紫花地丁VIGS体系的建立与VpPAL基因功能验证

doi:10.13560/j.cnki.biotech.bull.1985.2026-0211

摘要/Abstract

摘要：

目的构建紫花地丁（Viola philippica）VIGS体系并进行紫花地丁基因功能验证。方法以烟草脆裂病毒（TRV）为病毒载体，紫花地丁八氢番茄红素去饱和酶基因（phytoene desaturase, VpPDS）为报告基因，通过瞬时转化法侵染紫花地丁叶片，采用表型观察和RT-qPCR分析基因沉默的效果。通过测试不同靶基因片段和菌液浓度对该系统进行优化。结果 VpPDS基因沉默植株表现出光漂白表型，同时VpPDS基因相对表达量显著降低。菌液浓度增加可增强基因沉默效果，表达量最高降低89.0%，沉默株率达70.6%。通过沉默苯丙氨酸解氨酶（VpPAL）基因发现，冷胁迫后VpPAL沉默植株表现出叶片萎蔫及生长受阻现象，同时伴随活性氧（ROS）积累升高、丙二醛（MDA）含量增加、相对电导率（REC）上升、光合能力下降及黄酮类化合物积累减少。结论在紫花地丁中成功建立了稳定的VIGS系统，且通过该体系验证沉默VpPAL基因会降低紫花地丁的耐寒性。

关键词: 紫花地丁, 病毒诱导的基因沉默, 八氢番茄红素去饱和酶, 苯丙氨酸解氨酶, 烟草脆裂病毒

Abstract:

Objective This study aimed to establish a virus-induced gene silencing (VIGS) system in Viola philippica and perform gene function validation. Method This study is based on the tobacco rattle virus (TRV) vector and the phytoene desaturase gene (VpPDS) as a reporter. Leaves were infiltrated by transient transformation method, and silencing efficiency was assessed through phenotypic observation and RT-qPCR. The system was further optimized by testing different target gene fragments and bacterial culture concentrations. Result Photobleaching phenotypes were observed in VpPDS-silenced plants, accompanied by significant reductions in VpPDS gene expression. Increasing bacterial concentration enhanced silencing, with expression reduced by up to 89.0% and silence strain rate reaching 70.6%. The phenylalanine ammonia-lyase gene (VpPAL) was successfully silenced using this system. After cold stress,VpPAL-silenced plants exhibited leaf wilting and impaired growth. They also showed elevated reactive oxygen species (ROS) accumulation, higher malondialdehyde (MDA) content and relative electrical conductivity (REC), reduced photosynthetic capacity, and decreased flavonoid accumulation. Conclusion A stable VIGS system was successfully established in V. philippica, and silencing of the VpPAL gene via this system was verified to reduce cold tolerance in V. philippica.

Key words: Viola philippica, virus-induced gene silencing, phytoene desaturase (PDS), phenylalanine ammonia-lyase gene (PAL), tobacco rattle virus (TRV)

何龙娇, 张燕青, 张楠楠, 靳亚鑫, 宋芸, 乔永刚. 紫花地丁VIGS体系的建立与VpPAL基因功能验证[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0211.

HE Long-jiao, ZHANG Yan-qing, ZHANG Nan-nan, JIN Ya-xin, SONG Yun, QIAO Yong-gang. Establishment of a VIGS System and Functional Characterization of the VpPAL Gene in Viola philippica[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0211.

图/表 6

表1 紫花地丁VIGS沉默株率统计

Table 1 V. philippica VIGS silence strain rate data

处理组 Treatment group	OD₆₀₀	植株数 Total plants	存活株数 Number surviving	白化株数 Number of albino	死亡率 Death rate （%）	沉默株率 Silence strain rate （%）
CK	/	30	30	0	0	0.0
TRV	1	30	30	0	0	0.0
	2	30	29	0	3.3	0.0
	3	30	25	0	16.7	0.0
TRV2-VpPDS₁	1	42	42	14	0	33.3
	2	35	34	18	2.8	51.4
	3	31	28	21	9.6	67.7
TRV2-VpPDS₂	1	36	36	13	0	36.1
	2	30	29	16	3.3	53.3
	3	34	30	24	11.7	70.6
TRV2-VpPDS₃	1	32	32	8	0	25.0
	2	30	28	11	6.7	36.7
	3	35	31	15	11.4	42.8

图1 VpPDS扩增及阳性菌落鉴定A：扩增VpPDS基因CDS区；B：pTRV2-VpPDS1 （228 bp）与pTRV2-VpPDS3 （202 bp）阳性菌落鉴定；C：pTRV2-VpPDS2 （410 bp）阳性菌落鉴定；Marker均为2000A: Amplification of the VpPDS coding sequence (CDS). B: Identification of positive colony for pTRV2-VpPDS1 (228 bp)与pTRV2-VpPDS3 (202 bp). C: Identification of positive colony for pTRV2-VpPDS2 (410 bp). Marker are 2000

Fig. 1 VpPDS amplification and identification of positive colony

图2 紫花地丁VpPDS基因沉默植株的表型及基因相对表达分析A：对照组（CK）、空载体组（TRV）及VpPDS基因沉默组植株在侵染后20 d的表型；B：不同处理组中VpPDS基因的相对表达量分析。数据以3次生物学重复的平均值±标准差表示，不同字母表示组间存在显著差异（P<0.05）

Fig. 2 Phenotypes and gene relative expression analysis of VpPDS-silenced V. philippica plantsA: Phenotypes of control CK, TRV and VpPDS-silenced plants at 20 d post-infiltration; B: Relative expression analysis of the VpPDS gene in different treatment groups. Data are presented as mean ± SD from three biological replicates, different letters indicate statistically significant differences among the groups (P< 0.05)

图3 VpPAL基因的克隆及阳性菌落鉴定A：VpPAL基因全长编码序列（CDS）扩增产物的凝胶电泳图；B：pTRV2-VpPAL1阳性菌落鉴定。M：DNA Marker 2000

Fig. 3 Cloning of the VpPAL gene and identification of positive colonyA: Gel electrophoresis showing the amplified full-length coding sequence (CDS) of the VpPAL gene; B: Identification of positive colony for pTRV2-VpPAL1. M: DNA Marker 2000

图4 低温胁迫下VpPAL基因沉默植株的表型与相对表达量分析A：VpPAL基因相对表达水平；B：低温胁迫下VpPAL基因沉默植株的表型；C：冷处理前后VpPAL基因的表达水平。不同字母表示组间存在显著差异（P<0.05），**P<0.01

Fig. 4 Phenotype and relative expression analysis of the VpPAL-silenced plants under cold stressA: Relative expressions of VpPAL gene. B: Phenotype of the VpPAL-silenced plants under cold stress. C: Relative expression level of VpPAL gene before and after cold treatment. Different letters indicate statistically significant differences among the groups (P< 0.05), **P<0.01

图5 低温胁迫下VpPAL沉默植株活性氧染色及生理指标检测A：NBT染色；B：DAB染色；C：MDA含量；D：相对电导率；E：Fv/Fm比值；F：类黄酮含量。*P<0.05， **P<0.01

Fig. 5 Detection of reactive oxygen species staining and physiological indexes in VpPAL silencing plants under low temperature stressA: NBT staining. B: DAB staining. C: MDA content. D: Relative conductivity. E: Fv/Fm ratio. F: Flavonoids content. *P<0.05,**P<0.01

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