西花蓟马不同RNA干扰技术比较研究

李恒; 周知恩; 陈勇; 陆承聪; 张祥琴; 王谅; 陈艺欣; 田厚军; 林硕; 张洁; 游泳; 魏辉

doi:10.19303/j.issn.1008-0384.2019.10.010

西花蓟马不同RNA干扰技术比较研究

doi: 10.19303/j.issn.1008-0384.2019.10.010

李恒^{1, 2,},
周知恩¹,
陈勇²,
陆承聪²,
张祥琴^{1, 2},
王谅^{1, 2},
陈艺欣²,
田厚军²,
林硕²,
张洁³,
游泳²,
魏辉^{1, 2, ,}

1.
福建农林大学闽台作物有害生物生态防控国家重点实验室，福建　福州　350002
2.
福建省农业科学院植物保护研究所/福建省作物有害生物监测与治理重点实验室/农业农村部福州作物有害生物科学观测试验站，福建　福州　350003
3.
云南省农业科学院生物技术与种质资源研究所/云南省农业生物技术重点实验室/农业农村部西南作物基因资源与种质创制重点实验室，云南　昆明　650223

基金项目: 国家重点研发计划项目（2018YFD0201102、2018YFD0201209）；国家自然科学基金项目（31871936、31560499）；福建省科技重大专项（2017NZ0003-1-4）；福建省科技计划公益类专项（2017R1025-9、2018R1025-7、2019R1024-2、2019R1024-3）；福建省农业科学院科技项目（STIT2017-3-2、AGY2018-5、AC2017-6、YC2019003）；云南省应用基础研究计划面上项目（2016FB063）；云南省应用基础研究计划重点项目（2018FA020）；云南省中青年学术技术带头人后备人才项目（2015HB081）

详细信息

作者简介:
李恒（1993−），女，硕士研究生，研究方向：昆虫生态与害虫综合治理（E-mail：592995627@qq.com）

通讯作者:
魏辉（1972−），男，博士，研究员，研究方向：昆虫化学生态学（E-mail：weihui@faas.cn）

中图分类号: S 433.89
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出版历程
- 收稿日期: 2019-08-28
- 修回日期: 2019-09-29
- 刊出日期: 2019-10-01

Artificial RNA Interference on Frankliniella occidentalis

LI Heng^{1, 2
,},
ZHOU Zhi-en¹,
CHEN Yong²,
LU Cheng-cong²,
ZHANG Xiang-qin^{1, 2},
WANG Liang^{1, 2},
CHEN Yi-xin²,
TIAN Hou-jun²,
LIN Shuo²,
ZHANG Jie³,
YOU Yong²,
WEI Hui^{1, 2
, ,}

1.
State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Fujian Agriculture and Forestry University, Fuzhou, Fujian　350002, China
2.
Institute of Plant Protection, Fujian Academy of Agricultural Sciences/Fujian Key Laboratory for Monitoring and Integrated Management of Crop Pests/Fuzhou Scientific Observing and Experimental Station of Crop Pests, Ministry of Agriculture and Rural Affairs, Fuzhou, Fujian　350003, China
3.
Biotechnology and Germplasm Resource Institute, Yunnan Academy of Agricultural Sciences/Yunnan Provincial Key Laboratory of Agricultural Biotechnology/Key Laboratory for Gene Resources and Germplasm Innovation of Southwest Crops, Ministry of Agriculture and Rural Affairs, Kunming, Yunnan　650223, China

