球孢白僵菌对小菜蛾的侵染相关基因分析

doi:10.16409/j.cnki.2095-039x.2018.03.018

摘要/Abstract

摘要： 本文研究球孢白僵菌对小菜蛾的侵染相关基因，为提高球孢白僵菌的致病力提供基因靶点，从而提高球孢白僵菌对小菜蛾的防治效果。通过室内生物测定，筛选出球孢白僵菌对小菜蛾的高毒力菌株；采用新一代Solexa高通量测序技术对纯培养的球孢白僵菌和球孢白僵菌侵染小菜蛾48 h的虫菌混合样品分别进行转录组测序，筛选差异表达基因；结合生物信息学方法分析差异表达基因涉及的基因功能及细胞通路等；应用荧光定量PCR技术验证20个基因的差异表达。转录组测序结果显示，纯培养的球孢白僵菌与侵染小菜蛾幼虫48 h的球孢白僵菌转录组对比分析共得到8383个差异表达基因，其中显著差异表达基因716个，上调基因350个，下调基因366个。本研究筛选获得的差异表达基因，特别是上调表达的基因可能与球孢白僵菌对小菜蛾的侵染有关。GO二级分类表明，DEGs被注释到26个GO term中，包括11个生物学过程，8个细胞组分和7个分子功能；Pathway分析表明共有12个Pathway，93个上调基因和61个下调基因参与了这些途径。深入分析发现，胞外丝氨酸富集蛋白、细胞壁蛋白、胞外双加氧酶、铁转运蛋白、细胞壁半乳糖抗原蛋白等在侵染过程中与毒力相关的基因均具有显著性差异表达。研究结果为阐明球孢白僵菌对小菜蛾的侵染机制提供了基础。

关键词: 球孢白僵菌, 小菜蛾, 侵染相关基因, 转录组

Abstract: To provide genetic targets for increasing the pathogenicity of Beauveria bassiana, and improve the control efficiency of B. bassiana against Plutella xylostella, the infection related genes of B. bassiana on P. xylostella was studied in this paper. The high virulence strain of B. bassiana was selected through indoor bioassay. Solexa high-throughout sequencing technology was employed in the transcriptome analysis of the pure culture of B. bassiana and the mixture sample of P. xylostella infected by B. bassiana after 48 h. Differentially expressed genes (DEGs) and their functions, classifications and pathways were analyzed by bioinformatic methods. Twenty DEGs were further identified by fluorescence quantitative PCR. Paired analysis of DEG libraries derived from B. bassiana infected larvae for 48 h and pure-cultivated B. bassiana was conducted. 8383 DEGs were obtained, which included 716 significantly differentially expressed genes. There were 350 up-regulated genes and 366 down-regulated genes. The DEGs screened in this study, especially the up-regulated genes, should be related to the infection of B. bassiana on P. xylostella. GO annotation was used to analyze the DEGs and found 26 terms, which included biological processes (11), cellular component (8) and molecular function (7). 93 up-regulated genes and 61 down-regulated genes were enriched in 12 KEGG pathways. Further analysis showed that many DEGs were enriched in infection and virulence processes, including extracellular serine-rich protein, cell wall protein, extracellular dioxygenase,siderophore iron transporter mirB and antigeniccell wall galactomannoprotein. The results of this study provided a basis for elucidating the infection mechanism of B. bassiana on P. xylostella and improving the control efficiency of B. bassiana against P. xylostella from molecular level.

Key words: Beauveria bassiana, Plutella xylostella, dip-dye related gene, transcriptome

中图分类号:

S476.1

张腾飞, 张航航, 周红霞, 朱虹. 球孢白僵菌对小菜蛾的侵染相关基因分析[J]. 中国生物防治学报, 2018, 34(3): 388-397.

ZHANG Tengfei, ZHANG Hanghang, ZHOU Hongxia, ZHU Hong. Analysis of the Infection Related Genes of Beauveria bassiana against Plutella xylostella[J]. journal1, 2018, 34(3): 388-397.

