Identification of Insecticidal Protein Genotypes of Bacillus thuringiensis and Screening of Highly Virulent Strains Against Lepidopteran Pests

doi:10.7668/hbnxb.20195733

Abstract

Abstract:

Bacillus thuringiensis(Bt)produces multiple insecticidal proteins and serves as a vital biological control agent.This study aims to identify Bt strains with insecticidal activity against lepidopteran pests,thereby alleviating pest resistance pressure and expanding resource reserves.A total of 109 Bt strains were selected from soil of Hebei Province.The shape of parasporal crystals was observed under an oil microscope,and the insecticidal protein genotypes of the Bt strains were identified by PCR using 40 pairs of primers(including cry,cyt,and vip genotypes).The insecticidal activity bioassay was performed using five lepidopteran pests,including Plutella xylostella and Spodoptera litura.The molecular weight of crystal proteins was detected via SDS-PAGE.The shape of parasporal crystals from 90 strains was spherical,and the crystals from the remaining 19 strains were diamond-shaped.The detection rate of insecticidal protein genotypes reached 93.6%,with 29 distinct cry genotypes,1 cyt genotype,and 3 vip genotypes identified.A total of 91 strains possessed combinations of at least two insecticidal protein genotypes,among which 26 strains contained six or more,predominantly exhibiting cry+vip patterns.We found that 19 Bt strains showed high insecticidal activity against various lepidopteran pests such as P.xylostella. These strains primarily expressed proteins at 130,65 ku,encompassing lepidopteran-killing genotypes such as cry1,cry2,and cry15,consistent with PCR identification results.Here,we identified broad-spectrum insecticidal and highly toxic Bt strains against lepidopteran pests,and revealed its insecticidal genes.Our work provides an important candidate for the development of bioinsecticide.

Key words: Bacillus thuringiensis, Genotype, Lepidopteran pests, Insecticidal activity

CLC Number:

FENG Shuo, HUANG Guoqiang, CHENG Jiaxu, CAO Weiping, JIA Haimin, SONG Jian. Identification of Insecticidal Protein Genotypes of Bacillus thuringiensis and Screening of Highly Virulent Strains Against Lepidopteran Pests[J]. Acta Agriculturae Boreali-Sinica, 2026, 41(1): 184-193. doi: 10.7668/hbnxb.20195733.

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URL: http://www.hbnxb.net/EN/10.7668/hbnxb.20195733

http://www.hbnxb.net/EN/Y2026/V41/I1/184

Figures/Tables 7

References 29

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靶标害虫 Target insects	杀虫蛋白 Insecticidal protein
鳞翅目Lepidopterans	Cry1、Cry2、Cry7、Cry8、Cry9、Cry15、Cry22、Cry32、Cry51、Cry54、Cry56、Vip3
双翅目Dipterans	Cry1、Cry2、Cry4、Cry10、Cry11a、 Cry16、 Cry19、Cry20、Cry24、Cry27、Cry32、Cry39、Cry44、Cry47、Cry56、Cry60、Cry69、Cry80、Cyt1、Cyt2
鞘翅目Coleopterans	Cry1、Cry3、Cry7、Cry8、Cry9、Cry14、Cry18、Cry22、Cry23、Cry34、Cry35、Cry36、Cry37、Cry43、Cry55、Cyt1、Cyt2、Vip1、Vip2
半翅目Hemipterans	Cry2、Cry3、Cry11、Cry51、Cry78、Vip1、Vip2

靶标害虫 Target insects	杀虫蛋白 Insecticidal protein
鳞翅目Lepidopterans	Cry1、Cry2、Cry7、Cry8、Cry9、Cry15、Cry22、Cry32、Cry51、Cry54、Cry56、Vip3
双翅目Dipterans	Cry1、Cry2、Cry4、Cry10、Cry11a、 Cry16、 Cry19、Cry20、Cry24、Cry27、Cry32、Cry39、Cry44、Cry47、Cry56、Cry60、Cry69、Cry80、Cyt1、Cyt2
鞘翅目Coleopterans	Cry1、Cry3、Cry7、Cry8、Cry9、Cry14、Cry18、Cry22、Cry23、Cry34、Cry35、Cry36、Cry37、Cry43、Cry55、Cyt1、Cyt2、Vip1、Vip2
半翅目Hemipterans	Cry2、Cry3、Cry11、Cry51、Cry78、Vip1、Vip2

