TRV介导的小报春基因沉默技术体系的建立

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

摘要/Abstract

摘要：

小报春是报春花科报春花属的二年生草本花卉,具有较高的观赏价值和园林应用前景,是研究花柱二型的理想材料,建立快速高效的报春花属基因功能验证技术,是小报春基因功能研究中的关键问题。以小报春为材料,以八氢番茄红素脱氢酶（PDS）基因作为标记基因,探索TRV病毒载体在小报春中的最佳侵染对象、侵染液配方、菌液浓度和侵染方式,建立适用于小报春的VIGS体系。结果表明,用含有200 μmol/L乙酰丁香酮（AS）、10 mmol/L MgCl₂和10 mmol/L乙磺酸缓冲液（MES）的浸染液,将含有pTRV1和pTRV2-PfPDS的菌液OD₆₀₀值均调至1.0等体积混合后通过叶背注射方式侵染小报春,以TRV病毒载体上的引物对处理后的植株叶片进行PCR,在出现表型变化的植株和空载组中均检测到TRV1和TRV2的病毒载体,白化植株的PfPDS表达量显著低于空载组和对照组。建立的VIGS体系侵染效率达60%,沉默表型可持续12个月之久,并能在小报春植株的各部位（从叶片到萼片）均起到沉默作用。建立了小报春基因沉默体系,由于沉默效果持续时间长,所有基因都可以利用这一方法进行功能验证。

关键词: 小报春, 烟草脆裂病毒, 基因沉默

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

付偲僮, 司未佳, 刘颖, 程堂仁, 王佳, 张启翔, 潘会堂. TRV介导的小报春基因沉默技术体系的建立[J]. 生物技术通报, 2022, 38(4): 295-302.

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.

图/表 11

图1 TRV1和TRV2载体图谱

Fig.1 Vector map of TRV1 and TRV2

表1 侵染液菌液浓度及配方

Table 1 OD600 value and combinations of infiltration liquid

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

表2 病毒载体检测及表达量检测引物

Table 2 Primers for viral vector detection and gene expre-ssion level detection

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

图2 注射法侵染小报春后PDS基因沉默效果 A：对照;B：TRV2空载组;C：叶背注射PDS基因;D：叶柄注射PDS基因

Fig. 2 Silencing effect of PDS gene infection in P. forbesii A：CK. B：Empty TRV2 vector. C：Inject PDS gene at the back of leaf. D：Inject PDS gene at petiole

图3 PfPDS沉默后小报春的白化现象 A：PfPDS沉默后小报春整株白化现象;B：PfPDS沉默后小报春各部位白化现象

Fig.3 Whole plant albino after PfPDS gene silenced in P. forbessii A：Whole plant albinism after PfPDS gene silenced in P. forbessii. B：Albinism of all part after PfPDS gene silenced in P. forbessii vacuum infiltration

图4 真空渗透法侵染小报春后PDS基因的沉默效果 A：种子萌发长出下胚轴阶段;B：种子萌发长出子叶阶段;C：真空渗透侵染后叶片的表型变化

Fig. 4 Silencing effect of PDS gene in P. forbesii infected by vacuum infiltration method A：Stage of hypocotyl growth. B：Stage of cotyledon growth. C：Phenotypic changes of leaves infected by vacuum infiltration

表3 真空渗透法侵染小报春萌发种子和带子叶幼苗的效果

Table 3 Infection efficiency on P. forbesii when seeds with hypocotyl and cotyledons infected by vacuum infiltration

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

表4 不同OD600与侵染液配方侵染小报春的效率

Table 4 Infection efficiency of different OD600 values and different formula of infiltration liquid on P. forbesii

处理编号No.	侵染效率Infection efficiency/%	处理编号No.	侵染效率Infection efficiency/%
1	40	9	0
2	0	10	20
3	0	11	0
4	0	12	40
5	20	13	0
6	0	14	0
7	20	15	0
8	0	16	0

图5 基因沉默后小报春植株病毒载体检测结果 1-3：出现白化现象的小报春的不同植株

Fig.5 Detection results of virus vector in P. forbessii after gene silencing 1-3：Different albino plants

图6 使用不同方法基因沉默后小报春PDS基因的表达量检测 TRV2-PfPDS-叶片：叶背注射法得到的白化植株;TRV2-PfPDS-叶柄：叶柄注射法得到的白化植株;TRV2-PfPDS-真空：真空渗透法得到的白化植株

Fig.6 Detection results of PDS gene expression after gene silencing by different methods TRV2-PfPDS-leaf：Albino plant obtained by injection at the back of leaf. TRV2-PfPDS-petiole：Albino plant obtained by injection at petiole. TRV2-PfPDS-vacuum：Albino plant obtained by vacuum infiltration

图7 基因沉默后小报春植株不同部位PDS基因的表达量检测

Fig.7 Detection of PDS gene expression in different parts of P. forbessii after gene silencing

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