短小芽孢杆菌表达系统的优化研究
摘要
真菌病害是造成农作物产量损失的重要原因之一,利用天然的或经遗传改良
的微生物进行植物真菌病害的防治是当前国际上较为活跃的研究领域之一。本实
验室已从水稻叶片上分离出一株附生菌 DX01,经试验证明该菌具有天然拮抗某
些真菌的特性,若能进一步将其定向改造成具有更强抗真菌能力的工程菌,作为
生物农药的有效组分,从而可以减少因使用化学农药而造成的环境污染,无疑具
有重要的理论和现实意义。绿色荧光蛋白(GFP)基因作为标记基因,被广泛应用
于环境微生物学方面,由于其表达稳定,容易观测,因此,也可以作为启动子活
性的报告基因。为了进一步优化短小芽孢杆菌表达系统,以 GFP 作为报告基因,
对克隆自 DX01 菌株的启动子进行了一系列的研究。
采用 PCR 技术,亚克隆来自短小芽孢杆菌 (Bacillus brevis) DX01 菌株总基
因组中的启动子活性片段 F1,分别构建了大肠杆菌组成型表达载体
pUC118-F1gfp 和一个大肠杆菌-短小芽孢杆菌穿梭表达载体 pHY300-F1gfp,以缺
失启动子的绿色荧光蛋白 (GFP) 基因为报告基因,检测了该片段在大肠杆菌
DH5α 和短小芽孢杆菌 DX01 菌株中的启动活性,在荧光显微镜下,均观察到了
明亮的绿色荧光,证实活性片段 F1 具有组成型启动子的功能,gfp 基因在 2 种宿
主细胞中实现了组成型表达。该表达结果为 Western 印迹所证实。对 2 种宿主表
达细胞中 GFP 蛋白的表达量作了 FACS 分析,结果表明 gfp 基因在 DX01 细胞中
的表达强度约为 DH5α 中的 2 倍。
运用来自噬菌体 Mu 的转座酶 MuA,对启动子 F1 进行了随机插入突变,从
中筛选出 13 个 F1 的突变体。对突变体的氯霉素抗性检测表明,与 F1 对照相比,
7 个含相应突变体的菌株其氯霉素抗性水平有显著提高,其中突变体 Fm3 和
Fm10 抗性提高了 3.6 倍,可抗高达 900μg/mL 氯霉素,揭示这些启动子突变体有
着更高的启动活性;4 个突变体氯霉素抗性水平下降;2 个突变体抗性水平基本
不变。CAT-ELISA 检测的结果,也进一步证实了氯霉素抗性水平的变化与 CAT
酶活力的变化有着密切的关系。在启动子 F1 的 DNA 序列上,存在着不同插入
效应敏感位点,在这些位点附近的插入突变,可使启动活性急剧变化,并且相邻
的插入位点,其突变效应可能完全不同。FACS 分析表明,在大肠杆菌中表现为
启动活性增强的 F1 突变体,转入短小芽孢杆菌 DX01 后其活性仍表现为增强。
采用 RT-PCR 方法,克隆了萝卜抗真菌蛋白 1(Rs-AFP1)的 cDNA 全长,在该
VI
基因的上游连上了一个短小芽孢杆菌 47 株的中壁蛋白(MWP)信号肽,将 Rs-afp1
基因的终止密码子突变掉,其后接上绿色荧光蛋白(GFP)基因和 T7 终止子,分
别构建成AFP-GFP融合蛋白表达载体pUC118-F1SP' 和pHY300-F1SP' 。
Agfp Agfp
Western 杂交证实融合蛋白在大肠杆菌 DH5α 中得到了表达。
[附录部分]
杂交墨西哥落羽杉是我国已故著名林木育种家叶培忠教授于 1963 年通过墨
西哥落羽杉(母本)与柳杉(父本)杂交所获得的新种。目前,其主要分布在上海、
江苏等地。杂交墨杉苗期长势不突出,早年并未引起林木工作者的注意,故目前
这些杂交墨西哥落羽杉的分布情况已很难从原始档案中查找到完整的资料。为
此,采用随机引物扩增多态性 DNA(RAPD)技术,对 8 个可能是杂交墨西哥落羽
杉片林样本、其杂交母本?墨西哥落羽杉和杂交父本的同类?柳杉,共计 12 个
样本进行了亲缘关系鉴定和遗传多样性分析。从 31 个随机引物中筛选出 17 个引
物,共扩增出 164 条带。其中,多态带有 162 条,占全部条带的 98.8 %。检测结
果表明:在 8 号、11 号、12 号 3 个柳杉样本中,11 号样本与原杂交父本的亲缘
关系最近;1 号、4 号和 9 号三个片林样本是杂交墨杉群落的可能性最大;5 号
片林样本可能是未杂交的墨杉纯林;其他群落的样本是否为杂交墨杉尚不能通过
该实验得出明确结论。
The green fluorescent protein (GFP) of the jellyfish Aequorea Victoria is a useful
reporter molecule for monitoring gene expression in vivo in eukaryotic and
prokaryotic cells. A GFP vector was constructed for in situ detection of the rice
epiphyte Bacillus brevis strain DX01. The promoterless gfp-S65T gene was
transcriptionally fused to a strong B. brevis promoter pCP01 functional F1 fragment,
which was subcloned from the genome of strain DX01 by PCR technique, and then
respectively inserted into plasmid pUC118 and an Escherichia coli-Bacillus shuttle
vector pHY300PLK leading to result in expression vectors pUC118-F1gfp and
pHY300-F1gfp. The DX01 cells harboring the plasmid pHY300-F1gfp could produce
bright green fluorescence when they were examined by using fluorescent microscopy.
The results were confirmed by western blot analysis. The transcription from F1
remained constitutive although it was 280 bp shorter than in promoter pCP01.
