转录组和代谢组联合分析三株蜡蚧轮枝菌菌株毒力差异

doi:10.13560/j.cnki.biotech.bull.1985.2024-1281

摘要/Abstract

摘要：

目的研究蜡蚧轮枝菌（Lecanicillium lecanii）不同菌株的毒力差异，结合转录组学和代谢组学数据分析，筛选蜡蚧轮枝菌（L. lecanii）菌株毒力的关键性差异表达基因（DEGs）及次级代谢产物（DEMs）。方法采用RNA-seq和LC-MS/MS技术，对培养8 d和液体发酵8 d的3株蜡蚧轮枝菌的基因表达和次级代谢产物进行检测，同时对差异基因进行RT-qPCR验证。结果室内毒力结果表明，J-1次级代谢产物对桃蚜的毒力最好，与J-2和V-1存在极显著差异。转录组和代谢组结果表明，J-1_vs_J-2获得225个DEGs和59种DEMs，J-1_vs_V-1获得2 464个DEGs和75种DEMs。差异基因与差异代谢物经KEGG富集分析，主要富集在苯丙氨酸代谢、酪氨酸代谢及ABC转运蛋白中，得到79个DEGs和19种DEMs，高毒力菌株与低毒力菌株间，水杨酸、3-羟基苯乙酸、苯乳酸、马尿酸和Methyl beta-D-galactoside显著上调，去甲肾上腺素极显著下调，c75905.graph_c0、c78027.graph_c0、c77968.graph_c0、c78586.graph_c1、c74779.graph_c0和c78871.graph_c0显著上调。RT-qPCR结果表明，关键差异基因与转录组中表达趋势一致。结论转录组和代谢组联合分析发现可能参与调控蜡蚧轮枝菌菌株毒力的6个关键基因和6个显著富集差异代谢物，为构建高毒力菌株提供依据。

关键词: 蜡蚧轮枝菌, 转录组, 代谢组, 毒力

Abstract:

Objective To study the virulence differences among various strains of Lecanicillium lecanii, and to identify key differentially expressed genes (DEGs) and differential secondary metabolites (DEMs) associated with strain virulence through integrated transcriptomic and metabolomic data analyses. Method RNA-seq and LC-MS/MS techniques were used to detect the gene expression and secondary metabolites of three L. lecanii strains cultured for 8 d and liquid fermentation for 8 d, and the differential genes were verified by RT-qPCR. Result Indoor virulence results showed that J-1 secondary metabolites were the most virulent against Myzus persicae, with highly significant differences from J-2 and V-1. Transcriptome and metabolome results: 225 DEGs and 59 DEMs were obtained for J-1_vs_J-2, 2464 DEGs and 75 DEMs were obtained for J-1_vs_V-1. Differential genes and differential metabolites were enriched by KEGG analysis, mainly in phenylalanine metabolism, tyrosine metabolism and ABC transporter protein, 79 DEGs and 19 DEMs, salicylic acid, 3-hydroxyphenylacetic acid, phenyllactic acid, hippuric acid, and methyl beta-D-galactoside were significantly up-regulated and norepinephrine was highly significantly down-regulated between high and low virulence strains. c75905.graph_c0,c78027.graph_c0,c77968.graph_c0, c78586.graph_c1, c74779.graph_c0, and c78871.graph_c0 were significantly up-regulated. RT-qPCR results showed that the key differential genes were consistent with the expression trends in the transcriptome. Conclusion Six key genes and six significantly enriched differential metabolites that may be involved in regulating the virulence of L. lecanii strains are found by combined transcriptome and metabolome analysis, which may provide a basis for the construction of highly virulent strains.

Key words: Lecanicillium lecanii, transcriptomic, metabolomic, virulence

柴军发, 洪波, 贾彦霞. 转录组和代谢组联合分析三株蜡蚧轮枝菌菌株毒力差异[J]. 生物技术通报, 2025, 41(8): 311-321.

CHAI Jun-fa, HONG Bo, JIA Yan-xia. Combined Transcriptomic and Metabolomic Analysis of Virulence Differences among Three Lecanicillium lecanii Strains[J]. Biotechnology Bulletin, 2025, 41(8): 311-321.

