TRV病毒诱导大豆基因沉默体系优化及应用

doi:10.13560/j.cnki.biotech.bull.1985.2022-1449

摘要/Abstract

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。

关键词: 大豆, 病毒诱导的基因沉默, 烟草脆裂病毒

Abstract:

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.

Key words: soybean, virus-mediated gene silencing, tobacco rattle virus

李文辰, 刘鑫, 康越, 李伟, 齐泽铮, 于璐, 王芳. TRV病毒诱导大豆基因沉默体系优化及应用[J]. 生物技术通报, 2023, 39(7): 143-150.

LI Wen-chen, LIU Xin, KANG Yue, LI Wei, QI Ze-zheng, YU Lu, WANG Fang. Optimization and Application of Tobacco Rattle Virus-induced Gene Silencing System in Soybean[J]. Biotechnology Bulletin, 2023, 39(7): 143-150.

图/表 5

表1 引物序列

Table 1 Primer sequence

引物名称Primer name	引物序列Primer sequence（5'-3'）	引物长度Primer length/bp
GmATL3-1-F	taaggttaccGAATTCTGGATCCCGCGGTG	374
GmATL3-1-R	atgcccgggcCTCGAGAGGATGAGGATGAACCTTCC	374
GmATL3-2-F	taaggttaccGAATTCGGTGACTCTTCCGCG	296
GmATL3-2-R	atgcccgggcCTCGAGTACCAACACCGGTAGGGAG	296
GmPDS-F	taaggttaccGAATTCTCTCCGCGTCCTCTAAAAC	332
GmPDS-R	atgcccgggcCTCGAGTCCAGGCTTATTTGGCATAGC	332
qATL3-F	TGCCTTCATCTCTTCGCCAG	196
qATL3-R	CCGCACATTCCAAACCATCC	196
qPDS-F	CCGCTGCAAGCTTGGCTTTA	218
qPDS-R	CGACACGCAAGGGAGAGAAA	218
EF4-F	TGCCGCCAAGAAGAAGTGAT	149
EF4-R	GCGGACACTTCAAAATATAACTGGT	149

图1 GmATL3与GmPDS序列及沉默片段区域蓝色代表基因序列全长，GmATL3全长1 347 bp，GmPDS全长2 196 bp；紫色代表GmATL3 CDS编码区全长783 bp，GmPDS CDS编码区全长1 710 bp；红色代表保守结构区域，GmATL3保守结构区域全长132 bp，GmPDS保守结构区域全长1 665 bp；黑色代表沉默片段选择区域，GmATL3-1位于492-833 bp，GmATL3-2位于265-528 bp，GmPDS位于695-880 bp

Fig. 1 GmATL3 and GmPDS gene sequences and silent fragment regions Blue indicates the full length of the gene sequences, 1 347 bp for GmATL3 gene and 2 196 bp for GmPDS gene; purple indicates the full length of the CDS coding region, 783 bp for GmATL3 coding region and 1 710 bp for GmPDS CDS coding region; red represents the conserved structural region, 402 bp for GmATL3 and 1 665 bp for GmPDS, respectively; black represents silent fragment regions, 492-833 bp for GmATL3-1, 265-528 bp for GmATL3-2 and 695-880 bp for GmPDS

图2 GmATL3-1、GmATL3-2、GmPDS PCR凝胶电泳检测 1：GmATL3-1；2：GmATL3-2；3：GmPDS；M：DL2000 DNA marker

Fig. 2 GmATL3-1, GmATL3-2 and GmPDS PCR electrophoresis detection

图3 不同接种方法GmPDS沉默叶部表型观察

Fig. 3 Phenotypic observations of the leaves after GmPDS silenced via different inoculation methods

图4 3种接种方法GmATL3-1、GmATL3-2、GmPDS在根与叶中的相对表达量 A：注射接种；B：灌根接种；C：注射加灌根接种；D：1：叶部对照组；2：叶部试验组；3：根部对照组；4：根部试验组；M：DL2000 marker。**代表P ≤ 0.01，***代表P ≤ 0.001，ns代表差异不显著

Fig. 4 Relative expressions of GmATL3-1, GmATL3-2 and GmPDS in the roots and leaves by three inoculation methods A: Injection inoculation; B: root inoculation; C: injection plus root inoculation; D: 1: leaf control; 2: leaf test group; 3: root control; 4: root test group; M: DL2000 marker.** indicates P ≤ 0.01，*** indicates P ≤ 0.001 and ns indicates no signifraunt differenle

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