Establishing Tobacco Rattle Virus-mediated Gene Silencing System for Primula forbesii

doi:10.13560/j.cnki.biotech.bull.1985.2021-0924

Abstract

Abstract:

Primula forbesii is a biennial herbaceous flower,which has high ornamental value as a landscape plant. It is also an ideal material to study heterostyly. In this study,a rapid and efficient verification technology of gene function is established for P. forbesii laying a foundation for studying the function of genes in P. forbesii. The optimal infection object,infection solution formula,bacterial solution concentration and infection mode of tobacco rattle virus（TRV）vector in P. forbesii were explored,and the VIGS system suitable for P. forbesii was established. The results showed that the OD₆₀₀ value of bacterial solution containing TRV1 and PfPDS-TRV2 was adjusted to 1.0 in the infection solution of 200 μmol/L AS,10 mmol/L MgCl₂ and 10 mmol/L MES. After mixing,P. forbesii was infected by abaxial leaf injection. The treated plant leaves were used in PCR with primers on TRV virus vector,and the virus vectors of TRV1 and TRV2 were detected in the plants with phenotypic changes and the no-load group. The expression of PfPDS in albino plants was significantly lower than that in the no-load group and the control group. The infection efficiency of the established VIGS system was 60%,the silencing phenotype lasted for 12 months,and could play a silencing role from leaf to sepal. Due to the long duration of silencing effect,all genes can be verified by this method.

Key words: Primula forbesii, tobacco rattle virus, gene silencing

FU Si-tong, SI Wei-jia, LIU Ying, CHENG Tang-ren, WANG Jia, ZHANG Qi-xiang, PAN Hui-tang. Establishing Tobacco Rattle Virus-mediated Gene Silencing System for Primula forbesii[J]. Biotechnology Bulletin, 2022, 38(4): 295-302.

Figures/Tables 11

References 23

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处理编号 No.	菌液浓度 OD₆₀₀	侵染液配方 Formula of infiltration liquid
1	0.5	200 μmol/L AS+10 mmol/L MgCl₂+10 mmol/L MES
2	0.5	400 μmol/L AS+10 mmol/L MgCl₂+10 mmol/L MES
3	0.5	200 μmol/L AS+10 mmol/L MgCl₂+10 mmol/L MES+400 cysteine+5 mL/L Tween 20
4	0.5	400 μmol/L AS+10 mmol/L MgCl₂+10 mmol/L MES+400 cysteine+5 mL/L Tween 20
5	1.0	200 μmol/L AS+10 mmol/L MgCl₂+10 mmol/L MES
6	1.0	400 μmol/L AS+10 mmol/L MgCl₂+10 mmol/L MES
7	1.0	200 μmol/L AS+10 mmol/L MgCl₂+10 mmol/L MES+400 cysteine+5ml/L Tween 20
8	1.0	400 μmol/L AS+10 mmol/L MgCl₂+10 mmol/L MES+400 cysteine+5ml/L Tween 20
9	1.5	200 μmol/L AS+10 mmol/L MgCl₂+10 mmol/L MES
10	1.5	400 μmol/L AS+10 mmol/L MgCl₂+10 mmol/L MES
11	1.5	200 μmol/L AS+10 mmol/L MgCl₂+10 mmol/L MES+400 cysteine+5 mL/L Tween 20
12	1.5	400 μmol/L AS+10 mmol/L MgCl₂+10 mmol/L MES+400 cysteine+5 mL/L Tween 20
13	2.0	200 μmol/L AS+10 mmol/L MgCl₂+10 mmol/L MES
14	2.0	400 μmol/L AS+10 mmol/L MgCl₂+10 mmol/L MES
15	2.0	200 μmol/L AS+10 mmol/L MgCl₂+10 mmol/L MES+400 cysteine+5 mL/L Tween 20
16	2.0	400 μmol/L AS+10 mmol/L MgCl₂+10 mmol/L MES+400 cysteine+5 mL/L Tween 20