图/表 10

参考文献 37

[1]	张鹏飞, 张昕然, 张龙. 蜡蚧轮枝菌及其在有害生物防治中的应用研究进展 [J]. 环境昆虫学报, 2023, 45(4): 910-921.
	Zhang PF, Zhang XR, Zhang L. Lecanicillium spp. and its application research progress in pests control [J]. J Environ Entomol, 2023, 45(4): 910-921.
[2]	谷祖敏, 周飞, 陈思, 等. 蜡蚧轮枝菌VL17油剂配方筛选及室内毒力评价 [J]. 农药, 2013, 52(5): 337-339.
	Gu ZM, Zhou F, Chen S, et al. An oil formulation of verticillum lecanii VL17 and its bioassy in labortory [J]. Agrochemicals, 2013, 52(5): 337-339.
[3]	王联德, 黄建. 蜡蚧轮枝菌高产毒素菌株的筛选[J]. 福建农业大学学报:自然科学版, 2006, 35(2): 134-137.
	Wang LD, Huang J. Selection of Verticillum lecanii strains for high production of toxin[J]. Journal of Fujian Agriculture and Forestry University: Natural Science Edition, 2006, 35(2): 134-137.
[4]	吴佳, 李惠中, 邓权清, 等. 甘蔗鞭黑粉菌致病力差异菌株转录组分析 [J]. 华中农业大学学报, 2020, 39(3): 54-59.
	Wu J, Li HZ, Deng QQ, et al. Analyzing transcriptome of Sporisorium scitamineum strains with different pathogenicity [J]. J Huazhong Agric Univ, 2020, 39(3): 54-59.
[5]	蒋冬花, 丁曼青, 苏来婧, 等. 不同来源红曲霉菌株的鉴定和代谢产物多样性分析 [J]. 浙江师范大学学报: 自然科学版, 2020, 43(4): 410-416.
	Jiang DH, Ding MQ, Su LJ, et al. Identification of Monascus strains from different sources and diversity analysis of their metabolites [J]. J Zhejiang Norm Univ Nat Sci, 2020, 43(4): 410-416.
[6]	Zhang YP, Liu XM, Yin T, et al. Comparative transcriptomic analysis of two Saccharopolyspora spinosa strains reveals the relationships between primary metabolism and spinosad production [J]. Sci Rep, 2021, 11(1): 14779.
[7]	Koduru UD, Galidevara S, Reineke A, et al. Bioinformatic tools in the analysis of determinants of pathogenicity and ecology of entomopathogenic fungi used as microbial insecticides in crop protection [M]//Agricultural Bioinformatics. New Delhi: Springer India, 2014: 215-234.
[8]	李伟, 吴广畏, 杨玉萍, 等. 后基因组时代的真菌天然产物发现 [J]. 菌物学报, 2015, 34(5): 914-926.
	Li W, Wu GW, Yang YP, et al. Discovery of fungal natural product in the post‐genomic era [J]. Mycosystema, 2015, 34(5): 914-926.
[9]	张河山. 两个不同毒力小麦叶锈菌菌株的转录组分析 [D]. 保定: 河北农业大学, 2014.
	Zhang HS. Transcriptome analysis of two wheat leaf rust strains with different virulence [D]. Baoding: Hebei Agricultural University, 2014.
[10]	Wang JJ, Bai WW, 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]. J Invertebr Pathol, 2017, 145: 45-54.
[11]	Colvin D, Dhuri V, Verma H, et al. Enterococcus durans with mosquito larvicidal toxicity against Culex quinquefasciatus, elucidated using a proteomic and metabolomic approach [J]. Sci Rep, 2020, 10(1): 4774.
[12]	Prosser GA, Larrouy-Maumus G, de Carvalho LPS. Metabolomic strategies for the identification of new enzyme functions and metabolic pathways [J]. EMBO Rep, 2014, 15(6): 657-669.
[13]	汪汉成, 黄艳飞, 陈兴江, 等. 烟草赤星病菌嘧菌酯敏感与抗性菌株的代谢表型差异分析 [J]. 植物病理学报, 2018, 48(6): 822-832.
