TRV介导的溪荪VIGS转化体系的构建与鉴定

doi:10.7525/j.issn.1673-5102.2024.01.015

摘要/Abstract

摘要：

病毒诱导基因沉默（Virus-induced gene silencing，VIGS）技术为缺乏稳定遗传转化体系的植物进行基因功能分析提供了可能，为解决缺少稳定遗传转化体系的植物进行基因功能验证的需求，以单子叶植物溪荪（Iris sanguinea）为材料，克隆IsPDS基因的特异性片段并构建其VIGS重组载体pTRV2-IsPDS，通过注射法侵染溪荪叶片，结果显示：pTRV1和pTRV2-IsPDS的菌液OD₆₀₀调至1.8~2.0后重悬，重悬液OD₆₀₀调至0.8~1.0，通过注射器叶脉注射方式侵染溪荪叶片效果最佳。在室外温度为15~20 ℃的下午6~8时进行，用1 mL注射器针头扎伤溪荪叶片外表皮，沿着平行脉缓慢注射重悬液1 mL至溪荪叶片维管束中，14 d左右会出现明显的白化表型。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IsPDS表达量显著低于对照组。侵染液配制中通过提高携带病毒载体的农杆菌的浓度提高侵染效率，且接种后无需遮光。

关键词: 溪荪, IsPDS基因, VIGS体系

Abstract:

To analyze gene function in plants lacking stable genetic transformation system， virus-induced gene silencing（VIGS） was needed， and Iris sanguinea， a monocotyledon， was selected as materials. The specific fragment of IsPDS gene was isolated and the VIGS recombinant vector pTRV2-IsPDS was constructed and leaves were infected by injection. The results showed that the most effective infection was achieved by injecting its leaf veins with syringes when the OD₆₀₀ values of the resuspension were adjusted to 0.8-1.0 after the those of pTRV1 and pTRV2-IsPDS were adjusted to 1.8-2.0. The experiment was conducted when outdoors temperature was 15-20 ℃ from 6 p.m. to 8 p.m.， and a 1 mL syringe needle pricking the outer epidermis of I. sanguinea leaves and 1 mL of heavy suspension slowly injected along parallel veins into its vascular bundles. A clear albino phenotype might appear after about 14 days. TRV1 and TRV2 virus vectors were detected in the plants with phenotypic changes and in the no-load group. The expression of IsPDS in the albino plants was significantly lower than that in the no-load group and the control group. With the concentration of agrobacterium tumefaciens carrying virus vector increased in the preparation of infection solution， the infection efficiency of the whole experiment was improved， and no shading was needed after inoculation.

Key words: Iris sanguinea, IsPDS gene, VIGS system

中图分类号:

Q949.71+8.28

刘桂伶, 徐诺, 史恭发, 王玲. TRV介导的溪荪VIGS转化体系的构建与鉴定[J]. 植物研究, 2024, 44(1): 132-138.

Guiling LIU, Nuo XU, Gongfa SHI, Ling WANG. Construction and Identification of TRV-mediated VIGS Transformation System of Iris sanguinea[J]. Bulletin of Botanical Research, 2024, 44(1): 132-138.

图/表 3

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