松材线虫细胞色素<i>cyp</i>-13<i>A</i>11基因RNA干扰载体的构建及其功能分析

盛东萍; 张晓阳; 冯梦婷; 叶建仁; 邱秀文

doi:10.12171/j.1000-1522.20200025

松材线虫细胞色素cyp-13A11基因RNA干扰载体的构建及其功能分析

doi: 10.12171/j.1000-1522.20200025

盛东萍^{1, 2,},
张晓阳^{1, 2},
冯梦婷^{1, 2},
叶建仁³,
邱秀文^{1, 2, ,}

1.
九江学院江西长江经济带研究院，江西九江 332005
2.
江西农业大学生物科学与工程学院，江西南昌 330045
3.
江苏省有害生物入侵预防与控制重点实验室，南京林业大学林学院，江苏南京 210037

基金项目: 国家自然科学基金项目（31660205），江西省林业厅林业科技创新项目（201711）

详细信息

作者简介:
盛东萍。主要研究方向：森林病理学研究。Email：1973661421@qq.com　地址：332005 江西省九江市前进东路551号

责任作者:
邱秀文，博士，副教授。主要研究方向：森林病理学研究。Email：qiuxiuwen3@163.com　地址：同上

计量
- 文章访问数: 525
- HTML全文浏览量: 136
- PDF下载量: 43
- 被引次数: 0
出版历程
- 收稿日期: 2020-01-20
- 修回日期: 2020-04-09
- 网络出版日期: 2020-12-24
- 刊出日期: 2021-02-05

Construction and its function analysis of RNA interference vector of cytochrome cyp-13A11 gene in Bursaphelenchus xylophilus

Sheng Dongping^{1, 2
,},
Zhang Xiaoyang^{1, 2},
Feng Mengting^{1, 2},
Ye Jianren³,
Qiu Xiuwen^{1, 2
, ,}

1.
Jiangxi Yangtze River Economic Zone Research Institute of Jiujiang University, Jiujiang 332005, Jiangxi, China
2.
College of Bioscience and Bioengineering, Jiangxi Agricultural University, Nanchang 330045, Jiangxi, China
3.
Jiangsu Key Laboratory for Prevention and Management of Invasive Specie, College ofForest, Nanjing Forestry University, Nanjing 210037, Jiangsu, China