	Wang HC, Huang YF, Chen XJ, et al. Difference analysis between azoxystrobin-sensitive and-resistant isolates of Alternaria alternata causing tobacco brown spot in metabolic phenotypic characterization [J]. Acta Phytopathol Sin, 2018, 48(6): 822-832.
[14]	Zhang M, Gao X, Hao KQ, et al. Transcriptome analysis of Verticillium lecanii based on RNA-Seq technology [J]. Journal of biosafety, 2017, 26(3): 236-243.
[15]	Zheng P, Xia YL, Xiao GH, et al. Genome sequence of the insect pathogenic fungus Cordyceps militaris, a valued traditional Chinese medicine [J]. Genome Biol, 2011, 12(11): R116.
[16]	王龑, 刘阳, 刘伊宁. 产毒真菌基因组研究进展 [J]. 生物技术通报, 2015, 31(2): 26-34.
	Wang Y, Liu Y, Liu YN. Advances in studies on genomics of toxogenic fung [J]. Biotechnol Bull, 2015, 31(2): 26-34.
[17]	宋晓兵, 彭埃天, 凌金锋, 等. 昆虫病原真菌基因组学及多组学研究进展 [J]. 广东农业科学, 2021, 48(1): 17-25.
	Song XB, Peng AT, Ling JF, et al. Research progress in genomics and multi-omics of entomopathogenic fungi [J]. Guangdong Agric Sci, 2021, 48(1): 17-25.
[18]	Gao Q, Jin K, Ying SH, et al. Genome sequencing and comparative transcriptomics of the model entomopathogenic fungi Metarhizium anisopliae and M. acridum [J]. PLoS Genet, 2011, 7(1): e1001264.
[19]	Zhang YJ, Zhang JQ, Jiang XD, et al. Requirement of a mitogen-activated protein kinase for appressorium formation and penetration of insect cuticle by the entomopathogenic fungus Beauveria bassiana [J]. Appl Environ Microbiol, 2010, 76(7): 2262-2270.
[20]	Supakdamrongkul P, Bhumiratana A, Wiwat C. Characterization of an extracellular lipase from the biocontrol fungus, Nomuraea rileyi MJ, and its toxicity toward Spodoptera litura [J]. J Invertebr Pathol, 2010, 105(3): 228-235.
[21]	Zhang LB, Qiu TT, Guan Y, et al. Analyses of transcriptomics and metabolomics reveal pathway of vacuolar Sur7 contributed to biocontrol potential of entomopathogenic Beauveria bassiana [J]. J Invertebr Pathol, 2021, 181: 107564.
[22]	李晓娇. 蜡蚧轮枝菌(Verticillum lecanii)对柑橘粉虱(Diaelorude citri)基因表达的影响 [D]. 重庆: 西南大学, 2014.
	Li XJ. Effects of Verticillum lecanii on gene expression of Diaelorude citri [D]. Chongqing: Southwest University, 2014.
[23]	Hyun MW, Yun YH, Kim JY, et al. Fungal and plant phenylalanine ammonia-lyase [J]. Mycobiology, 2011, 39(4): 257-265.
[24]	Hyun MW, Kim SH. Metabolic fate of phenylalanine in the corn smut fungus Ustilago maydis [J]. Korean J Mycol, 2011, 39(3): 249-253.
[25]	何文静, 张亚东, 张超, 等. StSOBIR1类似基因沉默对马铃薯应答虫害的影响 [J]. 浙江大学学报: 农业与生命科学版, 2024, 50(4): 615-632.
	He WJ, Zhang YD, Zhang C, et al. Effects of silencing StSOBIR1-like gene on potato in response to herbivory [J]. Journal of Zhejiang University: Agriculture and Life Sciences, 2024, 50(4):615-632.
[26]	杜民杰. 水杨酸代谢在绿僵菌抗逆和致病中的作用 [D]. 重庆: 重庆大学, 2016.
	Du MJ. The role of salicylic acid metabolism in the resistance and pathogenesis of Metarhizium anisopliae [D]. Chongqing: Chongqing University, 2016.
