TRV病毒介导的基因沉默体系在连翘中的建立

doi:10.16420/j.issn.0513-353x.2024-0972

摘要/Abstract

摘要：

为建立连翘[Forsythia suspensa（Thunb.）Vahl]的基因功能验证体系，以连翘幼苗、果实、花芽为材料，FsPDS为报告基因分别在连翘营养器官和生殖器官中进行了病毒介导的基因沉默（virus-induced gene silencing，VIGS）体系的构建及优化，在连翘花芽中对异型花柱关键基因FsCYP90D1（FsPDS）的功能进行验证。结果显示试验组植株新生叶片出现典型的光漂白表型且FsPDS基因表达量显著下降，成功构建连翘幼苗FsPDS基因沉默体系，连翘最佳侵染液配方为：400 μmol · L^-1 AS + 10 mmol · L^-1 MgCl₂ + 10 mmol · L^-1 MES + 400 mg · L^-1 Cysteine + 5 mL · L^-1 Tween-20；从果实处理、接种方法、菌液浓度3个方面构建并优化连翘果实VIGS体系，降低了连翘果实中FsPDS基因表达水平；在连翘花芽中开展了FsCYP90D1基因沉默研究，试验组FsCYP90D1基因表达量降低，长花柱变短，验证其参与连翘花柱伸长。

关键词: 连翘, 病毒诱导的基因沉默, 烟草脆裂病毒, FsCYP90D1, 异型花柱

Abstract:

In order to establish the gene function verification technology of Forsythia suspensa，by using Forsythia seedlings，fruits，and flower buds as materials，the virus-induced gene silencing system was constructed and optimized in the vegetative and reproductive organs of F. suspensa with FsPDS as the reporter gene，and the function of the key gene FsCYP90D1 was verified in the flower buds of F. suspensa. The results showed that the new grown leaves of the experimental group exhibited typical photobleaching phenotype and the expression of FsPDS gene decreased significantly. The FsPDS gene silencing system of F. suspensa seedlings was successfully constructed and the optimal infection solution formula of F. suspensa was：400 μmol · L^-1 AS + 10 mmol · L^-1 MgCl₂ + 10 mmol · L^-1 MES + 400 mg · L^-1 Cysteine + 5 mL · L^-1 Tween-20. The VIGS system of F. suspensa fruit was constructed and optimized from three aspects of fruit treatment，inoculation method and bacterial liquid concentration，which reduced the expression level of FsPDS gene in F. suspensa fruit. The FsCYP90D1 gene silencing study was conducted in the flower buds of F. suspensa，with the experimental group showing decreased expression levels of the FsCYP90D1 gene and shortened floral columns，verifying the involvement of the FsCYP90D1 gene in the elongation of the Forsythia floral column.

Key words: Forsythia suspensa, virus-induced gene silencing, tobacco rattle virus, FsCYP90D1, heterostyly

李澳旋, 樊阿梅, 杜晓蓉, 何龙娇, 靳亚鑫, 李琦, 王德富, 宋芸, 乔永刚. TRV病毒介导的基因沉默体系在连翘中的建立[J]. 园艺学报, 2025, 52(12): 3373-3385.

LI Aoxuan, FAN Amei, DU Xiaorong, HE Longjiao, JIN Yaxin, LI Qi, WANG Defu, SONG Yun, QIAO Yonggang. Establishment of TRV Virus-Induced Gene Silencing System in Forsythia suspensa[J]. Acta Horticulturae Sinica, 2025, 52(12): 3373-3385.

图/表 10

参考文献 23

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基因Gene	引物序列（5′-3′） Primer sequence	扩增子长度/bp Amplicon length	退火温度/℃ Annealing temperature	备注 Remark
PDSBOS	F：aaggttaccgaattctctagaATGGAAAGATGATGATGGAGACTGG	309	60.5	PCR
PDSBOS	R：cgtgagctcggtaccggatccACATACGACTGTCCACCAATTATTG	309	64.5	PCR
FsPDSB	F：CTCCGGTCTTGGATAGTCAATG	100	55.3	RT-qPCR
FsPDSB	R：AGTCCAAACAAGCCACTAAAG	100	55.4	RT-qPCR
EF1αH	F：TGGAAATTTGAGACCACCAA	/	51.2	RT-qPCR
EF1αH	R：GCACAATCAGCCTGTGAAGT	/	55.7	RT-qPCR
FsCYP90D1B	F：aaggttaccgaattctctagaATGCAAAAACGTTTGTTCCATCT	328	60.1	PCR
FsCYP90D1B	R：cgtgagctcggtaccggatccCCAATAAATTCTTGAAATTGTTTCTTTA	328	60.3	PCR
FsCYP90D1	F：ATGCAAAAACGTTTGTTCCATCT	105	60.1	RT-qPCR
FsCYP90D1	R：CCAATAAATTCTTGAAATTGTTTCTTTA	105	60.3	RT-qPCR
TUB	F：GGATGGACGAGATGGAGTTTAC	93	57.2	RT-qPCR
TUB	R：CCTCATCTGCTGTTGCTTCT	93	58.0	RT-qPCR

