摘要
病毒诱导的基因沉默(virus-induced gene silencing,VIGS)技术已广泛用于植物基因功能研究,以烟草脆裂病毒(tobacco rattle virus,TRV)为载体的沉默体系介导大豆基因沉默效率有待明确,采用无缝克隆技术构建TRV-VIGS沉默体系,探索不同接种方法对大豆靶基因在不同组织间的沉默效率,为大豆基因功能研究提供依据。以八氢番茄红素去饱和酶(phytoene desaturase,GmPDS)及泛素连接酶(GmATL3)基因为靶基因,将含有pTRV1和重组载体菌液采用注射、灌根(agroinoculation)、注射与灌根相结合3种方法分别接种大豆中黄13,接种28 d观察沉默表型现象,并使用RT-qPCR技术检测根部与叶部基因相对表达量,明确不同方法沉默效果。注射接种的大豆叶边缘及叶内出现黄化褪绿,灌根接种与注射加灌根接种的叶片表面出现褪绿斑点及褶皱褪绿表型。RT-qPCR结果表明,3种接种方法对沉默GmPDS效果接近100%;注射接种对GmATL3的沉默效率在叶部为80%-95%,根部为40%-60%;灌根与注射加灌根接种,根部沉默效率为70%-90%,叶部沉默效率为15%-50%。不同接种方法产生不同程度沉默表型,且对不同内源基因沉默效率不同。接种方法对不同组织沉默效率存在差异,注射方法对叶片沉默效率最高,注射加灌根结合的方法对根部沉默效率最高。
Virus-induced gene silencing(VIGS)technology has been widely used in plant gene function research.The efficiency of gene silencing in soybean mediated by tobacco rattle virus(TRV)-based vector system remains to be clarified.The seamless cloning technique was used to construct a TRV-VIGS system to explore the silencing efficiency in soybean target genes in different tissues with different inoculation methods,providing a basis for gene function studies in soybean.Taken phytoene desaturase(GmPDS)and ubiquitin ligase(GmATL3)as target genes,pTRV1 and the recombinant vectors solution were inoculated into soybean(Glycine max)plants Zhonghuang13 by three methods:injection,agroinoculation and a combination of injection plus agroinoculation.The silencing phenotypes were observed at 28 d after inoculation,and the relative expressions of the genes in the roots and leaves were quantified by RT-qPCR to determine the silencing effectiveness of the different methods.Silencing of GmPDS resulted in yellowing and chlorosis on the edges and inside of the leaves,while chlorosis spots and fold chlorosis phenotypes in the leaves surface occurred.The results of RT-qPCR showed that silencing efficiency of GmPDS of all three inoculation methods was close to 100%.The silencing efficiency of GmATL3 by the injection was 80%-95% in the leaves and 40%-60% in the roots;the silencing efficiency of the root inoculation and injection plus root inoculation was 70%-90% in roots and 15%-50% in leaves.Different inoculation methods caused different levels of silencing phenotypes and silencing efficiencies for different endogenous genes.The silencing efficiency of an inoculation method on different tissues was different,and the highest silencing efficiency on the leaves and roots were injection method and injection plus agroinoculation,respectively.
作者
李文辰
刘鑫
康越
李伟
齐泽铮
于璐
王芳
LI Wen-chen;LIU Xin;KANG Yue;LI Wei;QI Ze-zheng;YU Lu;WANG Fang(College of Life Sciences and Agroforestry,Heilongjiang Provincial Key Laboratory of Resistance Gene Engineering and Protection of Biodiversity in Cold Areas,Qiqihar University,Qiqihar 161006;Heilongjiang Academy of Agricultural Sciences Livestock&Veterinary Branch,Qiqihar 161005)
出处
《生物技术通报》
CAS
CSCD
北大核心
2023年第7期143-150,共8页
Biotechnology Bulletin
基金
国家自然科学基金项目(31801725)
黑龙江省教育厅基本科研业务费科研项目(135309361)
齐齐哈尔大学研究生创新科研项目。
