柚木驼蛾高毒力Bt菌株筛选及cry杀虫基因鉴定

doi:10.11707/j.1001-7488.LYKX20240665

Abstract

Abstract:

Objective: This study aims to screen efficient strains of Bacillus thuringiensis (Bt) strains with high efficiency against Hyblaea puera (Cramer), providing excellent biocontrol resources for its green prevention and control. Method: In this study, the bioactivity of 10 Bt strains against 3rd instar larvae of H. puera was determined. The similarities and differences between highly and low virulent strains were investigated in terms of parasporal crystal morphology, cry genotypes, and insecticidal crystal proteins using phase contrast microscope and scanning electron microscope (SEM), PCR-RFLP analysis combined with target gene fragment sequencing, and SDS-PAGE methods. Result: 1) All 10 Bt strains showed insecticidal activity against 3rd instar larvae of H. puera, among which 3 highly virulent strains, FJ-3, YN-1 and YN-2, had highly virulent and caused up to or close to 100% mortality to the insects within 48 hour, with LC₅₀ of the three strains were 1.51×10⁷ cfu·mL^?1、6.20×10⁶ cfu·mL^?1和2.22×10⁶ cfu·mL^?1, respectively, and the LT₅₀ of 22.14 h, 19.87 h and 17.04 h, respectively. 2) Through scanning electron microscopy observation, it was found that highly virulent strains were able to produce a variety of the parasporal crystals (3–4 types), mainly in bipyramidal and mosaic-shaped, while low virulent strains produced 1–2 types of crystals, mostly spherical and irregular. There were also differences in cry genotypes and insecticidal crystal protein molecular weight between the high and low virulent strains. Additionally, the cry genotypes of all the above-mentioned three highly virulent strains were cry1Aa, cry2Ab and cry1Ia. Conclusion: In this study, Bt strains FJ-3, YN-1, and YN-2 with strong pathogenicity against H. puera in mangrove ecosystems have been screened, and the analysis of their parasporal crystal characteristics and cry genotypes provides a reference for the molecular screening, exploration, and utilization of high-quality biocontrol resources in future research.

Key words: Hyblaea puera, mangrove, Bacillus thuringiensis (Bt), characteristics of parasporal crystals, cry genotype

CLC Number:

S763.42

Jicheng Zhao,Qinghua Wang,Yunpeng Liu,Longwa Zhang,Dezhi Kong,Yaojun Zhu,Liangjian Qu. Screening of Highly Virulent Bt Strains Against Hyblaea puera (Cramer) and Identification of cry Insecticidal Genes[J]. Scientia Silvae Sinicae, 2025, 61(11): 45-55.

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基因类型 Gene type	引物名称 Primer name	引物序列 Primer sequence（5'→3'）	限制性酶切位点 Restriction enzyme site
基因类型 Gene type	引物名称 Primer name	引物序列 Primer sequence（5'→3'）	A、B
cry1	L5un1	ATGGATAACAATCCGAACATC
	L3un1	TTCCTCCATAAGGAGTAATTCC
	K5un2	AGGACCAGGATTTACAGGAGG	XbaⅠ、PstⅠ
	K3un2	GCTGTGACACGAAGGATATAGCCAC
	K5un3	CAATGCGTACCTTACAATTGTTTAAGTAAT	EcoRⅠ、PstⅠ
	K3un3	CCTCCTGTAAATCCTGGTCCT
cry2	S5un2	GGAAGAACTACTATTTGTGAGTC	MspⅠ、HindⅡ
cry2	S3un2	AATAGTTTGAATTACCGCGAGC
cry9	S5un9	AGGACCAGGATTTACAGGAGG	XbaⅠ、PstⅠ
cry9	S3un9	CCCAATGCGAAAGAACTAAG
cry1I	S5uni	GCTGTCTACCATGATTCGCTTG	BstBⅠ、BanⅠ
cry1I	S3uni	CAGTGCAGTAACCTTCTCTTGC

菌株 Strain	种类 Types	伴孢晶体形态及大小 Morphology and size of parasporal crystals						7天累计校正死亡率 7-day cumulative corrected mortality (%)
菌株 Strain	种类 Types	菱形 Bipyramidal/μm	镶嵌形 Mosaic/μm	不规则形 Irregular/μm	卵圆形 Ovoid/μm	方形 Cubic/μm	球形 Spherical/μm	7天累计校正死亡率 7-day cumulative corrected mortality (%)
FJ-1	2	—	—	(1.32±0.36)× (0.98±0.23)	—	—	(1.08±0.138)× (1.07±0.08)	37.50±4.17
FJ-2	1	—	—	—	—	—	(1.07±0.07)× (1.07±0.11)	40.28±2.41
FJ-3	3	(1.81±0.08) × (0.78±0.11)	(1.87±0.09)× (0.84±0.11)	—	(1.98±0.10)× (0.93±0.07)	—	—	100.00
HN-1	2	(1.76±0.15)× (0.81±1.11)	—	—	(1.90±0.10)× (0.88±0.10)	—	—	81.94±2.41
HN-2	3	(1.73±0.25)× (0.81±0.14)	—	(1.45±0.28)× (1.06±0.32)	—	—	(1.03±0.11)× (1.06±0.09)	83.33
HN-3	2	(1.76±0.16)× (0.91±0.17)	—	—	(1.82±0.16)× (0.88±0.10)	—	—	88.90±4.81
YN-1	4	(1.65±0.25)× (0.78±0.11)	—	(1.20±0.12)× (0.93±0.17)	—	(1.17±0.22)× (1.19±0.19)	(1.31±0.22)× (1.25±0.18)	100.00
YN-2	4	(1.79±0.14）× （0.80±0.12）	(1.83±0.13)× (0.75±0.12)	—	(1.76±0.13)× (0.68±0.09)	—	(1.09±0.09)× (1.11±0.11)	100.00
YN-3	1	—	—	—	—	—	(1.08±0.09)× (1.08±0.07)	18.06±2.41
S-X	3	(1.73±0.09)× (0.77±0.13)	—	(1.42±0.35)× (1.40±0.24)	—	—	(1.13±0.06)× (1.11±0.09)	25.00