GFP-expressing cells were also sorted by fluorescence-activated cell sorting (FACS)
analysis. The fluorescence intensity of cell populations of DX01::pHY300-F1gfp was
two-fold higher than that of DH5a::pUC118-F1gfp, which implied that the GFP
expression vector pHY300-F1gfp was highly efficient in B. brevis DX01. Though F1
showed no host cell specificity, it displayed higher transcriptional activity in B. brevis
DX01 than in E. coli DH5 a.
To improve the activity of F1, insertion mutagenesis of F1 based on in vitro
transposition reaction was performed. 227 insertion clones were screened from an
insertion library, 13 mutants were identified and sequenced. Their MIC of Cam and
CAT synthesis were assayed. Seven mutants with enhanced transcription activity in E.
coli DH5α were obtained. The MICs of Cam of Fm3 and Fm10 were 900 μg/mL,
which is 2.6 fold higher than that of the control. Four mutants showed lower MIC of
Cam, and two kept unaltered. In general, the MIC of Cam was positively related to
CAT activity in E. coli DH5α cells. The insertions in DNA sequence of promoter F1
resulted in different effects. Remarkably, the insertions in the neighbor sites led to
diametrically opposing effects. The results of FACS analysis confirmed that the
enhanced promoters showed similar high activities in B. brevis strain DX01.
The anti-fungal protein 1 (Rs-AFP1) cDNA was derived from radish cultivar
“Chuanxinhong” by reverse transcription PCR, and then the promoter F1 and the
VIII
middle-wall-protein signal peptide of B. brevis strain 47 were fused to its 5'end in a
correct orientation respectively. Subsequently, the terminator codon of the Rs-afp1
gene was mutated and a T7 terminator was fused to its 3' resulting in the two
end
expression vectors pUC118-F1SP' Agfp and pHY300-F1SP' Agfp. The fused protein
was confirmed expressed in E. coli DH5α by Western blot analysis.
RAPD analyses were conducted on genetic polymorphisms of twelve fir genotypes
to identify their relationships. These genotypes included eight supposed hybrid
Taxodium mucronatum Tenore forest samples and the hybrid female-parent?T.
mucronatum Tenore and the same class of hybrid male-parent? Cryptomeria fortunei
Hooibrenk and sugi C. Japonica(L.f.) D.Don. 17 of 31 primers were selected to use
for separate amplification reactions with DNA from 12 genotypes. 164 RAPD
fragments were produced and 98.8 % of them showed polymorphism. The results
revealed that the genetic relationship of sample No.11 in three Cryptomeria genotypes
is the closest to the original male-parent; the samples No.1,4 and 9 are most possibly
the true hybrid T. mucronatum Tenore populations; the sample No.5 may be the false
hybrid. Conclusions could not be drawn from the data in hand for the rest samples.