摘要

摘要: 目的西花蓟马Frankliniella occidentalis (Pergande)是一种重要的外来入侵害虫，其繁殖能力强、寄主范围广、抗药性强，给我国蔬菜、花卉等经济作物造成了严重危害。本研究旨在评估膜饲喂和显微注射dsActin对西花蓟马Actin基因的沉默效率，以期为蓟马类等小型昆虫基因功能的研究提供方法和依据。方法通过膜饲喂和显微注射的方法将体外合成的dsActin导入西花蓟马二龄若虫体内，用RT-qPCR方法检测Actin基因的mRNA表达；通过单头饲养方法观察统计西花蓟马成虫体长、成虫翅膀或胸腹部畸形率及死亡率。结果 dsActin膜饲喂后24 、48、72 h，Actin基因的相对表达量分别为对照组的97%、91%和98%；通过显微注射将dsActin注入西花蓟马体腔，在注射后24、48、72 h，Actin基因的表达量分别为对照组的68%、56%和53%。注射dsActin的西花蓟马在第24～120 h 死亡率为44%～98%，显著高于dsGFP对照组；此外，dsActin组个体体长仅为dsGFP对照组的90%，且翅膀或胸腹部出现畸形率为41%。结论显微注射dsActin能显著沉默西花蓟马Actin基因mRNA水平的表达，并引起西花蓟马个体不正常发育和死亡，从而建立蓟马类小型昆虫RNAi体系。
- 西花蓟马 /
- RNA干扰 /
- 膜饲喂法 /
- 显微注射法
Abstract: Objective Application of dsActin to control the proliferating, multiple-hosting, insecticide-resistant invasive western flower thrips on vegetables and ornamental crops in China was investigated. Method Synthetic dsActin or dsGFP (as control) was introduced into the 2^nd-instar nymphs of Frankliniella occidentalis (Pergande) by either membrane-feeding or microinjection. The treated thrips were collected for mRNA expression detection by RT-qPCR at 24, 48 and 72 h after the treatment. Effects of 0.5 μg·μL⁻¹ dsRNA on the body length, wing or thorax malformation, and mortality of the insects were monitored in an individual culture experiment conducted in the laboratory. Result The relative expression of Actin mRNA in the thrips was similar to that of control 24, 48 and 72 h after membrane-feeding. But the expressions in the injection group decreased to 68% in 24 h, 56% in 48 h, and 53% in 72 h. Significantly higher mortality rate (44%-98% in 24-120 h), shorter body length (90% of control), and higher bodily deformation rate (41%) were observed in the dsRNA-treated thrips than control. Conclusion The Actin mRNA expression in F. occidentalis was significantly suppressed by dsActin injection at the test level to induce malformations or death in the insects. It suggested that an in vivo RNA interference method was made available for studying the gene functions and control of thrips.
- Frankliniella occidentalis /
- RNA interference /
- membrane-feeding /
- microinjection

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图 1 西花蓟马Actin基因的RT-PCR产物

Figure 1. RT-PCR products of Actin gene of F. occidentalis

下载: 全尺寸图片幻灯片

图 2 西花蓟马Actin基因核酸序列和预测的氨基酸序列

Figure 2. Nucleotide and predicted amino acid sequences of Actin genes of F. occidentalis

下载: 全尺寸图片幻灯片

图 3 dsRNA在饲喂过程中的稳定性

Figure 3. Stability of dsRNA during membrane-feeding of F. occidentalis

下载: 全尺寸图片幻灯片

图 4 dsRNA对西花蓟马Actin相对表达量的影响

注：不同字母表示差异显著（Tukey HSD test, P＜0.05）。

Figure 4. Repression of Actin by dsRNA treatment

Note：Different lowercase letters mean significant difference (Tukey HSD test, P＜0.05).

下载: 全尺寸图片幻灯片

图 5 dsRNA对西花蓟马死亡率及生长发育的影响

注：*或者不同的字母表示差异显著（Tukey HSD test, P＜0.05）

Figure 5. Effects of dsRNA on growth and mortality of F. occidentalis

Note：Asterisks or different lowercase letters mean significant difference (Tukey HSD test at P < 0.05).

下载: 全尺寸图片幻灯片

表 1 引物信息

Table 1. Primers applied

引物名称 Primer name	序列（5′-3′） Sequence (5′-3′)
Actin/F	ATGTGTGACGACGATGTTGC
Actin/R	TTAGAAGCACTTGCGGTGGACG
T7Actin/F	TAATACGACTCACTATAGGGTTCGTGGGCATGGAATC TTGCGGTAT
T7Actin/R	TAATACGACTCACTATAGGGCGGACTCGTCGTACTCG TCCTTGGAGA
T7GFP/F	TAATACGACTCACTATAGGGCGAGGAGCTGTTCACC GG
T7GFP/R	TAATACGACTCACTATAGGGTCCTCGATGTTGTGGCGG
q18S/F	TTTTATGGTGGTGTTGTTGTGG
q18S/R	CAAGGGCTTTGGGTAATGG
qActin /F	TGGTCGGTATGGGACAGAAGGA
qActin/R	TCGGTGAGCAGGACAGGGTG

下载: 导出CSV

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