引物名称 Primers	序列(5'—3') Sequence(5'—3')	引物名称 Primers	序列(5'—3') Sequence(5'—3')
cry1-F	AGGACCAGGATTTACAGGAGG	cry24/25-F	AAGATTGGGTGAATGAGTGTG
cry1-R	GCTGTGACACGAAGGATATAGCCAC	cry24/25-R	CAGGGATTTGTGTGATTTCATC
cry2-F	GGAAGAACTACTATTTGTGATGC	cry27/29-F	ATGAATATGAAATCTTAGATGC
cry2-R	AATAGTTTGAATTACCGCGAGC	cry27/29-R	CGTTGTACCACGTTGTACAATG
cry3-F	CGAACAATCGAAGTGAACATGATAC	cry26/28-F	TACCACATTGATCAAGCGGC
cry3-R	GTCTACACTTTTATGTGTCCAAGT	cry26/28-R	GATAATGGTCCCACTTCTATC
cry4/10-F	GTGTCAAGAGAACCAACAGTATG	cry30-F	AAGATTGGCTCAATATGTGTC
cry4/10-R	ACTAAGTCTCCTCCTGTATGACCAG	cry30-R	GATTATCAGGATCTACACTAG
cry5-F	TTACGTAAATTGGTCAATCAAGCAAA	cry31-F	GATATAAATAGTATGCGAAATAC
cry5-R	AAGACCAAATTCAATACCAGGGTT	cry31-R	TTATGAAACAGGACTAAAAATAAT
cry6-F	TGGCGTAGAGGCTGTTCAAGTA	cry32-F	TTAGCACGTGTGAAAAAACGCGAA
cry6-R	TGTCGAGTTCATCATTAGCAGTGT	cry32-R	TTCGTGCTGTTACACGTAACAC
cry7-F	TGGGAGATTTATGAAGAAGTAG	cry33-F	ATGGCTATTATTGATTTTAACC
cry7-R	GCTTGTTTTGCTCGTGTATAAATC	cry33-R	CTACATCGGATTAATAATTGGA
cry8-F	GTCCGAATAATCAGAATGAATATG	cry34/35-F	CAGCACGTGAAGTACACATTG
cry8-R	CGTTTTGCCTCTCTCACTGCATC	cry34/35-R	CTGTACCTCCACCTACTTCTG
cry9-F	GGCTGGAAGGCAAGTAACGG	cry36-F	AAAGTACTAGATTTCAAAAAATA
cry9-R	GAGCCGCTTCACAGCAATCC	cry36-R	TGGTGTTTTCCATATTGATAAAT
cry11-F	CCAGCATTAATAGCAGTAGCTCC	cry37-F	ATGACAGTATATATAACGCAACTT
cry11-R	AAGCATTTGTACTTGAAGTTG	cry37-R	TTATGCTGGAGTCAAGGAATAC
cry12-F	CTCCCCCAACATTCCATCC	cry40-F	CTATATGTGATTTATTATCTATAATT
cry12-R	AATTACTTACACGTGCCATACCTG	cry40-R	ACCACATGCCGCATTTGATAATCTAT
cry13-F	CTTTGATTATTTAGGTTTAGTTCA	cry42-F	CTTTGGCCTACAATACACAAGC
cry13-R	TTGTAGTACAGGCTTGTGATTC	cry42-R	TTAAAGCTGTCACGATTTGATAG
cry14-F	ATAATGCGCGACCTACTGTTGT	cry43-F	ACTTTACAGTCCCAATAAGTATCCG
cry14-R	TGCCGTTATCGCCGTTATT	cry43-R	GTAAACCCGTCTGATACCAAGT
cry15-F	ATCTGGGGTTACCGTTTCTGC	cry44-F	AATATGCCAAACAGATATCCTTTT
cry15-R	CGTCGTTGCTGTTCCTCTCC	cry44-R	ACTTGCATAACGAATTCGAACACG
cry16/17-F	CAAGAACAGTTTATTCAGATCC	cry47-F	TGGGAAATTTTTATGGAACAAGT
cry16/17-R	TTATAGTCTTGGGTGGCAGC	cry47-R	CCAACTGGATCTGTATAAACTTC
cry18-F	CCGAGGCGATTTGGATAGAT	cyt1-F	TTCGTTTAGCAAATGCATTTCAA
cry18-R	TGCCGGTGTAAACAAAGAAGG	cyt1-R	TTGAAATGCATTTGCTAAACGAA
cry19-F	AGGGGAGTCCAGGTTATGAGTTAC	cyt2-F	ATTTGAGTTTCTAAATTTGTAAA
cry19-R	ATTTCCCTAGTTAGTTCGGTTTTT	cyt2-R	TTTACAAATTTAGAAACTCAAAT
cry20-F	CAATCCCTGGCTTCACTCGT	vip1-F	GTGGGAAATAACGTACTGAA
cry20-R	CCGCGGGCATTAGGATT	vip1-R	CCCACCATCTATAACAGTAAT
cry21-F	ATACAGGGATAGGATTTCAAG	vip2-F	TTATTTTAATGGCATTTATGGAT
cry21-R	ATCCCCATTTTCTATAAGTGTCT	vip2-R	GCAGGTGTAATTTCAGTAAGT
cry22-F	GCATTTCATAGAGGATCAATTGG	vip3-F	GCACCACACGTGATAATAC
cry22-R	TTCGATTGTTTTTTCATCACTTTC	vip3-R	GATCCCATCTGTATCCGT

