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浙江大学学报（农业与生命科学版）
浙江大学学报（农业与生命科学版）  2019, Vol. 45 Issue (2): 189-195    DOI: 10.3785/j.issn.1008-9209.2018.08.141
植物保护     
食用菌异迟眼蕈蚊苏云金芽孢杆菌筛选及杀虫蛋白基因鉴定
王帆帆1,2(),曲绍轩2,林金盛2,李辉平2,侯立娟2,蒋宁2,骆昕2,马林2,3(),韩巨才1()
1. 山西农业大学农学院，山西 太谷 030801
2. 江苏省农业科学院蔬菜研究所，南京 210014
3. 江苏省高效园艺作物遗传改良重点实验室，南京 210014
Screening of Bacillus thuringiensis and identification of insecticidal crystal protein gene against Bradysia difformis in mushroom cultivation
Fanfan WANG1,2(),Shaoxuan QU2,Jinsheng LIN2,Huiping LI2,Lijuan HOU2,Ning JIANG2,Xin LUO2,Lin MA2,3(),Jucai HAN1()
1. College of Agriculture, Shanxi Agricultural University, Taigu 030801, Shanxi, China
2. Vegetable Research Institute, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China
3. Jiangsu Key Laboratory for Horticultural Crop Genetic Improvement, Nanjing 210014, China
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摘要：

苏云金芽孢杆菌（Bacillus thuringiensis, Bt）杀虫谱广，活性高，可以很好地应用于双翅目眼蕈蚊科的防治。为了筛选出对食用菌异迟眼蕈蚊(Bradysia difformis)有高毒力的苏云金芽孢杆菌菌株，并进行杀虫蛋白基因鉴定，采用醋酸钠-抗生素法，从江苏省南京市紫金山土壤中分离产晶体芽孢杆菌，通过室内毒力测定和对食用菌菌丝影响试验逐步进行筛选，同时进行Bt杀虫蛋白基因型聚合酶链式反应-限制性片段长度多态性(polymerase chain reaction-restricted fragment length polymorphism, PCR-RFLP)扩增鉴定。结果从分离到的17株产晶体芽孢杆菌中筛选出7个对异迟眼蕈蚊杀虫活性的校正死亡率在50%以上且不影响食用菌菌丝生长的菌株。经16S rDNA基因序列同源性分析，这7株菌与公共数据库中苏云金芽孢杆菌菌株的同源性可达99%以上，初步认定为苏云金芽孢杆菌(B. thuringiensis)。Bt杀虫蛋白基因型鉴定发现，这7个菌株含有cry4/10、cry11、cyt1、cyt2等4个不同的毒蛋白基因型。筛选出的这7株Bt菌株对于防治食用菌异迟眼蕈蚊具有较好的开发利用潜力。
关键词： 异迟眼蕈蚊;  苏云金芽孢杆菌;  杀虫活性;  杀虫晶体蛋白基因    
Abstract:

Bacillus thuringiensis (Bt) is exhibiting the high ability and wide insecticidal spectrum in the Sciaridae pest control. This experiment was conducted to screen out effective Bt strains and identify their insecticidal crystal protein (ICP) genotypes against Bradysia difformis in the mushroom cultivation. The bacillus strains that produced crystals were separated from the soil of Zijin Mountain of Nanjing by sodium acetate-antibiotic method. The strains that showed insecticidal activity against larvae of B. difformis and inhibitory effect to the hyphae of mushroom were screened by using indoor toxicity measurement method and effect test on hyphae, separately. Then ICPs were identified based on polymerase chain reaction-restricted fragment length polymorphism (PCR-RFLP) analysis. Seven strains that showed high insecticidal activity (with the corrected mortality over 50%) against B. difformis and no influence on Pleurotus ostreatus were screened from 17 strains obtained from Nanjing. The 16S rDNA analysis results showed that the seven strains were homologous to B. thuringiensis with 99% identity. The PCR-RFLP system results revealed that the seven strains contain cry4/10, cry11, cyt1, and cyt2 genes. The Bt strains have potential for application to the biocontrol of B. difformis in mushroom cultivation.
Key words: Bradysia difformis    Bacillus thuringiensis    insecticidal activity    insecticidal crystal protein gene
收稿日期: 2018-08-14 出版日期: 2019-04-25
CLC:  S 436.462  
基金资助: 国家基金项目:现代农业产业技术体系建设专项资金(CARS20)
通讯作者: 马林,韩巨才     E-mail: wff164229@163.com;malin1590@sina.com;sxndhjc@163.com
作者简介: 王帆帆（https://orcid.org/0000-0001-8898-7070），E-mail： wff164229@163.com|马林（https://orcid.org/0000-0002-6725-7981），Tel： +86-25-84390875，E-mail： malin1590@sina.com|韩巨才（https://orcid.org/0000-0002-5897-5025），E-mail： sxndhjc@163.com
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王帆帆
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引用本文:

王帆帆,曲绍轩,林金盛,李辉平,侯立娟,蒋宁,骆昕,马林,韩巨才. 食用菌异迟眼蕈蚊苏云金芽孢杆菌筛选及杀虫蛋白基因鉴定[J]. 浙江大学学报（农业与生命科学版）, 2019, 45(2): 189-195.

Fanfan WANG,Shaoxuan QU,Jinsheng LIN,Huiping LI,Lijuan HOU,Ning JIANG,Xin LUO,Lin MA,Jucai HAN. Screening of Bacillus thuringiensis and identification of insecticidal crystal protein gene against Bradysia difformis in mushroom cultivation. Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(2): 189-195.

链接本文:

http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2018.08.141    或    http://www.zjujournals.com/agr/CN/Y2019/V45/I2/189

基因 Gene

引物

Primer

序列(5′→3′)

Sequence (5′→3′)

 基因 Gene

引物

Primer

序列(5′→3′)

Sequence (5′→3′)
cry2 S5un2 GGAAGAACTACTATTTGTGATGC cry30 S5un30 AAGATTGGCTCAATATGTGTC
S3un2 AATAGTTTGAATTACCGCGAGC S3un30 GATTATCAGGATCTACACTAG
cry4/10 S5un4 GTGTCAAGAGAACCAACAGTATG cry32 S5un32ab GACAAAATCGAATTCATTCC
S3un4 ACTAAGTCTCCTCCTGTATGACCAG S3un32ab GTGTCTCTGTATAGTCTGCAC
cry11 S5un11 GGTAATCAGCCTGCTACTATGG cry32 S5un32cd GATCCTACTGGATCAAGTCAAG
S3un11 GTTGCTTGATCTGGTGTATCTTC S3un32cd TTCGTGCTGTTACACGTAACAC
cry16-17 S5un16-17 CAAGAACAGTTTATTCAGATCC cry40 S5un40 GTATGTCAGATGAACATTCTC
S3un16-17 TTATAGTCTTGGGTGGCAGC S3un40 GCTAGCATATCCTTGCATAC
cry19 S5un19 GAATGAATATGACATCTTAGATGC cyt1 cyt1F CCTCAATCAACAGCAAGGGTTATT
S3un19 GTGTAAATGCAAACGAATAACCATG cyt1R TGCAAACAGGACATTGTATGTGTAATT
cry24-25 S5un24-25 AAGATTGGGTGAATGAGTGTG cyt2 cyt2F ATTACAAATTGCAAATGGTATTCC
S3un24-25 CAGGGATTTGTGTGATTTCATC cyt2R TTTCAACATCCACAGTAATTTCAAATGC
cry27-29 S5un27-29 ATGAATATGAAATCTTAGATGC
S3un27-29 CGTTGTACCACGTTGTACAATG
表1  杀虫晶体蛋白基因型鉴定引物序列
图1   Bt菌株芽孢、晶体形态
图2   Bt菌株对异迟眼蕈蚊幼虫的室内毒力测定结果
图3   Bt菌株对平菇菌丝生长的影响
图4   7个Bt菌株的16S rDNA系统发育树

毒蛋白基因型

ICP genotype

 菌株 Strains
69-5 70-2 70-4 71-1 72-3 83-1 214-6 Bti
cry2 － － － － － － － －
cry4/10 + + － － － － － +
cry11 + + + + + + + +
cry16-17 － － － － － － － －
cry19 － － － － － － － －
cry24-25 － － － － － － － －
cry27-29 － － － － － － － －
cry30 － － － － － － － +
cry32ab － － － － － － － －
cry32cd － － － － － － － －
cry40 － － － － － － － －
cyt1 + + + + － + + +
cyt2 + + － + + － + +
表2  Bt菌株杀虫蛋白基因型的鉴定
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