摘要

摘要: 目的构建松材线虫cyp-13A11基因RNA干扰载体，研究cyp-13A11基因的功能，为松材线虫病的生物防治提供理论依据。方法本文通过设计特异性引物，扩增松材线虫cyp-13A11基因片段，构建至pEASY-T1干扰载体上并转入Trans1-T1大肠杆菌菌株。采用浸泡法对松材线虫进行RNA干扰，通过qRT-PCR检测cyp-13A11基因的表达，将干扰后的线虫分别接种至长满灰葡萄孢菌丝的培养皿中和黑松苗上，测定RNA干扰后线虫的取食、繁殖、致病力和干扰效率等指标。结果成功构建了带有pEASY-T1干扰载体的Trans1-T1菌株并且能够合成cyp-13A11 dsRNA，通过RNA干扰抑制了松材线虫cyp-13A11基因的表达。松材线虫RNA干扰后接种至灰葡萄孢第3天，cyp-13A11 dsRNA处理松材线虫的取食面积较小，ddH₂O处理松材线虫取食面积达到了整体的2/3；接种第6天，cyp-13A11 dsRNA处理松材线虫的取食面积为整体的1/3，而ddH₂O处理松材线虫已将灰葡萄孢菌丝取食殆尽。ddH₂O处理松材线虫的繁殖数量比cyp-13A11 dsRNA处理高4.28倍。松材线虫接种至黑松苗第10天，ddH₂O处理黑松发病率为22.2%，而cyp-13A11 dsRNA处理黑松发病率为5.6%；接种至20天，ddH₂O处理的黑松发病率为44.4%，cyp-13A11 dsRNA处理的黑松发病率为33.3%；直至接种30天，ddH₂O和cyp-13A11 dsRNA处理的黑松全部枯萎，发病率达到100%。结论成功构建了松材线虫cyp-13A11基因RNA干扰载体，cyp-13A11基因表达沉默后影响了松材线虫的取食，降低了松材线虫的繁殖能力和致病力。
- 松材线虫 /
- cyp-13A11基因 /
- 载体构建 /
- RNA干扰 /
- 功能分析
Abstract: Objective In order to reveal the function of cyp-13A11 gene in Bursaphelenchus xylophilus, the RNA interference vector was constructed and the function of cyp-13A11 gene of Bursaphelenchus xylophilus was analyzed to provide theoretical basis for biological control of pine wilt diseases. Method Specific primers were designed to amplify the cyp-13A11 gene fragment of Bursaphelenchus xylophilus. The products of amplification were constructed into pEASY-T1 interference vector, which was further transferred into Trans1-T1 escherichia coli strain. RNA interference efficiency was determined by qRT-PCR. The feeding reproduction and pathogenicity of pine wood nematodes were investigated after the nematodes soaked in cyp-13A11 dsRNA solution were inoculated on Botrytis cinerea and pine trees, respectively. Result Trans1-T1 strain with pEASY-T1 interference vector was successfully constructed and cyp-13A11 dsRNA was synthesized. RNA interference inhibited the expression of cyp-13A11 gene in Bursaphelenchus xylophilus. In addition, the feeding area was less in cyp-13A11 dsRNA treatment than that in ddH₂O treatment, as two thirds of the Botrytis cinerea were fed by nematodes in ddH₂O treatment on day 3. After 6 days, one third of Botrytis cinerea was fed by nematodes in cyp-13A11 dsRNA treatment, while almost all of the Botrytis cinerea were exhausted by nematodes in ddH₂O treatment. The reproduction number of nematodes in ddH₂O treatment was 4.28 times higher than that in cyp-13A11 dsRNA treatment. Furthermore, the wilting rates in both ddH₂O and cyp-13A11 dsRNA treatments were 22.2% and 5.6% after Bursaphelenchus xylophilus being inoculated on pine trees for 10 days, respectively. The wilting rates of pine trees in both ddH₂O and cyp-13A11 dsRNA treatments were 44.4% and 33.3% after 20 days, respectively. Obviously, the wilting rates were 100% in both treatments after 30 days. Conclusion The RNA interference vector of cyp-13A11 gene in pine wood nematode was successfully constructed. Silencing of cyp-13A11 gene exhibited an important effect on feeding of Bursaphelenchus xylophilus, as well as reduced the reproduction ability and pathogenicity of Bursaphelenchus xylophilus.
- Bursaphelenchus xylophilus /
- cyp-13A11 gene /
- vector construction /
- RNA interference /
- function analysis

HTML全文

图 1 松材线虫cyp-13A11基因的克隆

Figure 1. Cloning of cyp-13A11 gene from Bursaphelenchus xylophilus

下载: 全尺寸图片幻灯片

图 2 Trans1-T1表达菌株PCR验证

Figure 2. PCR verification of Trans1-T1 expression strain

下载: 全尺寸图片幻灯片

图 3 cyp-13A11基因dsRNA合成结果

Figure 3. Double-stranded RNA synthesis of cyp-13A11 gene

下载: 全尺寸图片幻灯片

图 4 RNA干扰后cyp-13A11基因表达量

不同字母表示不同处理间差异显著性(P < 0.01)。下同。Different letters indicate significant differences between treatments (P < 0.01). The same below.

Figure 4. Expression level of cyp-13A11 gene after RNAi treatment

下载: 全尺寸图片幻灯片

图 5 松材线虫在灰葡萄孢上的取食情况

A. 接种3 d；B. 接种6 d。对照组：ddH₂O浸泡松材线虫；RNAi处理组：cyp-13A11 dsRNA浸泡松材线虫。下同。A，inoculated for three days; B，inoculated for six days. Control group，Bursaphelenchus xylophilus soaked in ddH₂O; RNAi treatment group，Bursaphelenchus xylophilus soaked in cyp-13A11 dsRNA. The same below.