引物名称 Primers	序列(5'—3') Sequence(5'—3')	引物名称 Primers	序列(5'—3') Sequence(5'—3')
cry1-F	AGGACCAGGATTTACAGGAGG	cry24/25-F	AAGATTGGGTGAATGAGTGTG
cry1-R	GCTGTGACACGAAGGATATAGCCAC	cry24/25-R	CAGGGATTTGTGTGATTTCATC
cry2-F	GGAAGAACTACTATTTGTGATGC	cry27/29-F	ATGAATATGAAATCTTAGATGC
cry2-R	AATAGTTTGAATTACCGCGAGC	cry27/29-R	CGTTGTACCACGTTGTACAATG
cry3-F	CGAACAATCGAAGTGAACATGATAC	cry26/28-F	TACCACATTGATCAAGCGGC
cry3-R	GTCTACACTTTTATGTGTCCAAGT	cry26/28-R	GATAATGGTCCCACTTCTATC
cry4/10-F	GTGTCAAGAGAACCAACAGTATG	cry30-F	AAGATTGGCTCAATATGTGTC
cry4/10-R	ACTAAGTCTCCTCCTGTATGACCAG	cry30-R	GATTATCAGGATCTACACTAG
cry5-F	TTACGTAAATTGGTCAATCAAGCAAA	cry31-F	GATATAAATAGTATGCGAAATAC
cry5-R	AAGACCAAATTCAATACCAGGGTT	cry31-R	TTATGAAACAGGACTAAAAATAAT
cry6-F	TGGCGTAGAGGCTGTTCAAGTA	cry32-F	TTAGCACGTGTGAAAAAACGCGAA
cry6-R	TGTCGAGTTCATCATTAGCAGTGT	cry32-R	TTCGTGCTGTTACACGTAACAC
cry7-F	TGGGAGATTTATGAAGAAGTAG	cry33-F	ATGGCTATTATTGATTTTAACC
cry7-R	GCTTGTTTTGCTCGTGTATAAATC	cry33-R	CTACATCGGATTAATAATTGGA
cry8-F	GTCCGAATAATCAGAATGAATATG	cry34/35-F	CAGCACGTGAAGTACACATTG
cry8-R	CGTTTTGCCTCTCTCACTGCATC	cry34/35-R	CTGTACCTCCACCTACTTCTG
cry9-F	GGCTGGAAGGCAAGTAACGG	cry36-F	AAAGTACTAGATTTCAAAAAATA
cry9-R	GAGCCGCTTCACAGCAATCC	cry36-R	TGGTGTTTTCCATATTGATAAAT
cry11-F	CCAGCATTAATAGCAGTAGCTCC	cry37-F	ATGACAGTATATATAACGCAACTT
cry11-R	AAGCATTTGTACTTGAAGTTG	cry37-R	TTATGCTGGAGTCAAGGAATAC
cry12-F	CTCCCCCAACATTCCATCC	cry40-F	CTATATGTGATTTATTATCTATAATT
cry12-R	AATTACTTACACGTGCCATACCTG	cry40-R	ACCACATGCCGCATTTGATAATCTAT
cry13-F	CTTTGATTATTTAGGTTTAGTTCA	cry42-F	CTTTGGCCTACAATACACAAGC
cry13-R	TTGTAGTACAGGCTTGTGATTC	cry42-R	TTAAAGCTGTCACGATTTGATAG
cry14-F	ATAATGCGCGACCTACTGTTGT	cry43-F	ACTTTACAGTCCCAATAAGTATCCG
cry14-R	TGCCGTTATCGCCGTTATT	cry43-R	GTAAACCCGTCTGATACCAAGT
cry15-F	ATCTGGGGTTACCGTTTCTGC	cry44-F	AATATGCCAAACAGATATCCTTTT
cry15-R	CGTCGTTGCTGTTCCTCTCC	cry44-R	ACTTGCATAACGAATTCGAACACG
cry16/17-F	CAAGAACAGTTTATTCAGATCC	cry47-F	TGGGAAATTTTTATGGAACAAGT
cry16/17-R	TTATAGTCTTGGGTGGCAGC	cry47-R	CCAACTGGATCTGTATAAACTTC
cry18-F	CCGAGGCGATTTGGATAGAT	cyt1-F	TTCGTTTAGCAAATGCATTTCAA
cry18-R	TGCCGGTGTAAACAAAGAAGG	cyt1-R	TTGAAATGCATTTGCTAAACGAA
cry19-F	AGGGGAGTCCAGGTTATGAGTTAC	cyt2-F	ATTTGAGTTTCTAAATTTGTAAA
cry19-R	ATTTCCCTAGTTAGTTCGGTTTTT	cyt2-R	TTTACAAATTTAGAAACTCAAAT
cry20-F	CAATCCCTGGCTTCACTCGT	vip1-F	GTGGGAAATAACGTACTGAA
cry20-R	CCGCGGGCATTAGGATT	vip1-R	CCCACCATCTATAACAGTAAT
cry21-F	ATACAGGGATAGGATTTCAAG	vip2-F	TTATTTTAATGGCATTTATGGAT
cry21-R	ATCCCCATTTTCTATAAGTGTCT	vip2-R	GCAGGTGTAATTTCAGTAAGT
cry22-F	GCATTTCATAGAGGATCAATTGG	vip3-F	GCACCACACGTGATAATAC
cry22-R	TTCGATTGTTTTTTCATCACTTTC	vip3-R	GATCCCATCTGTATCCGT

