马铃薯甲虫RNA干扰高效致死基因的筛选

doi:10.6048/j.issn.1001-4330.2019.07.012

新疆农业科学 ›› 2019, Vol. 56 ›› Issue (7): 1283-1293.DOI: 10.6048/j.issn.1001-4330.2019.07.012

• 土壤肥料·贮藏加工·植物保护·畜牧水产 • 上一篇下一篇

马铃薯甲虫RNA干扰高效致死基因的筛选

米热古丽·胡尔西达¹, 付开赟^2,3, 徐晴玉⁴, 吐尔逊·艾合买提², 丁新华², 何江², 郭文超⁵

1.新疆农业大学农学院,乌鲁木齐 830052;
2.新疆农业科学院植物保护研究所/农业部西北荒漠绿洲作物有害生物综合治理重点实验室,乌鲁木齐 830091;
3. 新疆农业大学博士后流动站,新疆农业科学院博士后工作站,乌鲁木齐 830052;
4.南京农业大学植物保护学院,南京 210095;
5.新疆农业科学院微生物应用研究所,乌鲁木齐 830091

收稿日期:2019-01-29 出版日期:2019-07-20 发布日期:2019-09-05
通信作者: 郭文超(1966-),男,河北人,研究员,博士生导师,研究方向为农业入侵有害生物综合防控,(E-mail) gwc1966@163.com付开赟(1987-),男,浙江人,助理研究员,研究方向为农业入侵有害生物综合防控,(E-mail)fukaiyun87@163.co
作者简介:米热古丽·胡尔西达(1990-),女,新疆人,硕士研究生,研究方向为农业害虫综合防治,(E-mail)402237103@qq.com
基金资助:
国家重点研发计划“马铃薯化肥农药减施技术集成研究与示范(2018YFD0200800)”;新疆维吾尔自治区公益性科研院所基本科研业务费专项项目“基于马铃薯甲虫蛋白酶体的RNA干扰高效致死基因挖掘及初步应用(KYGY2016120)”;新疆维吾尔自治区优秀博士后;新疆维吾尔自治区“百名青年博士引进计划”

Screening of Highly Effective Lethal Genes Interfered with RNA in Leptinotarsa decemlineata and Study on the Evaluation Indexes of Its Lethal Factors

Mireguli Huerxida¹, FU Kai-yun^2,3, XU qing yu⁴, Tursun Ahmat², DING Xin-hua², HE Jiang², GUO Wen-chao⁵

1.College of Agronomy, Xinjiang Agricultural University, Urumqi 830052, China;
2. Key Laboratory of Intergraded Management of Harmful Crop Vermin in China North-Western Oasis, Ministry of Agriculture, P. R. China / Research Institute of Plant Protection, Xinjiang Academy of Agricultural Sciences, Urumqi 830091,China;
3. Post-doctoral Mobile Station of Xinjiang Agricultural University/ Post-doctoral Workstation of Xinjiang Academy of Agricultural Sciences Urumqi 830052, China;
4. College of Plant Protection, Nanjing Agricultural University,Nanjing 210095, China;
5. Research Institute of Applied Microbiology, Xinjiang Academy of Agricultural Sciences, Urumqi 830091, China

Received:2019-01-29 Online:2019-07-20 Published:2019-09-05
Correspondence author: GUO Wen-chao(1966-),male, born in Hebei, Professor, doctoral supervisor, Research direction:Integrated managementof Agricutural invasive pests , (E-mail)gwc1966@163.com
FU Kai-yun(1987-), male, born in Zhejiang, Professor associate, Research direction:Integrated management of Agricutural invasive pests, (E-mail) fukaiyun87@163.com
Supported by:
Supported by the National Key R & D Program of China "Integrated Research and Demonstration of Potato Fertilizer and Pesticide Reduction Technology" (2018YFD0200800); Basic Scientific R & D Program of Public Welfare Research Institutes of Xinjiang Uygur Autonomous Region of China "RNA Interference Efficient Lethal Gene Mining Based on Leptinotarsa decemlineata Proteasome and Its Preliminary Application" (KYGY2016120); Excellent PhD Funding Project of Xinjiang Uygur Autonomous Region "Introduction Scheme for 100 Young Doctors of Xinjiang Uygur Autonomous Region"

