烟草脆裂病毒介导的茶树VIGS体系的构建

doi:10.16420/j.issn.0513-353x.2024-0075

摘要/Abstract

摘要：

以茶树（Camellia sinensis）八氢番茄红素脱氢酶（phytoene desaturase）基因CsPDS和咖啡碱合成酶1（caffeine synthase 1）基因TCS1为指示基因，本氏烟草（Nicotiana benthamiana）为载体增殖积累烟草脆裂病毒，探究侵染液浓度、培养温度、不同载体和培养时间对增殖病毒的影响。并探究不同接种方式和不同茶树品种对构建VIGS（Virus-Induced Gene Silencing）体系的影响。结果显示，用乙酰丁香酮（Acetosyringone，AS）浓度为200 μmol · L^-1、菌液浓度OD₆₀₀ = 1.0的侵染液接种烟草后，23 ℃培养有利于病毒在烟草中增殖。使用‘舒茶早’的叶片通过注射接种构建茶树的VIGS体系并成功利用该体系进行以GFP为指示基因探究外源基因表达的可能性。

关键词: 茶树, 病毒诱导的基因沉默, 烟草脆裂病毒, 八氢番茄红素脱氢酶基因, 咖啡碱合成酶基因, 绿色荧光蛋白基因

Abstract:

In this study，Camellia sinensis phytoene desaturase gene（CsPDS）and tea caffeine synthase gene（TCS1）as indicator genes and Nicotiana benthamiana as a vector were employed to enrich tobacco rattle virus. The effects of the concentration of infiltration solution，incubation temperature，different vectors and incubation time on the enriched viruses were investigated，with N. benthamiana used as the vector to enrich the viruses. The effects of different inoculation methods and cultivars on the construction of a virus-induced gene silencing（VIGS）system were also investigated in tea plant. The results demonstrated that inoculation of tobacco with acetosyringone（AS）at a concentration of 200 μmol · L^-1 bacteriophage with an optical density（OD₆₀₀）of 1.0 was conducive to virus enrichment in tobacco when incubated at 23 ℃. The leaves of‘Shuchazao’were used to construct the VIGS system of tea plant by injection inoculation, and the system was successfully used to explore the possibility of foreign gene expression with GFP as the indicator gene.

Key words: Camellia sinensis, virus-induced gene silencing, tobacco rattle virus, phytoene desaturase gene, caffeine synthase 1 gene, green fluorescent protein gene

胡锦瑜, 刘桂芝, 陈兰, 黄梦迪, 苏芹, 谭月萍, 刘硕谦, 田娜. 烟草脆裂病毒介导的茶树VIGS体系的构建[J]. 园艺学报, 2024, 51(11): 2710-2724.

HU Jinyu, LIU Guizhi, CHEN Lan, HUANG Mengdi, SU Qin, TAN Yueping, LIU Shuoqian, TIAN Na. Construction of Virus-Mediated Genetic Transformation System of Camellia sinensis[J]. Acta Horticulturae Sinica, 2024, 51(11): 2710-2724.

图/表 12

表1 本研究中所使用的引物信息

Table 1 Information on primers used in this study

作用 Function	引物名称 Primer name	引物序列 Primer sequence
载体构造 Tectonic carrier	TRV2-CsPDS2	F：5′-gtgagtaaggttaccgaattcTGCTGACATGTCAGTGGCATG-3′ R：5′-cgtgagctcggtaccggatccGTCACCAGACAAATAGAACCCTTCT-3′
	TRV2-TCS1-2	F：5′-gtgagtaaggttaccgaattcGAAATGATTTCAATACCCTCTTCAAA-3′ R：5'-cgtgagctcggtaccggatccCATTGTGAAATCTTCATGAAATTGAG-3′
	C2b-GFP2	F：5′-gttgacagtagtggtgtcgacATGGTGAGCAAGGGCGAGG-3′ R：5′-ttcgggacatgcccggtcgacTTACTTGTACAGCTCGTCCATGCC -3′
	pTRV2-GFP2	F：5′-atttcctttaccattgacgtcATGGTGAGCAAGGGCGAGG -3′ F：5′-gctaccaacgacactgacgtcTTACTTGTACAGCTCGTCCATGCC-3′
病毒检测 Virus detection	pTRV1	F：5′-GGTATTTGGTGGCAACGACT-3′ R：5'-ACCGCTGTGCTTTGATTTCT-3′
	TRV2-CsPDS1	F：5′-TTGTTACTCAAGGAAGCACGAT-3′ R：5′-cgtgagctcggtaccggatccGTCACCAGACAAATAGAACCCTTCT-3′
	TRV2-TCS1-1	F：5′- TTGTTACTCAAGGAAGCACGAT-3′ R：5′-cgtgagctcggtaccggatccCATTGTGAAATCTTCATGAAATTGAG-3′
	C2b-GFP1	F：5′- TTGTTACTCAAGGAAGCACGAT-3′ F：5′-gttgacagtagtggtgtcgacATGGTGAGCAAGGGCGAGG-3′
	pTRV2-GFP1	F：5′- TTGTTACTCAAGGAAGCACGAT-3′ F：5′-gctaccaacgacactgacgtcTTACTTGTACAGCTCGTCCATGCC-3′
qRT-PCR	qCsPDS	F：5′-GCTACGATGAAGGAACTGGCAA-3′ R：5′-TGCAATGGACGACAAGGTTCAC-3′
	qTCS1	F：5′- CCTCTTCAAAGGCCTGTCGTCT-3′ R：5′- CAAGCCTTCTCTGCTTGTGA-3′
	CsGAPDH	F：5′-TTGGCATCGTTGAGGGTCT-3′ R：5′-CAGTGGGAACACGGAAAGC-3′