菌株编号 Strain	基因型 Genotype	晶体形状 Shape
WJ501	—	圆球形
WJ502	cry15	圆球形
WJ503	cry32、cry47	圆球形
WJ504	cry15、cry20	圆球形
WJ505	cry15、cry20	圆球形
WJ506	cry1、cry15、cry32	圆球形
AB18	cry1、cry2、cry20	圆球形
AB35	cyt2、vip1	圆球形
AB48	cry1、cry22、cry47、vip1	圆球形
AB65	cry2	圆球形
AB69	cry1、cry4/10、cry11、cry15、cry47	圆球形
AB81	cry20、cry26/28	圆球形
AB82	cry9、cry15、cry20、cry32、vip2	圆球形
AB83	cry9、cry26/28、cry44、cry47	圆球形
AB84	cry2、cry9、cry15、vip3	圆球形
AB85	cry15、cry20、vip3	圆球形
AB86	cry15、vip3	圆球形
AB87	cry16/17、cry26/28	圆球形
AB88	cry15、cry44、cry47	圆球形
AB89	cry14、vip3	圆球形
AB90	cry15	圆球形
AB91	—	圆球形
AB92	cry15	圆球形
AB93	cry1、cry2、cry9、cry12、cry15、cry20、cry34/35、vip1	菱形
AB94	cry4/10、cry5、cry7、cry8、cry9、cry15、 cry20、cry32、vip2	圆球形
AB95	cry20	圆球形
AB96	cry1、cry2	菱形
AB97	cry1、cry2、cry26/28、cry44	菱形
AB98	cry1、cry2	菱形
AB99	cry2	圆球形
AB100	cry1、cry2	圆球形
AB101	cry2、cry15	圆球形
AB102	cry15、vip3	圆球形
AB103	cry9、cry15、vip2、vip3	圆球形
AB104	cry8、cry47、vip1	圆球形
AB105	cry1、cry2、cry15、cry37	菱形
AB106	cry8、cry13、cry15、cry24/25、cry36、vip1	菱形
AB107	cry2、cry7、cry15、cry20、vip2、vip3	圆球形
AB108	cry15	圆球形
AB109	cry1、cry8、cry15、cry20、cry44、vip1	圆球形
AB110	—	圆球形
AB111	cry9、cry20、cry47、vip1	圆球形
AB112	cry20	圆球形
AB113	cry8、cry9、cry15、cry20、cry26/28	圆球形
AB114	cry1、cry2	菱形
AB115	cry2、cry7、cry9、cry13、cry14、cry15、cry18、cry20、cry26/28、vip2、vip3	圆球形
AB116	cry1、cry2、cry7、cry9、cry15、cry20、cry42、vip1	菱形
AB117	cry1、cry2、cry24/25、vip1	菱形
AB118	cry15、vip1	圆球形
AB119	cry2、cry14、cry15、cry20、cry26/28、vip3	圆球形
AB120	cry15	圆球形
AB121	cry11、cry15	圆球形
AB122	cry15、cry18、cry24/25、cry44、cry47、vip3	圆球形
AB123	cry1、cry2、cry15、vip1	圆球形
AB124	cry7、cry18、cry32	圆球形
AB125	cry7、cry18、cry26/28、cry44、cry47、vip1	圆球形
AB126	cry30、cry32	圆球形
AB127	cry1、cry44	圆球形
AB128	cry1、cry2、cry14、cry20、cry22	圆球形
AB129	cry1、cry2、cry9、cry18、cry22、vip1	菱形