基因Gene	引物序列（5′-3′） Primer sequence	扩增子长度/bp Amplicon length	退火温度/℃ Annealing temperature	备注 Remark
PDSBOS	F：aaggttaccgaattctctagaATGGAAAGATGATGATGGAGACTGG	309	60.5	PCR
PDSBOS	R：cgtgagctcggtaccggatccACATACGACTGTCCACCAATTATTG	309	64.5	PCR
FsPDSB	F：CTCCGGTCTTGGATAGTCAATG	100	55.3	RT-qPCR
FsPDSB	R：AGTCCAAACAAGCCACTAAAG	100	55.4	RT-qPCR
EF1αH	F：TGGAAATTTGAGACCACCAA	/	51.2	RT-qPCR
EF1αH	R：GCACAATCAGCCTGTGAAGT	/	55.7	RT-qPCR
FsCYP90D1B	F：aaggttaccgaattctctagaATGCAAAAACGTTTGTTCCATCT	328	60.1	PCR
FsCYP90D1B	R：cgtgagctcggtaccggatccCCAATAAATTCTTGAAATTGTTTCTTTA	328	60.3	PCR
FsCYP90D1	F：ATGCAAAAACGTTTGTTCCATCT	105	60.1	RT-qPCR
FsCYP90D1	R：CCAATAAATTCTTGAAATTGTTTCTTTA	105	60.3	RT-qPCR
TUB	F：GGATGGACGAGATGGAGTTTAC	93	57.2	RT-qPCR
TUB	R：CCTCATCTGCTGTTGCTTCT	93	58.0	RT-qPCR

侵染材料 Infiltration material	OD₆₀₀	侵染方法 Infiltration mode	侵染材料 Infiltration material	OD₆₀₀	侵染方法 Infiltration mode
完整果实 Complete fruit	0.5	真空2 min Vacuum 2 min	损伤果实 Damaged fruit	0.5	真空2 min Vacuum 2 min
		真空5 min Vacuum 5 min			真空5 min Vacuum 5 min
		真空10 min Vacuum 10 min			真空10 min Vacuum 10 min
		共培养12 h Co-culture for 12 h			共培养12 h Co-culture for 12 h
		共培养24 h Co-culture for 24 h			共培养24 h Co-culture for 24 h
	1.0	真空2 min Vacuum 2 min		1.0	真空2 min Vacuum 2 min
		真空5 min Vacuum 5 min			真空5 min Vacuum 5 min
		真空10 min Vacuum 10 min			真空10 min Vacuum 10 min
		共培养12 h Co-culture for 12 h			共培养12 h Co-culture for 12 h
		共培养24 h Co-culture for 24 h			共培养24 h Co-culture for 24 h
	1.5	真空2 min Vacuum 2 min		1.5	真空2 min Vacuum 2 min
		真空5 min Vacuum 5 min			真空5 min Vacuum 5 min
		真空10 min Vacuum 10 min			真空10 min Vacuum 10 min
		共培养12 h Co-culture for 12 h			共培养12 h Co-culture for 12 h
		共培养24 h Co-culture for 24 h			共培养24 h Co-culture for 24 h

侵染材料 Infiltration material	OD₆₀₀	侵染方法 Infiltration mode	侵染材料 Infiltration material	OD₆₀₀	侵染方法 Infiltration mode
完整果实 Complete fruit	0.5	真空2 min Vacuum 2 min	损伤果实 Damaged fruit	0.5	真空2 min Vacuum 2 min
		真空5 min Vacuum 5 min			真空5 min Vacuum 5 min
		真空10 min Vacuum 10 min			真空10 min Vacuum 10 min
		共培养12 h Co-culture for 12 h			共培养12 h Co-culture for 12 h
		共培养24 h Co-culture for 24 h			共培养24 h Co-culture for 24 h
	1.0	真空2 min Vacuum 2 min		1.0	真空2 min Vacuum 2 min
		真空5 min Vacuum 5 min			真空5 min Vacuum 5 min
		真空10 min Vacuum 10 min			真空10 min Vacuum 10 min
		共培养12 h Co-culture for 12 h			共培养12 h Co-culture for 12 h
		共培养24 h Co-culture for 24 h			共培养24 h Co-culture for 24 h
	1.5	真空2 min Vacuum 2 min		1.5	真空2 min Vacuum 2 min
		真空5 min Vacuum 5 min			真空5 min Vacuum 5 min
		真空10 min Vacuum 10 min			真空10 min Vacuum 10 min
		共培养12 h Co-culture for 12 h			共培养12 h Co-culture for 12 h
		共培养24 h Co-culture for 24 h			共培养24 h Co-culture for 24 h

AS浓度/（μmol · L^-1） AS concentration	OD₆₀₀	侵染组 Infestation group	有效侵染率/% Infection efficiency
/	/	对照Control	0
200	0.5	TRV	0
	0.5	TRV-FsPDS	42.31
	1	TRV	7.69
	1	TRV-FsPDS	48.00
	1.5	TRV	3.70
	1.5	TRV-FsPDS	47.83
400	0.5	TRV	8.33
	0.5	TRV-FsPDS	55.00
	1	TRV	12.50
	1	TRV-FsPDS	60.87
	1.5	TRV	7.69
	1.5	TRV-FsPDS	50.00
600	0.5	TRV	12.00
	0.5	TRV-FsPDS	50.00
	1	TRV	11.54
	1	TRV-FsPDS	42.11
	1.5	TRV	7.69
	1.5	TRV-FsPDS	37.50