的微生物进行植物真菌病害的防治是当前国际上较为活跃的研究领域之一。本实
验室已从水稻叶片上分离出一株附生菌 DX01,经试验证明该菌具有天然拮抗某
些真菌的特性,若能进一步将其定向改造成具有更强抗真菌能力的工程菌,作为
生物农药的有效组分,从而可以减少因使用化学农药而造成的环境污染,无疑具
有重要的理论和现实意义。绿色荧光蛋白(GFP)基因作为标记基因,被广泛应用
于环境微生物学方面,由于其表达稳定,容易观测,因此,也可以作为启动子活
性的报告基因。为了进一步优化短小芽孢杆菌表达系统,以 GFP 作为报告基因,
对克隆自 DX01 菌株的启动子进行了一系列的研究。
采用 PCR 技术,亚克隆来自短小芽孢杆菌 (Bacillus brevis) DX01 菌株总基
因组中的启动子活性片段 F1,分别构建了大肠杆菌组成型表达载体
pUC118-F1gfp 和一个大肠杆菌-短小芽孢杆菌穿梭表达载体 pHY300-F1gfp,以缺
失启动子的绿色荧光蛋白 (GFP) 基因为报告基因,检测了该片段在大肠杆菌
DH5α 和短小芽孢杆菌 DX01 菌株中的启动活性,在荧光显微镜下,均观察到了
明亮的绿色荧光,证实活性片段 F1 具有组成型启动子的功能,gfp 基因在 2 种宿
主细胞中实现了组成型表达。该表达结果为 Western 印迹所证实。对 2 种宿主表
达细胞中 GFP 蛋白的表达量作了 FACS 分析,结果表明 gfp 基因在 DX01 细胞中
的表达强度约为 DH5α 中的 2 倍。
运用来自噬菌体 Mu 的转座酶 MuA,对启动子 F1 进行了随机插入突变,从
中筛选出 13 个 F1 的突变体。对突变体的氯霉素抗性检测表明,与 F1 对照相比,
7 个含相应突变体的菌株其氯霉素抗性水平有显著提高,其中突变体 Fm3 和
Fm10 抗性提高了 3.6 倍,可抗高达 900μg/mL 氯霉素,揭示这些启动子突变体有
着更高的启动活性;4 个突变体氯霉素抗性水平下降;2 个突变体抗性水平基本
不变。CAT-ELISA 检测的结果,也进一步证实了氯霉素抗性水平的变化与 CAT
酶活力的变化有着密切的关系。在启动子 F1 的 DNA 序列上,存在着不同插入
效应敏感位点,在这些位点附近的插入突变,可使启动活性急剧变化,并且相邻
的插入位点,其突变效应可能完全不同。FACS 分析表明,在大肠杆菌中表现为
启动活性增强的 F1 突变体,转入短小芽孢杆菌 DX01 后其活性仍表现为增强。
采用 RT-PCR 方法,克隆了萝卜抗真菌蛋白 1(Rs-AFP1)的 cDNA 全长,在该
VI
基因的上游连上了一个短小芽孢杆菌 47 株的中壁蛋白(MWP)信号肽,将 Rs-afp1
基因的终止密码子突变掉,其后接上绿色荧光蛋白(GFP)基因和 T7 终止子,分
别构建成AFP-GFP融合蛋白表达载体pUC118-F1SP' 和pHY300-F1SP' 。
Agfp Agfp
Western 杂交证实融合蛋白在大肠杆菌 DH5α 中得到了表达。
[附录部分]
杂交墨西哥落羽杉是我国已故著名林木育种家叶培忠教授于 1963 年通过墨
西哥落羽杉(母本)与柳杉(父本)杂交所获得的新种。目前,其主要分布在上海、
江苏等地。杂交墨杉苗期长势不突出,早年并未引起林木工作者的注意,故目前
这些杂交墨西哥落羽杉的分布情况已很难从原始档案中查找到完整的资料。为
此,采用随机引物扩增多态性 DNA(RAPD)技术,对 8 个可能是杂交墨西哥落羽
杉片林样本、其杂交母本?墨西哥落羽杉和杂交父本的同类?柳杉,共计 12 个
样本进行了亲缘关系鉴定和遗传多样性分析。从 31 个随机引物中筛选出 17 个引
物,共扩增出 164 条带。其中,多态带有 162 条,占全部条带的 98.8 %。检测结
果表明:在 8 号、11 号、12 号 3 个柳杉样本中,11 号样本与原杂交父本的亲缘
关系最近;1 号、4 号和 9 号三个片林样本是杂交墨杉群落的可能性最大;5 号
片林样本可能是未杂交的墨杉纯林;其他群落的样本是否为杂交墨杉尚不能通过
该实验得出明确结论。
The green fluorescent protein (GFP) of the jellyfish Aequorea Victoria is a useful
reporter molecule for monitoring gene expression in vivo in eukaryotic and
prokaryotic cells. A GFP vector was constructed for in situ detection of the rice
epiphyte Bacillus brevis strain DX01. The promoterless gfp-S65T gene was
transcriptionally fused to a strong B. brevis promoter pCP01 functional F1 fragment,
which was subcloned from the genome of strain DX01 by PCR technique, and then
respectively inserted into plasmid pUC118 and an Escherichia coli-Bacillus shuttle
vector pHY300PLK leading to result in expression vectors pUC118-F1gfp and
pHY300-F1gfp. The DX01 cells harboring the plasmid pHY300-F1gfp could produce
bright green fluorescence when they were examined by using fluorescent microscopy.
The results were confirmed by western blot analysis. The transcription from F1
remained constitutive although it was 280 bp shorter than in promoter pCP01.
GFP-expressing cells were also sorted by fluorescence-activated cell sorting (FACS)