[27]	El-Sherbeni AEE, Khaleid MS, El All AbdAllah SA, et al. Effect of some insecticides alone and in combination with salicylic acid against aphid, Aphis gossypii, and whitefly Bemisia tabaci on the cotton field [J]. Bull Natl Res Cent, 2019, 43(1): 57.
[28]	Torres MJ, Rocha VF, Audisio MC. Laboratory evaluation of Lactobacillus johnsonii CRL1647 metabolites for biological control of Musca domestica [J]. Entomologia Exp Applicata, 2016, 159(3): 347-353.
[29]	刘敏, 韩海斌, 刘爱萍, 等. 茶足柄瘤蚜茧蜂滞育和非滞育蛹中与能量代谢相关的差异表达蛋白 [J]. 昆虫学报, 2020, 63(6): 708-716.
	Liu M, Han HB, Liu AP, et al. Differentially expressed proteins associated with energy metabolism in diapause and non-diapause pupae of Lysiphlebus testaceipes (Hymenoptera: Braconidae) [J]. Acta Entomol Sin, 2020, 63(6): 708-716.
[30]	张航航. 球孢白僵菌高毒力菌株的筛选及其与小菜蛾互作相关基因的转录组分析 [D]. 合肥: 安徽农业大学, 2017.
	Zhang HH. Screening of Beauveria bassiana strains with high virulence and transcriptome analysis of genes related to their interaction with Plutella xylostella [D]. Hefei: Anhui Agricultural University, 2017.
[31]	Perlin MH, Andrews J, Toh SS. Essential letters in the fungal alphabet: ABC and MFS transporters and their roles in survival and pathogenicity [J]. Adv Genet, 2014, 85: 201-253.
[32]	廖景燕. 真菌源ABCC转运蛋白对提高植物真菌毒素耐受性的作用研究 [D]. 重庆: 西南大学, 2018.
	Liao JY. Effect of fungal ABCC transporter on improving plant mycotoxin tolerance [D]. Chongqing: Southwest University, 2018.
[33]	安一博, 马玲, 韩静, 等. 链格孢菌毒素胁迫下的棘孢木霉转录组构建及生防基因差异表达 [J]. 东北林业大学学报, 2020, 48(11): 105-110, 116.
	An YB, Ma L, Han J, et al. Transcriptome construction and biocontrol gene differential expression analysis of Trichoderma asperellum under Alternaria alternata toxin stress [J]. Journal of Northeast Forestry University, 2020, 48(11): 105-110, 116.
[34]	张亚洲. 水杨酸影响小麦与禾谷镰刀菌互作机制 [D]. 雅安: 四川农业大学, 2020.
	Zhang YZ. Mechanism of salicylic acid affecting the interaction between wheat and Fusarium graminearum [D]. Ya’an: Sichuan Agricultural University, 2020.
[35]	Laib DE, Benzara A, Akkal S, et al. The anti-acetylcholinesterase, insecticidal and antifungal activities of the entophytic fungus Trichoderma sp. isolated from Ricinus communis L. against Locusta migratoria L. and Botrytis cinerea Pers.: Fr [J]. Acta Sci Nat, 2020, 7(1): 112-125.
[36]	Hou CX, Qin GX, Liu T, et al. Transcriptome analysis of silkworm, Bombyx mori, during early response to Beauveria bassiana challenges [J]. PLoS One, 2014, 9(3): e91189.
[37]	汪永松, 耿涛, 卢芙萍, 等. 球孢白僵菌Bbchitinase 1和Bbchitinase 2在侵染宿主过程中的不同作用 [J]. 热带作物学报, 2021, 42(4): 1092-1098.
	Wang YS, Geng T, Lu FP, et al. Different roles of Beauveria bassiana bbchitinase 1 and bbchitinase 2 in host infection [J]. Chin J Trop Crops, 2021, 42(4): 1092-1098.