处理编号 No.	菌液浓度 OD₆₀₀	侵染液配方 Formula of infiltration liquid
1	0.5	200 μmol/L AS+10 mmol/L MgCl₂+10 mmol/L MES
2	0.5	400 μmol/L AS+10 mmol/L MgCl₂+10 mmol/L MES
3	0.5	200 μmol/L AS+10 mmol/L MgCl₂+10 mmol/L MES+400 cysteine+5 mL/L Tween 20
4	0.5	400 μmol/L AS+10 mmol/L MgCl₂+10 mmol/L MES+400 cysteine+5 mL/L Tween 20
5	1.0	200 μmol/L AS+10 mmol/L MgCl₂+10 mmol/L MES
6	1.0	400 μmol/L AS+10 mmol/L MgCl₂+10 mmol/L MES
7	1.0	200 μmol/L AS+10 mmol/L MgCl₂+10 mmol/L MES+400 cysteine+5ml/L Tween 20
8	1.0	400 μmol/L AS+10 mmol/L MgCl₂+10 mmol/L MES+400 cysteine+5ml/L Tween 20
9	1.5	200 μmol/L AS+10 mmol/L MgCl₂+10 mmol/L MES
10	1.5	400 μmol/L AS+10 mmol/L MgCl₂+10 mmol/L MES
11	1.5	200 μmol/L AS+10 mmol/L MgCl₂+10 mmol/L MES+400 cysteine+5 mL/L Tween 20
12	1.5	400 μmol/L AS+10 mmol/L MgCl₂+10 mmol/L MES+400 cysteine+5 mL/L Tween 20
13	2.0	200 μmol/L AS+10 mmol/L MgCl₂+10 mmol/L MES
14	2.0	400 μmol/L AS+10 mmol/L MgCl₂+10 mmol/L MES
15	2.0	200 μmol/L AS+10 mmol/L MgCl₂+10 mmol/L MES+400 cysteine+5 mL/L Tween 20
16	2.0	400 μmol/L AS+10 mmol/L MgCl₂+10 mmol/L MES+400 cysteine+5 mL/L Tween 20

引物名称 Primer name	序列 Sequence（5'-3'）	用途 Purpose
TRV1-F	TTACAGGTTATTTGGGCTAG	病毒载体检测 Virus vector detection
TRV1-R	CCGGGTTCAATTCCTTATC	病毒载体检测 Virus vector detection
TRV2-F	TGTTTGAGGGAAAAGTAG- AGAACGT	病毒载体检测 Virus vector detection
TRV2-R	TTACCGATCAATCAAGAT- CAGTCGA	病毒载体检测 Virus vector detection
PfPDS F	CTGCACATTTGGGTAAG	qRT-PCR
PfPDS-R	TGTCTTAGGTGGAAAGG	qRT-PCR

引物名称 Primer name	序列 Sequence（5'-3'）	用途 Purpose
TRV1-F	TTACAGGTTATTTGGGCTAG	病毒载体检测 Virus vector detection
TRV1-R	CCGGGTTCAATTCCTTATC	病毒载体检测 Virus vector detection
TRV2-F	TGTTTGAGGGAAAAGTAG- AGAACGT	病毒载体检测 Virus vector detection
TRV2-R	TTACCGATCAATCAAGAT- CAGTCGA	病毒载体检测 Virus vector detection
PfPDS F	CTGCACATTTGGGTAAG	qRT-PCR
PfPDS-R	TGTCTTAGGTGGAAAGG	qRT-PCR

处理时期 Time of treatment	处理条件Condition of treatment		侵染效率 Infection efficiency/%
处理时期 Time of treatment	压强 Pressure/kpa	侵染时间 Infection time	侵染效率 Infection efficiency/%
刚伸出胚轴的种子	-25	30 s	5
刚伸出胚轴的种子	-25	2 min	10
刚伸出胚轴的种子	-25	5 min	10
刚伸出胚轴的种子	-50	30 s	0
刚伸出胚轴的种子	-50	2 min	0
刚伸出胚轴的种子	-50	5 min	10
刚伸出胚轴的种子	-75	30 s	0
刚伸出胚轴的种子	-75	2 min	5
刚伸出胚轴的种子	-75	5 min	5
两片子叶的幼苗	-25	30 s	30
两片子叶的幼苗	-25	2 min	40
两片子叶的幼苗	-25	5 min	40
两片子叶的幼苗	-50	30 s	25
两片子叶的幼苗	-50	2 min	5
两片子叶的幼苗	-50	5 min	5
两片子叶的幼苗	-75	30 s	55
两片子叶的幼苗	-75	2 min	40
两片子叶的幼苗	-75	5 min	45