soybean NAC proteins were analyzed by bioinformatic methods,and the subcellular localization,the subcellular localization,gene covariance analysis and promoter region element analysis were also performed.GmNAC-1 was cloned,pCAMBIA3300 plasmid overexpressing GmNAC-1 was constructed,and transformed into tobacco by agrobacterium injection method for subcellular localization.qRT-PCR was performed on roots,stems and leaves of resistant and susceptible soybean after SCN3 stress.Results showed that NAC family proteins were electrically neutral in general.The proportion of irregularly curled secondary and tertiary structures was the highest.Subcellular localization suggested that NAC family proteins were present in the nucleus.The proteins were hydrophilic in general,and there was no chromosomal preference,serine played a major role in maintaining the function of the proteins,the proteins were soluble proteins in general.Collinearity analysis showed that there were some homology of NAC genes in different species.qRT-PCR results showed that the GmNAC-1 gene was up-regulated in both resistant and susceptible soybeans after SCN3 stress,but the up-regulation of GmNAC-1 was more obvious in resistant than in susceptible soybean roots.The results of subcellular localization indicated that GmNAC-1 was localized in the nucleus,which was consistent with the bioinformatics prediction.In this study,we screened out the differentially expressed gene GmNAC-1,which was involved in SCN adversity stress by RNA-seq,and the results provided a basis for further research on cyst nematode resistance genes in resistant soybean.

关 键 词：大豆胞囊线虫 GmNAC-1 RNA-SEQ 荧光定量PCR 生物信息学 

分 类 号：S565.1[农业科学—作物学]