参考文献

[1] Talekar N S, Shelton A M. Biology, ecology and management of the diamondback moth[J]. Annual Review of Entomology, 1993, 38:275-301.
[2] Capinera J L. Diamondback moth Plutella xylostella (Linnaeus) (Lepidoptera:Plutellidae)[M]. Springer Netherlands, 2008, 1202-1206.
[3] Tonnang H E Z, Nedorezov L V, Ochanda H, et al. Assessing the impact of biological control of Plutella xylostella through the application of Lotka-Volterra model[J]. Ecological Modeling, 2009, 220(1):60-70.
[4] 刘霞, 牛芳, 王开运. 小菜蛾抗药性研究现状及防治措施[J]. 农药科学与管理, 2013, 2:51-55.
[5] 林冠伦. 生物防治导论[M]. 扬州:江苏科学技术出版社, 1998, 250-260.
[6] Godonoua I, James B, Atcha-Ahowe C, et al. Potential of Beauveria bassiana and Metarhizium anisopliae isolates from Benin to control Plutella xylostella L. (Lepidoptera:Plutellidae)[J]. Crop Protection, 2009, 28(3):220-224.
[7] Siongers C, Coosemans J. Biological control of greenhouse whitefly (Trialeurodes vaporariorum) with fungal insecticides[J].Communications in Agricultural and Applied Biological Sciences, 2003, 68(4):239-247.
[8] 张奂, 张仙红, 张未仲, 等. 玫烟色拟青霉对菜青虫的侵染及致病作用[J]. 植物保护, 2007, 33(2):64-67.
[9] Boucias D G, Pendland J C, StLeger J P. Nonspecific factors involved in attachment of entomopathogenic deuteromycetes to host inseci eutile[J]. Applied and Environmental Microbiology, 1988, 54(7):1795-1805.
[10] Gao Q, Jin K, Ying S H, et al. Genome sequencing and comparative transcriptomics of the model entomopathogenic fungi Metarhizium anisopliae and M. acridum[J]. PLoS Genet, 2011, 7(1):e1001264.
[11] Wang J J, Bai W W, Zhou W, et al. Transcriptomic analysis of two Beauveria bassiana strains grown on cuticle extracts of the silkworm uncovers their different metabolic response at early infection stage[J]. Journal of Invertebrate Pathology, 2017, 145(5):45-54.
[12] Lacey L A, Frutos R, Kaya H K, et al. Insect pathogens as biological control agents:do they have a future[J]. Biological Control, 2001, 21(3):230-248.
[13] Golizadeh A, Kamali K, Fathipour Y, et al. Temperature-dependent of diamondback moth, Plutella xylostella (Lepidoptera:Plutellidae) on two brassicaceous host plants[J]. Insect Science, 2007, 14(4):309-316.
[14] 袁盛勇, 孔琼, 王丽波, 等. 球孢白僵菌MZ041016菌株对小菜蛾的室内毒力测定[J]. 江苏农业科学, 2007(6):74-75.
[15] Cho E M, Kirkland B H, Holder D J, et al. Phage display cDNA cloning and expression analysis of hydrophobins from the entomopathogenic fungus Beauveria (Cordyceps) bassiana[J]. Microbiology, 2007, 153(10):3438-3447.
[16] 李瑶, 范晓军. 昆虫几丁质酶及其在害虫防治中的应用[J]. 应用昆虫学报, 2011, 48(5):1489-1494.
[17] 李茂业. 对褐飞虱高毒力的真菌菌株筛选及其应用研究[D]. 合肥:安徽农业大学, 2012.
[18] Sampson M N, Gooday G W. Involvement of chitinases of Bacillus thuringiensis during pathogenesis in insects[J]. Microbiology, 1998, 144(8):2189.
[19] 冯明光. 胞外蛋白酶和脂酶活性作为球孢球孢白僵菌毒力指标的可靠性[J]. 微生物学报, 1998, 38(6):461-467.
[20] 侯成香, 覃光星, 耿涛, 等. 感染球孢球僵菌的家蚕免疫应答差异表达基因分析[J]. 昆虫学报, 2014, 57(1):13-24.
[21] 魏苹. 赤拟谷盗海藻糖酶基因调控几丁质合成及能量代谢的功能研究[D]. 杭州:杭州师范大学, 2013.
[22] 唐斌, 魏苹, 陈洁, 等. 昆虫海藻糖酶的基因特性及功能研究进展[J]. 昆虫学报, 2012, 55(11):1315-1321.