基于三角梅的病毒诱导基因沉默体系的建立与优化

doi:10.13560/j.cnki.biotech.bull.1985.2022-1266

摘要/Abstract

摘要：

三角梅是紫茉莉科灌木，具有较高的观赏价值，是研究花色叶色调控的理想材料。建立快速高效的基因验证体系，是三角梅基因功能研究的关键。以三角梅‘金心双色’品种为材料，选用八氢番茄红素脱氢酶基因（PDS）为指示基因，探索不同接种部位和基因片段长度对烟草脆裂病毒（TRV）诱导三角梅叶片内源PDS mRNA沉默效果的影响，建立适用于三角梅的病毒诱导基因沉默体系（VIGS）。结果表明，摩擦注射嫩叶法相比顶端嫩茎沉默效果更明显；基因沉默片段长度在336 bp和457 bp均可诱导PDS基因沉默，后者效果更加明显。初步构建了三角梅VIGS体系，为后续基因功能研究提供了技术参考。

关键词: 三角梅, 八氢番茄红素脱氢酶, 烟草脆裂病毒, 基因沉默

Abstract:

Bougainvillea peruviana ‘Thimma’, belonging to the family Nyctaginaceae, has high ornamental values in landscape. It is an ideal material to study the regulation of flower and leaf coloration. It is vital to establish a rapid and efficient gene verification system in B. peruviana. In this study, phytoene desaturase gene（PDS）was used as an indicator gene, we explored the effects of different inoculation sites and fragment length on the silencing effects of endogenous PDS mRNA in B. peruviana leaves induced by tobacco rattle virus（TRV）and established an optimized virus-induced gene silencing system（VIGS）for B. peruviana. The results showed that the silencing effect of leaf friction injection was more significant than that of tender stems friction injection. Additionally, PDS gene silencing was induced by both the inserted fragment of 336 bp and 457 bp, and the latter one was more significant. The VIGS system of B. peruviana is preliminarily constructed, which can be applied to the following gene function research.

Key words: Bougainvillea peruviana ‘Thimma’, phytoene desaturase（PDS）, tobacco rattle virus（TRV）, virus-induced gene silencing（VIGS）

刘珍银, 段郅臻, 彭婷, 王童欣, 王健. 基于三角梅的病毒诱导基因沉默体系的建立与优化[J]. 生物技术通报, 2023, 39(7): 123-130.

LIU Zhen-yin, DUAN Zhi-zhen, PENG Ting, WANG Tong-xin, WANG Jian. Establishment and Optimization of Virus-induced Gene Silencing System in Bougainvillea peruviana ‘Thimma’[J]. Biotechnology Bulletin, 2023, 39(7): 123-130.

图/表 8

表1 本文中使用的引物序列

Table 1 Primers used in this study

引物 Primer	引物序列 Primer sequence（5'-3'）	长度 Length/bp
BpPDS336-EcoRI-F	TAAGGTTACCGAATTCTGCATTTTGATTGCTTTG	368
BpPDS336-EcoRI-R	GCCTTCTAGAGAATTCAATGACTGGAACCCCTACTA	368
BpPDS457-EcoRI-F	TAAGGTTACCGAATTCGGTTTGAGTGTCCAAGAGTG	489
BpPDS457-EcoRI-R	GCCTTCTAGAGAATTCTAATGACTGGAACCCCTACT	489
pTRV1-F	CCTGGTGCTCAGTACATAACTTTCC	257
pTRV1-R	TCTTTGCTTACATCGTCCTCTTTCA	257
pTRV2-F	ACGCTGTTTGAGGGAAAAGTAG	321
pTRV2-R	CCGATCAATCAAGATCAGTCGAGA	321
BpActin-RT-F	TATGAACTTCCTGATGGG	125
BpActin-RT-R	GAATTGTAGGTTGTCTCG	125
BpPDS-RT-F	ATTCGCCTCGTCCTGATA	103
BpPDS-RT-R	ATGGGTTTGTGACCTGCA	103

图1 三角梅叶片总RNA

Fig. 1 Total RNA of B. peruviana ‘Thimma’

图2 目的基因片段扩增 M: DL2000 DNA marker，下同

Fig. 2 Amplification of target genes M: DL2000 DNA marker, the same below

图3 目的基因TRV2载体扩增检测

Fig. 3 Detection of target genes on constructed TRV2 vector

图4 沉默三角梅BpPDS叶片表型 A：空白对照；B：阴性对照；C, D：pTRV2-BpPDS336沉默处理；E, F：pTRV2-BpPDS457沉默处理。圆圈部位代表侵染部位

Fig. 4 Phenotype of BpPDS silenced B. peruviana ‘Thimma’ A: Control check; B: negative control; C, D: pTRV2-BpPDS336 silencing treatment; E, F: pTRV2-BpPDS457 silencing treatment. The circle indicates infection treatment part

图5 pTRV1和pTRV2病毒载体PCR检测 pTRV1引物检测：1、3、5、7、9、11、13、15、17；pTRV2引物检测：2、4、6、8、10、12、14、16、18

Fig. 5 PCR amplification of virus veetor pTRV1 and pTRV2 pTRV1 detection: 1, 3, 5, 7, 9, 11, 13, 15 and 17; pTRV2 detection: 2, 4, 6, 8, 10, 12, 14, 16 and 18

表2 不同处理三角梅叶片光合色素含量

Table 2 Content of photosynthetic pigments in the leaves of B. peruviana ‘Thimma’ under different treatments

处理 Treatment	叶绿素a含量 Chlorophyll a content/（mg·g^-1）	叶绿素b含量 Chlorophyll b content/（mg·g^-1）	叶绿素含量 Chlorophyll content/（mg·g^-1）	类胡萝卜素含量 Carotenoid content/（mg·g^-1）
空白对照	0.327 ± 0.021 a	0.214 ± 0.030 a	0.541 ± 0.052 a	0.114 ± 0.004 a
阴性对照	0.300 ± 0.007 a	0.153 ± 0.010 b	0.470 ± 0.025 a	0.114 ± 0.010 a
pTRV2-BpPDS336	0.230 ± 0.023 b	0.090 ± 0.010 c	0.310 ± 0.060 b	0.087 ± 0.003 b
pTRV2-BpPDS457	0.190 ± 0.016 c	0.053 ± 0.010 d	0.260 ± 0.035 b	0.086 ± 0.020 b

图6 RT-qPCR检测BpPDS基因相对表达量 *表示在（P<0.05）水平上有显著差异；ns代表无显著差异

Fig. 6 RT-qPCR detection of relative expression of BpPDS * indicates significant difference（P<0.05）, ns indicates no significance

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