AB130	cry15、cry24/25	圆球形
AB131	cry7、cry8、cry9、cry15、cry18、cry20、cry26/28、vip2、vip3	圆球形
AB132	cry9、cry15、cry20、cry24/25、cry32、vip2、vip3	圆球形
AB133	cry2、cry7、cry44、vip2	圆球形
AB134	cry9、cry12、cry15、cry26/28	菱形
AB135	cry7、cry8、cry26/28、vip3	圆球形
AB136	cry8、cry47、vip1	圆球形
AB137	—	圆球形
AB138	cry20、vip1	圆球形
AB139	cry8、cry20、cry37	圆球形
AB140	cry8、cry15、vip1、vip3	圆球形
AB141	cry8、cry20、cry44	圆球形
AB142	cry47、vip2	圆球形
AB143	cry7、cry8、cry9、cry20、cry21、cry24/25、vip1	圆球形
AB144	cry1、cry2、cry7、cry15、cry18、cry22、cry32	菱形
AB145	cry9、cry15、cry16/17	圆球形
AB146	cry8、cry9、cry15、cry20、cry32	圆球形
AB147	cry7、cry8、cry20、cry32、vip2、vip3	圆球形
AB148	cry2、cry7、cry8、cry9、cry15、cry20、cry32、cry47、vip3	圆球形
AB149	cry1、cry8、cry9、cry15、cry18、cry20、cry21、cry24/25、cry37、cry42、cry44、vip1	菱形
AB150	cry4/10、cry8、cry20、vip2	圆球形
AB151	cry20、cry37、vip2	圆球形
AB152	—	圆球形
AB153	cry47	菱形
AB154	cry20、cry26/28、cry32	圆球形
AB155	cry8、cry20、cry26/28	圆球形
AB156	cry4/10、cry7、cry9、cry15、cry20、cry26/28、cry32、vip3	圆球形
AB157	—	圆球形
AB158	vip1	圆球形
AB159	cry2、cry15、cry18、cry20、cry22、cry32	菱形
AB160	cry4/10、cry8、cry9、cry15、cry18、cry20、cry26/28	圆球形
AB161	cry8、cry9、cry20、cry32、vip3	圆球形
AB162	cry20、cry44、cry47、vip1	圆球形
AB163	cry2、cry4/10、cry8、cry32、vip1	圆球形
AB164	cry1、cry2、cry15、vip1	菱形
AB165	cry15、cry20、cry32	圆球形
AB166	cry9、cry15、cry20、cry32、cry36、cry37、cry47、vip3	圆球形
AB167	cry8、cry20、cry37、vip1	圆球形
AB168	—	菱形
AB169	cry8、cry20、cry26/28	圆球形
AB170	cry47、vip1	圆球形
AB171	cry15、cry30、cry40	圆球形
AB172	cry8、cry20、cry26/28、cry30、cry37、cry47、vip1	圆球形
AB173	cry11、cry15	圆球形
AB174	cry1、cry2、cry18、vip1	菱形
AB175	cry1、cry9、cry16/17、cry22	圆球形
AB176	cry1、cry2、cry15、cry20、cry22、cry30、cry32、cry40、vip1	菱形
AB177	cry1、cry9、cry15、cry26/28	圆球形
AB178	cry8、cry9、cry15、cry47	圆球形

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