Page 5 - imbr-Vol.01-No.01

昆虫分子生物学研究
(
网络版
), 2012
,
1
,
2
,
8
-
15
Kunchong Fenzi Shengwuxue Yanjiu (Online), 2012, Vol.1, No.2, 8
-
15
8
研究报告
Research Report
对鳞翅目昆虫小菜蛾高毒力的苏云金芽孢杆菌的鉴定
梁照东
1,2
,
钟昊
1,2
,
周燕
2
,
李有志
1
1
广西大学生命科学与技术学院
,
南宁
, 530005;
2
海南省热带农业资源研究所
,
三亚
, 572025
通讯作者
:
crystal.z.chow@gmail.com;
作者
昆虫分子生物学研究
, 2012
,
1
,
2
doi: 10.5376/imbr.cn.2012.01.0002
收稿日期:
2012
05
01
接受日期:
2012
07
26
发表日期:
2012
07
28
本文首次以英文发表在
Bt Research (2011
年第
2
卷第
1
期第
1-9
)
上。现依据版权所有人授权的许可协议,采用
Creative Commons Attribution
License
协议对其进行授权,用中文再次发表与传播。只要对原作有恰当的引用,版权所有人允许并同意第三方无条件的使用与传播。如果读者对中
文含义理解有歧义,请以英文原文为准。
建议最佳引用格式:
引用格式
(
中文
)
梁照东等
, 2012,
对鳞翅目昆虫小菜蛾高毒力的苏云金芽孢杆菌的鉴定
,
昆虫分子生物学研究
(
online) Vol.1 No.2 pp.8-15 (doi: 10.5376/imbr.cn.2012.01.0002)
引用格式
(
英文
)
Liang et al., 2012, Identification of
Bacillus thuringiensis
Isolates
Highly Toxic to
Plutella xylostella
,
Kunchong Fenzi Shengwuxue Yanjiu (online) Vol.1
No.2 pp.8-15 (doi: 10.5376/imbr.cn.2012.01.0002)
摘 要
本研究利用从黑龙江凉水国家自然保护区的土壤中分离出来的
31
Bt
野生菌株作为研究菌株,对重要的鳞翅目农业
害虫小菜蛾进行高毒力菌株的筛选。经生物活性定性测定,筛选出对小菜蛾活性较高的
10
Bt
菌株。镜鉴观察表明,这些野
生菌株在芽孢形成期均产生对鳞翅目小菜蛾有毒性的菱形晶体伴孢蛋白。
cry
基因型鉴定表明,这
10
株菌株均含有
cry
l
类基因。
进一步
SDS-PAGE
分析表明,这
10
株菌株表达约
130
kD
的蛋白。对菌株的生长曲线进行分析,发现
2
~14 h
Bt
菌株处于对数
生长期,
16
~30 h
Bt
菌株处于稳定期,
32
h
Bt
菌株进入衰亡期。其中,菌株
S2685
-
1
S2737
-
3
产生大约
130
kD
的晶体蛋白起
始时间分别为培养
22
小时及
20
小时,前者到培养
26
h
时蛋白表达量基本稳定,后者其表达量随时间而逐步增加,到
22
h
时蛋
白的表达量趋于稳定状态。对小菜蛾的半致死浓度
(
LC50)
和半致死时间
(
LT50)
测定显示,
S2685
-
1
对小菜蛾的
LC50
17.113
ng/mL
LT50
37.343
h
S2737
-
3
对小菜蛾的
LC50
25.782
ng/mL
LT50
36.825
h
而标准菌株
HD
-
73
对小菜蛾的
LC50
15.414
ng/mL
LT50
35.917
h
菌株
S2685
-
1
S2737
-
3
具有较突出的半致死浓度以及半致死时间,两者对小菜蛾有很明显的杀虫活性。从
本研究的结果我们认为,
Bt
菌株
S2685
-
1
S2737
-
3
是两株杀虫活性较为突出,具有一定应用潜力的野生
Bt
菌株,可望作为标
准菌株
HD
-
73
的替换菌株,用于作为生产防治鳞翅目害虫的
Bt
生物农药的出发菌株。
关键词
苏云金芽孢杆菌
;
小菜蛾
;
Bt
毒蛋白
;
生物测定
Identification of
Bacillus thuringiensis
Isolates Highly Toxic to
Plutella xylostella
Liang Zhaodong
1,2
,
Zhong Hao
1,2
,
Zhou Yan
2
,
Li Youzhi
1
1
College of Life Science and Technology, Guangxi University, Nanning, 530004;
2
Hainan Institute of Tropical Agricultural Resources (HITAR), Sanya, 572025
Corresponding author: crystal.z.chow@gmail.com;
Authors
Abstract
In this research, we employed 31
Bacillus thuringiensis
isolates from the soils collected in the Liang Shui Natural
Reserve of Heilongjiang province to identify the highly toxic to
Plutella xylostella
,
an important Lepidopteran pest in agriculture.
The results showed that ten of thirty one
Bacillus thuringiensis
isolates have high toxicity to
Plutella xylostella
via the bioassay.
Microscopic observation for ten seleted isolates was carried out; the results confirmed that these isolates can generate bi-diamond crystal
protein which might be crystalline toxin. The further genotypic analysis by PCR and SDS-PAGE indicated that the selected 10
Bt
isolates
were consistent with the genotype of
cry
1
class toxin genes that can generate 130 kD crystal protein in molecular weight. Whilst, The
growth curves of the ten isolates exhibited that the bacteria will be in the logarithmic growth phase after culturing at 2 hours or 14 hours, in
the stationary phase after culturing 16 hours or 30 hours and in the decline phase after culturing 32 hours . Among them, strains
S2685
-
1
and S2737
-
3
were further accredited to generate the bi-diamond crystal protein of about 130 kD in molecular weight after
being cultured for 22 hours and 20 hours, respectively, the former expressing toxin goes to stable at 26 hour in culture, whereas the
later expressing levels gradually increases and stabilizes until 22 hours. The trials of median lethal concentration (LC50) and median
lethal time (LT50) of two dominant isolates, S2685
-
1
and S2737
-
3,
for Diamondback moth (
Plutella xylostella
)
were carried out in