名称 Name	CAS	纯度 Purity	品牌 Brands
甲醇	67-56-1	≥99.0%	Thermo
乙腈	75-05-8	≥99.9%	Thermo
甲酸	64-18-6	LC-MS grade	TCI
甲酸铵	540-69-2	≥99.9%	Sigma
H₂O			Millipore
Succinic acid-2，2，3，3，-d4	14493-42-6		sigma
Cholic acid-2，2，3，4，4，-d5	53007-09-3		sigma
DL-Tryptophan-2，3，3-d3	340257-61-6		CDN
DL-Methionine-3，3，4，4-d4	93709-61-6		CDN
L-PHENYLALANINE （RING-D5）	63-91-2	98%	CIL
CHOLINE CHLORIDE（TRIMETHYL-D9）	/	98%	CIL

名称 Name	CAS	纯度 Purity	品牌 Brands
甲醇	67-56-1	≥99.0%	Thermo
乙腈	75-05-8	≥99.9%	Thermo
甲酸	64-18-6	LC-MS grade	TCI
甲酸铵	540-69-2	≥99.9%	Sigma
H₂O			Millipore
Succinic acid-2，2，3，3，-d4	14493-42-6		sigma
Cholic acid-2，2，3，4，4，-d5	53007-09-3		sigma
DL-Tryptophan-2，3，3-d3	340257-61-6		CDN
DL-Methionine-3，3，4，4-d4	93709-61-6		CDN
L-PHENYLALANINE （RING-D5）	63-91-2	98%	CIL
CHOLINE CHLORIDE（TRIMETHYL-D9）	/	98%	CIL

基因ID Gene ID	正向引物Forward primer （5′-3′）	反向引物 Reverse primer （5′-3′）
GAPDH	F： CCCCAAACACCTCATCCTCA	R： ATGCCAACCTTGACGGGAG
c78027.graph_c0	F： CAATCCGGCCCGACTACTTCT	R： TTACATGGCAACAACTGGTGAGG
c77500.graph_c2	F： TCTGAGAGTGTCCCGTCGAT	R： GCGAGACTCGATCCAGAGTG
c78385.graph_c0	F： CGCAGTTCAACTTGTGGTGG	R： AACGAGACGGCAACATGAGT
c74339.graph_c0	F： CATGGGCGGTTCGATACTCA	R： ATAGGTCCAGCGGTCAGAGT
c77841.graph_c0	F： ATCACGCTTCCACATTCACTCAC	R： CTTGAAATCCTGGAGGTTGATGTAG
c80667.graph_c0	F： CGTGGTGATAGTAATGAGAACGGAC	R： GGCGTGTCATGCTACGTTCATAT
c71547.graph_c0	F： GGCCAATCAATGGGACGAAA	R： CACATCGCCGACCAACCAA

基因ID Gene ID	正向引物Forward primer （5′-3′）	反向引物 Reverse primer （5′-3′）
GAPDH	F： CCCCAAACACCTCATCCTCA	R： ATGCCAACCTTGACGGGAG
c78027.graph_c0	F： CAATCCGGCCCGACTACTTCT	R： TTACATGGCAACAACTGGTGAGG
c77500.graph_c2	F： TCTGAGAGTGTCCCGTCGAT	R： GCGAGACTCGATCCAGAGTG
c78385.graph_c0	F： CGCAGTTCAACTTGTGGTGG	R： AACGAGACGGCAACATGAGT
c74339.graph_c0	F： CATGGGCGGTTCGATACTCA	R： ATAGGTCCAGCGGTCAGAGT
c77841.graph_c0	F： ATCACGCTTCCACATTCACTCAC	R： CTTGAAATCCTGGAGGTTGATGTAG
c80667.graph_c0	F： CGTGGTGATAGTAATGAGAACGGAC	R： GGCGTGTCATGCTACGTTCATAT
c71547.graph_c0	F： GGCCAATCAATGGGACGAAA	R： CACATCGCCGACCAACCAA

样本名称 Sample	Read number	Clean bases	碱基质量值≥Q30 Base quality value（%）	GC含量 GC content（%）
J-1-1	19 650 825	5 859 575 818	94.76	55.46
J-1-2	22 287 859	6 648 086 524	95.63	55.51
J-1-3	21 026 399	6 275 725 238	95.40	55.51
J-2-1	22 649 343	6 759 978 410	95.32	55.33
J-2-2	19 611 279	5 792 883 170	96.13	54.98
J-2-3	20 383 277	6 077 510 742	95.28	55.53
V-1-1	20 089 720	5 971 239 342	94.51	55.26
V-1-2	19 968 516	5 946 912 808	95.82	55.62
V-1-3	24 919 447	7 407 074 958	95.32	54.73