analysis. The fluorescence intensity of cell populations of DX01::pHY300-F1gfp was
two-fold higher than that of DH5a::pUC118-F1gfp, which implied that the GFP
expression vector pHY300-F1gfp was highly efficient in B. brevis DX01. Though F1
showed no host cell specificity, it displayed higher transcriptional activity in B. brevis
DX01 than in E. coli DH5 a.
To improve the activity of F1, insertion mutagenesis of F1 based on in vitro
transposition reaction was performed. 227 insertion clones were screened from an
insertion library, 13 mutants were identified and sequenced. Their MIC of Cam and
CAT synthesis were assayed. Seven mutants with enhanced transcription activity in E.
coli DH5α were obtained. The MICs of Cam of Fm3 and Fm10 were 900 μg/mL,
which is 2.6 fold higher than that of the control. Four mutants showed lower MIC of
Cam, and two kept unaltered. In general, the MIC of Cam was positively related to
CAT activity in E. coli DH5α cells. The insertions in DNA sequence of promoter F1
resulted in different effects. Remarkably, the insertions in the neighbor sites led to
diametrically opposing effects. The results of FACS analysis confirmed that the
enhanced promoters showed similar high activities in B. brevis strain DX01.
The anti-fungal protein 1 (Rs-AFP1) cDNA was derived from radish cultivar
“Chuanxinhong” by reverse transcription PCR, and then the promoter F1 and the
VIII
middle-wall-protein signal peptide of B. brevis strain 47 were fused to its 5'end in a
correct orientation respectively. Subsequently, the terminator codon of the Rs-afp1
gene was mutated and a T7 terminator was fused to its 3' resulting in the two
end
expression vectors pUC118-F1SP' Agfp and pHY300-F1SP' Agfp. The fused protein
was confirmed expressed in E. coli DH5α by Western blot analysis.
RAPD analyses were conducted on genetic polymorphisms of twelve fir genotypes
to identify their relationships. These genotypes included eight supposed hybrid
Taxodium mucronatum Tenore forest samples and the hybrid female-parent?T.
mucronatum Tenore and the same class of hybrid male-parent? Cryptomeria fortunei
Hooibrenk and sugi C. Japonica(L.f.) D.Don. 17 of 31 primers were selected to use
for separate amplification reactions with DNA from 12 genotypes. 164 RAPD
fragments were produced and 98.8 % of them showed polymorphism. The results
revealed that the genetic relationship of sample No.11 in three Cryptomeria genotypes
is the closest to the original male-parent; the samples No.1,4 and 9 are most possibly
the true hybrid T. mucronatum Tenore populations; the sample No.5 may be the false
hybrid. Conclusions could not be drawn from the data in hand for the rest samples.
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