摘要/Abstract

摘要： 【目的】基于备选基因RNA干扰后的致死效果评价,筛选出高效致死基因。【方法】通过在赤拟谷盗中已的报导和相似性搜索获得马铃薯甲虫致死基因Ldpp1alpha-96a、LdRpt3、Ldalpha snap、LdSrp54k、LdRpn6、LdRop、LdGawky、Ldshi、Ldcact、Ldinr-a,以及阳性对照基因LdATPaseA和LdATPaseE,构建dsRNA表达载体,经饲喂马铃薯甲虫二龄幼虫不同浓度的dsRNA,分析对幼虫的20%取食剂量AD₂₀、化蛹中量PD₅₀和幼虫7 d致死中量LD₅₀,经分析和评估,筛选出高效致死基因。【结果】12个基因的dsRNA对马铃薯甲虫幼虫的 AD₂₀在1.18±0.10(μg / mL)~ 61.07±10.17(μg / mL);PD₅₀在0.06 ±0.01(μg / mL)~ 31.20 ±1.89(μg / mL);LD₅₀在1.39±0.13(μg / mL)~ 228.13±6.72(μg / mL)。其中 AD₂₀最高与最低的基因分别为Ldinr-a、LdRpn6 ;PD₅₀最高与最低的基因分别为Ldinr-a、LdGawky;LD₀最高与最低的基因分别为Ldinr-a、Ldsrp54k。【结论】12个基因的dsRNA对马铃薯甲虫幼虫均有一定的抑制取食、抑制化蛹及致死活性。其致死活性随浓度增高而增大,表现出剂量依赖效应,与对照组存在显著差异。通过综合比较AD₂₀和LD₅₀,Ldalpha snap、LdRpn6、LdSrp54k、LdRpt3个基因的毒力较ATPaseE和ATPaseA更强,具有较高的生物活性。提出并推荐AD₂₀和LD₅₀是评价备选基因dsRNA对幼虫致死效果的关键指标。

关键词: 马铃薯甲虫; RNA干扰; 致死基因; dsRNA; LD₅₀

Abstract: 【Objective】 To screen out highly effective lethal genes based on evaluation of lethal effect of alternative gene RNA interference. 【Method】 Ten dsRNA expression vectors of Ldpp1alpha-96a, LdRpt3, Ldalpha snap, LdSrp54k, LdRpn6, LdRop, LdGawky,Ldshi, Ldcact, Ldinr-aand 2 positive control genes LdATPaseA and LdATPaseE were constructed, dsRNA were fed to the second instar larvae of Colorado Potato beetle with gradient concentrations, which was used to analyze AD₂₀, 20% antifeedant dose, PD₅₀, medium pupation dose, and LD₅₀, medium lethal dose, to screen highly effective lethal genes and evaluate lethal efficiency. 【Result】 The results showed that: AD₂₀ of the above 12 genes for the larvae of potato beetle ranged from 1.18±0.10(μg / mL)-61.07±10.17(μg / mL); the PD₅₀ ranged from 0.06 ±0.01(μg / mL)-31.20 ±1.89(μg / mL); and the AD₂₀ ranged from 1.39±0.13(μg / mL)-228.13±6.72(μg / mL). The highest and lowest genes of AD₂₀ were Ldinr-a and LdRpn6, the highest and lowest genes of PD₅₀ were Ldinr-a and LdGawky, and the highest and lowest genes of LD₅₀ were Ldinr-a and Ldsrp54k, respectively.【Conclusion】All of the 12 alternative genes have an effect on inhibition of feeding, pupation and lethal activity. Their lethal activity increased with the increase of concentration, showing a dose-dependent effect, which was significantly different from the control group. By comparison of AD₂₀ and LD₅₀, 5 genes of Ldcact, Ldalpha-snap,LdRpn6,LdSrp54k and LdRpt3 are the most effective. It is suggested and recommended that AD₂₀ and LD₅₀ are the key indexes to evaluate the lethal effect of alternative genes dsRNA on larvae.

Key words: Colorado potato beetle; RNA interference; lethal genes; dsRNA; LD₅₀

中图分类号:

S435.32

米热古丽·胡尔西达, 付开赟, 徐晴玉, 吐尔逊·艾合买提, 丁新华, 何江, 郭文超. 马铃薯甲虫RNA干扰高效致死基因的筛选[J]. 新疆农业科学, 2019, 56(7): 1283-1293.

Mireguli Huerxida, FU Kai-yun, XU qing yu, Tursun Ahmat, DING Xin-hua, HE Jiang, GUO Wen-chao. Screening of Highly Effective Lethal Genes Interfered with RNA in Leptinotarsa decemlineata and Study on the Evaluation Indexes of Its Lethal Factors[J]. Xinjiang Agricultural Sciences, 2019, 56(7): 1283-1293.

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马铃薯甲虫RNA干扰高效致死基因的筛选

Screening of Highly Effective Lethal Genes Interfered with RNA in Leptinotarsa decemlineata and Study on the Evaluation Indexes of Its Lethal Factors

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