大豆胞囊线虫相关基因GmSBPC的克隆及表达模式分析  

Cloning and Expression Pattern Analysis of GmSBPC Associated with Soybean Cyst Nematodes

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

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,哈尔滨150030

出　　处：《植物遗传资源学报》2024年第6期1001-1013,共13页Journal of Plant Genetic Resources

基　　金：国家自然科学基金项目(U22A20473);黑龙江省重点基金项目(ZD2022C002);黑龙江省重点研发项目(JD22A015);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：以黑农37(感)和东农L10(抗)大豆胞囊线虫3号生理小种胁迫RNA-seq数据,筛选出差异表达基因GmSBPC,对该基因编码蛋白的空间结构、蛋白理化性质、亲疏水性等进行生物信息学分析。利用抗病东农L10根系c DNA克隆GmSBPC。将含有pCAMBIA1302-GmSBPC重组载体转化至大肠杆菌DH5α、农杆菌GV3101(psoup-p19)进行亚细胞定位分析。重组pCAMBIA3300-Gm SBPC转至根癌农杆菌K599进行大豆毛状根侵染。线虫土种植东农L10(抗)、东农50(感),大豆胞囊线虫胁迫处理0 d、3 d、6 d、9 d、12 d、15 d分别取根、茎、叶进行qRT-PCR分析基因表达模式。结果表明,GmSBPC蛋白编码146个氨基酸,为不溶性蛋白,α螺旋区占28.08%、延伸结构占15.75%、无规则卷曲占56.16%。亚细胞定位结果表明基因定位在细胞核中。过表达毛状根相比野生型大豆单位面积内线虫数目减少。大豆胞囊线虫胁迫下该基因在东农50和东农L10根系的表达模式为先升高后降低,整体表达水平东农L10根系>东农50根系,东农L10根系中12 d表达量最高,该时期为线虫侵染大豆的二龄幼虫时期,因此判定该基因对线虫胁迫存在响应应答反应,推测该基因参与大豆胞囊线虫的胁迫反应。这些研究结果有助于进一步探讨SBPC基因在大豆抗胞囊线虫过程中的生理功能。This study used RNA-seq data from physiological race 3 of soybean cyst nematode,Heinong 37(susceptible)and Dongnong L10(resistant),to screen for the differentially expressed gene GmSBPC.Bioinformatics analysis was conducted on the spatial structure,protein physicochemical properties,hydrophilicity,and hydrophobicity of the protein encoded by this gene.Cloning GmSBPC using cDNA from the root system of disease resistant Dongnong L10.Transform the recombinant vector containing pCAMBIA1302-GmSBPC into Escherichia coli DH5α,Agrobacterium GV3101(psoup-p19)for subcellular localization analysis.Recombinant pCAMBIA3300-GmSBPC was transferred to Agrobacterium tumefaciens K599 for soybean hairy root infection.Planting Dongnong L10(resistant)and Dongnong 50(susceptible)in nematode soil,soybean cyst nematode stress treatment was performed on roots,stems,and leaves at 0,3,6,9,12,and 15 days for qRT-PCR analysis of gene expression patterns.The results indicate that the GmSBPC protein encodes 146 amino acids and is an insoluble protein,αspiral zone accounts for 28.08%,extended structure accounts for 15.75%,and irregular curl accounts for 56.16%.The subcellular localization results indicate that the gene is located in the nucleus.Overexpression of hairy roots reduces the number of nematodes per unit area compared to wild-type soybeans.The gene expression pattern of the roots of Dongnong 50 and Dongnong L10 under soybean cyst nematode stress was initially increased and then decreased,with the overall expression level being higher in Dongnong L10 roots than in Dongnong 50 roots.The highest expression level was observed in Dongnong L10 roots after 12 days,which is the second instar larval stage of soybean infection by nematodes.Therefore,it is determined that this gene has a response to nematode stress,and it is speculated that this gene is involved in the stress response of soybean cyst nematodes.These research results contribute to further exploring the physiological function of SBPC gene in soybean resistance to cyst nematodes.

关 键 词：大豆 GmSBPC QRT-PCR 亚细胞 

分 类 号：S435.651[农业科学—农业昆虫与害虫防治]