Figure 5. Feeding condition of Bursaphelenchus xylophilus on Botrytis cinerea

下载: 全尺寸图片幻灯片

图 6 RNA干扰对松材线虫繁殖的影响

Figure 6. Effects of RNA interference on the reproduction of Bursaphelenchus xylophilus

下载: 全尺寸图片幻灯片

图 7 不同接种处理第20天黑松发病情况

Figure 7. Symptoms of Pinus thunbergii seedlings after 20 days inoculation

下载: 全尺寸图片幻灯片

表 1 不同接种处理黑松的发病情况

Table 1. Disease status of Pinus thunbergii under different treatments

接种时间 Inoculating time/d	黑松发病率 Wilting rate of Pinus thunbergii/%
接种时间 Inoculating time/d	对照组1 Control group 1	RNAi处理组 RNAi treatment group	对照组2 Control group 2
10	0	5.6	22.2
20	0	33.3	44.4
30	0	100.0	100.0
注：对照组1：cyp-13A11dsRNA接种至黑松苗；RNAi：cyp-13A11 dsRNA干扰后的线虫悬液接种至黑松苗；对照组2：ddH₂O浸泡后的线虫悬液接种至黑松苗。Notes: control group 1, cyp-13A11 dsRNA is inoculated into Pinus thunbergii; RNAi, the suspension of nematodes after interference with cyp-13A11 dsRNA is inoculated into Pinus thunbergii; control group 2, the ddH₂O soaked nematode suspension is inoculated into Pinus thunbergii.

下载: 导出CSV

参考文献(24)