菌株 Strain	斜率 Slope	致死中浓度Median lethal concentration(LC₅₀)/(cfu·mL^?1)	95%置信区间 95% confidence interval/(cfu·mL^?1)	χ²
FJ-3	0.71±0.06	1.51×10⁷	9.15×10⁶~2.52×10⁷	2.88
YN-1	0.66±0.06	6.20×10⁶	3.59×10⁶~1.66×10⁷	2.01
YN-2	0.95±0.09	2.22×10⁶	1.43×10⁶~3.36×10⁶	1.30

菌株 Strain	回归方程 Regression equation	相关系数 Correlation coefficient	致死中时间Median lethal time (LT₅₀)/h	95%置信区间 95% confidence interval/h
FJ-3	Y=3.2X?4.24	0.99	22.14	15.05~30.53
YN-1	Y=3.9X?5.03	0.97	19.86	12.16~27.89
YN-2	Y=3.5X?4.35	0.98	17.04	11.69~22.17

菌株 Strain	基因 Gene	测序所用引物 Primers used	测序片段大小 Sequence length/bp	基因型 Genotypes	GeneBank序列号 Sequence ID	最大相似性 Identity (%)
FJ-1	cry1	L5un1-L3un1	3 443	cry1Aa	CP010092	99.38
FJ-2	—	—	—	—	—	—
FJ-3	cry1	L5un1-L3un1	3 451	cry1Aa	MK184461	98.77
	cry2	S5un2-S3un2	1 167	cry2Ab	MK184479	99.40
	cry1I	S5uni-S3uni	1 488	cry1Ia	AF517127	99.93
HN-1	cry1	L5un1-L3un1	3 456	cry1Ab	KT692985	97.51
	cry1I	S5uni-S3uni	1 488	cry1Ia	JQ228428	99.80
HN-2	cry1	L5un1-L3un1	3 452	cry1Ab	KT900192	95.51
	cry1I	S5uni-S3uni	1 492	cry1Ia	JQ228424	99.73
HN-3	cry1	L5un1-L3un1	3 444	cry1Ab	KT900192	99.88
	cry2	S5un2-S3un2	1 172	cry2Ad	DQ991239	98.64
	cry1I	S5uni-S3uni	1 489	cry1Ib	HM355853	99.93
YN-1	cry1	L5un1-L3un1	3 454	cry1Aa	MK184461	99.22
	cry2	S5un2-S3un2	1 174	cry2Ab	MK184479	99.66
	cry1I	S5uni-S3uni	1 488	cry1Ia	AF517127	99.93
YN-2	cry1	L5un1-L3un1	3 446	cry1Aa	KF938682	98.87
	cry2	S5un2-S3un2	1 169	cry2Ab	EU909454	98.12
	cry1I	S5uni-S3uni	1 488	cry1Ia	MN729491	99.93
YN-3	—	—	—	—	—	—
S-X	cry1	K5un3-K3un3	1 376	cry1Ea	OL899341	98.90
	cry2	S5un2-S3un2	1 174	cry2Ab	JN651494	99.06
	cry1I	S5uni-S3uni	1 488	cry1Ia	HQ439787	99.93

Screening of Highly Virulent Bt Strains Against Hyblaea puera (Cramer) and Identification of cry Insecticidal Genes

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菌株 Strain	cry基因型 cry genotypes	晶体蛋白分子量 Molecular weight of ICPs/kDa	杀虫晶体形态 Morphology of ICPs
FJ-1	cry1Aa	140, 80, 60	不规则形Irregular，球形Spherical
FJ-2	—	80, 60	球形Spherical
FJ-3	cry1Aa, cry2Ab, cry1Ia	140，60	菱形Bipyramidal，镶嵌形Mosaic，卵圆形Ovoid
HN-1	cry1Ab, cry1Ia	140, 80, 60	菱形Bipyramidal，卵圆形Ovoid
HN-2	cry1Ab, cry1Ia	140, 80, 60	菱形Bipyramidal，不规则形Irregular，球形Spherical
HN-3	cry1Ab, cry2Ad, cry1Ib	140, 80, 60	菱形Bipyramidal，卵圆形Ovoid
YN-1	cry1Aa, cry2Ab, cry1Ia	140, 80, 60	菱形Bipyramidal，不规则形Irregular，方形Cubic，球形Spherical
YN-2	cry1Aa, cry2Ab, cry1Ia	140, 80	菱形Bipyramidal，镶嵌形Mosaic，卵圆形Ovoid，球形Spherical
YN-3	—	80	球形Spherical
S-X	cry1Ea, cry2Ab, cry1Ia	140, 80, 60	菱形Bipyramidal，不规则形Irregular，球形Spherical

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