图1 注射含pTRV2-CsPDS载体的侵染液中不同浓度AS（μmol · L-1）和农杆菌（OD600）的对烟草的感染效果

Fig. 1 Effects of different concentrations of As（μmol · L-1）and Agrobacterium tumefaciens（OD600）in infected solution containing pTRV2-CsPDS vector on tobacco infection

图2 温度对接种含pTRV2-CsPDS载体侵染液的烟草中病毒增殖的影响

Fig. 2 Effect of temperature on the proliferation of tobacco virus inoculated with infection solution containing pTRV2-CsPDS vector

图3 不同载体接种后不同天数烟草的表型变化

Fig. 3 Phenotypic changes of tobacco in different days after inoculation

图4 携带pTRV2-CsPDS、pTRV2-c2b-CsPDS的烟草研磨液的不同接种方法对茶苗生长的影响

Fig. 4 Effects of different inoculation methods of tobacco grinding liquid carrying pTRV2-CsPDS and pTRV2-c2b-CsPDS on tea plants growth

图5 携带pTRV2-CsPDS、pTRV2-c2b-CsPDS的烟草研磨液接种不同茶树品种的电泳图 M：DL2000 marker。1：龙井43；2：舒茶早；3：龙井长叶；4：槠叶齐；5：铁观音；6：福鼎大白；7：碧香早。

Fig. 5 Electrophoresis of different tea varieties inoculated with tobacco grinding liquid carrying pTRV2-CsPDS and pTRV2-c2b-CsPDS M：DL2000 marker. 1：Longjing 43；2：Shuchazao；3：Longjing Changye；4：Zhuyeqi；5：Tieguanyin；6：Fuding Dabai；7：Bixiangzao.

图6 茶树VIGS体系中病毒RT-PCR电泳检测 M：DL2000 marker；WT：未注射病毒。1 ~ 7：pTRV1的上、下游引物；8 ~ 13：RNA2载体上游引物和目的基因的下游引物。

Fig. 6 RT-PCR electropherogram for the detection of viruses in the VIGS system of tea plants M：DL2000 marker；WT：Virus not injected. 1-7：Forward primer and reverse primer of pTRV1；8-13：Forward primer of RNA2 vector and reverse primer of target gene.

表2 茶树感染率

Table 2 Infection rates of tea plants

接种的载体 Inoculated vector	接种茶树数 Number of vaccinations	感染数 Number of infections	感染率/% Infection
pTRV1 + pTRV2-CsPDS	50	16	32
pTRV1 + pTRV2-c2b-CsPDS	50	21	42
pTRV1 + pTRV2-TCS1	50	15	30
pTRV1 + pTRV2-c2b-TCS1	50	19	38

图7 茶苗接种TCS1病毒沉默载体后不同时间接种叶片和40 d时不同部位新生芽和叶中咖啡碱含量变化 WT：未接种；**表示极显著性差异（P < 0.01）。

Fig. 7 Changes of caffeine content in new buds and leaves of different parts at different time and 40 d after tea seedlings were inoculated with TCS1 gene virus silencing vector WT：Unvaccinated；**indicates extremely significant difference（P < 0.01）.

图8 茶树VIGS体系中TCS1和CsPDS的相对表达量变化 WT：未接种；**表示极显著性差异（P < 0.01）。

Fig. 8 Changes in the relative expression of TCS1 and CsPDS in the VIGS system of tea plants WT：Unvaccinated；** indicates extremely significant difference（P < 0.01）.

图9 茶树VIGS体系接种40 d的表型变化

Fig. 9 Phenotypic changes of tea plants inoculated with VIGS system for 40 days

图10 烟草接种含pTRV2-GFP侵染液后荧光图（a）和GFP的RT-PCR电泳图（b）

Fig. 10 Fluorescence diagram（a）and RT-PCR electrophoresis diagram（b）of tobacco inoculated with infection solution containing pTRV2-GFP

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