[1]	Xia Y, Qi Y, Yu X, et al. Nematicidal effect against Bursaphelenchus xylophilus of harmine quaternary ammonium derivatives, inhibitory activity and molecular docking studies on acetylcholinesterase[J]. European Journal of Plant Pathology, 2019, 153(1): 239−250. doi: 10.1007/s10658-018-1559-8.
[2]	朱克恭, 姚仕义. 松材线虫病在我国蔓延的严重性[J]. 林业科技开发, 1992(3):6−7. Zhu K G, Yao S Y. The spread of pine wood nematode disease in China is serious[J]. China Forestry Science and Technology, 1992(3): 6−7.
[3]	程瑚瑞, 林茂松, 黎伟强. 南京松树线虫研究[J]. 南京农业大学学报, 1983, 6(4):84. Cheng H R, Lin M S, Li W Q. Study on Bursaphelenchus xylophilus in Nanjing[J]. Journal of Nanjing Agricultural College, 1983, 6(4): 84.
[4]	Gao R, Shi J, Huang R, et al. Effects of pine wilt disease invasion on soil properties and Masson pine forest communities in the Three Gorges Reservoir Region, China[J]. Ecology and Evolution, 2015, 5(8): 1702−1716. doi: 10.1002/ece3.1326
[5]	理永霞, 张星耀. 松材线虫病致病机理研究进展[J]. 环境昆虫学报, 2018, 40(2):231−241. Li Y X, Zhang X Y. Research advance of pathogenic mechanism of pine wilt disease[J]. Journal of Environmental Entomology, 2018, 40(2): 231−241.
[6]	Kikuchi T, Cotton J A, Dalzell J J, et al. Genomic insights into the origin of parasitism in the emerging plant pathogen Bursaphelenchus xylophilus[J]. PLoS Pathogens, 2011, 7(9): e1002219. doi: 10.1371/journal.ppat.1002219.
[7]	Coon M J. Cytochrome P450: nature’s most versatile biological catalyst[J]. Annual Reviews Pharmacol Toxicol, 2005, 45: 1−25. doi: 10.1146/annurev.pharmtox.45.120403.100030.
[8]	Thomas J H. Rapid birth-death evolution specific to xenobiotic cytochrome P450 genes in vertebrates[J]. PLoS Genetics, 2007, 3(5): e67. doi: 10.1371/journal.pgen.0030067.
[9]	Scott J G, Liu N, Wen Z. Insect cytochromes P450: diversity, insecticide resistance and tolerance to plant toxins[J]. Comparative Biochemistry and Physiology Part C: Pharmacology, Toxicology and Endocrinology, 1998, 121(1−3): 147−155. doi: 10.1016/S0742-8413(98)10035-X.
[10]	郭亭亭, 姜辉, 高希武. 昆虫细胞色素P450基因的多样性, 进化及表达调控[J]. 昆虫学报, 2009, 52(3):301−311. Guo T T, Jiang H, Gao X W. Diversity, evolution and expression regulation of cytochrome P450 monooxygenase genes in insects[J]. Acta Entomologica Sinica, 2009, 52(3): 301−311.
[11]	邹丰才, 宋慧群, 陈宁, 等. RNA干扰技术及其在寄生线虫基因功能研究中的应用[J]. 中国兽医科学, 2006, 36(1):80−84. Zou F C, Song H Q, Chen N, et al. RNA interference technique and its application to studies of gene functions of parasitic nematodes[J]. Veterinary Science in China, 2006, 36(1): 80−84.
[12]	陈贵元, 谭德勇. RNA干扰的研究现状与应用前景[J]. 大理学院学报, 2012, 11(10):52−55. Chen G Y, Tan D Y. The research status and application prospect of RNA interference[J]. Journal of Dali University, 2012, 11(10): 52−55.
[13]	Fire A, Xu S Q, Montgomery M K, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J]. Nature, 1998, 391: 806. doi: 10.1038/35888.
[14]	郭葆玉. RNA干扰技术[J]. 药物生物技术, 2008, 15(2):137−141. Guo B Y. RNA interference technology[J]. Pharmaceutical Biotechnology, 2008, 15(2): 137−141.
[15]	夏欢, 邹声雷, 李超程, 等. 家兔Zfx基因shRNA干扰载体构建及验证[J]. 现代畜牧兽医, 2019(5): 14−19. Xia H, Zou S L, Li C H, et al. The shRNA interference vector construction and validation from Lepus Zfx gene[J]. Modern Journal of Animal Husbandry and Veterinary Medicine, 2019 (5): 14−19.
[16]	王越, 张苏芳, 徐瑶, 等. 美国白蛾几丁质酶细菌表达的RNA干扰载体构建及其介导的RNA干扰[J]. 林业科学研究, 2019, 32(2):1−8. Wang Y, Zhang S F, Xu Y, et al. Construction of the expression vector and RNAi mediated by bacteria expressed dsRNA of chitinase gene from Hyphantria cunea[J]. Forest Research, 2019, 32(2): 1−8.
[17]	陈鸿鹏, 谭晓风, 谢耀坚, 等. 油茶FAD2 基因的植物表达载体和RNA干扰载体构建及其功能分析[J]. 植物研究, 2015, 35(3):347−354. Chen H P, Tan X F, Xie Y J, et al. Constructing and function of plant expression vector and RNA interference vector of a FAD2 gene from Camellia oleifera[J]. Bulletin of Botanical Research, 2015, 35(3): 347−354.
[18]	王守先, 牛宝龙, 沈卫锋, 等. 松材线虫RNA聚合酶基因的RNA干扰研究[J]. 浙江农业科学, 2007, 49(6):690−693. Wang S X, Niu B L, Shen W F, et al. RNA interference of the RNA polymerase gene in Bursaphelenchus xylophilus[J]. Journal of Zhejiang Agricultural Sciences, 2007, 49(6): 690−693.
[19]	Qiu X, Wu X, Huang L, et al. Specifically expressed genes of the nematode Bursaphelenchus xylophilus involved with early interactions with pine trees[J]. PLoS ONE, 2013, 8(10): e78063. doi: 10.1371/journal.pone.0078063.
[20]	Futai K. Pine wood nematode, Bursaphelenchus xylophilus[J]. Annual Review of Phytopathology, 2013, 51: 61−83. doi: 10.1146/annurev-phyto-081211-172910.
[21]	Benenati G, Penkov S, Müller-Reichert T, et al. Two cytochrome P450s in Caenorhabditis elegans are essential for the organization of eggshell, correct execution of meiosis and the polarization of embryo[J]. Mechanisms of Development, 2009, 126(5−6): 382−393. doi: 10.1016/j.mod.2009.02.001.
[22]	Kikuchi T, Shibuya H, Aikawa T, et al. Cloning and characterization of pectate lyases expressed in the esophageal gland of the pine wood nematode Bursaphelenchus xylophilus[J]. Molecular Plant-Microbe Interactions, 2006, 19(3): 280−287. doi: 10.1094/MPMI-19-0280.
[23]	Studencka M, Wesołowski R, Opitz L, et al. Transcriptional repression of Hox genes by C. elegans HP1/HPL and H1/HIS-24[J]. PLoS Genetics, 2012, 8(9): e1002940. doi: 10.1371/journal.pgen.1002940.
[24]	王璇, 理永霞, 刘振宇, 等. 松材线虫CYP450基因与松树蒎烯类物质代谢的相关性[J]. 林业科学, 2017, 53(6):105−110. Wang X, Li Y X, Liu Z Y, et al. Relationship between the cytochrome P450 gene of pine wood nematode and the accumulation of pine pinene[J]. Scientia Silvae Sinicae, 2017, 53(6): 105−110.