根癌农杆菌vbp2基因启动子转录调控的探析

doi:10.13560/j.cnki.biotech.bull.1985.2021-0151

摘要/Abstract

摘要：

根癌农杆菌（Agrobacterium tumefaciens）能够将自身质粒上的部分DNA片段（简称T-DNA）以T-复合物的形式转运、整合至宿主基因组,而用作高效的植物转基因工具。vbp2是编码VBP蛋白的3个同源基因之一,VBP蛋白通过与T-复合物上的VirD2结合参与T-DNA的转运过程。根据生物信息学预测,vbp2基因的启动子区可能含有多个转录调控元件。通过同源重组的方法,将A. tumefaciens染色体上的vbp2基因替换成编码红色荧光蛋白的rfp基因,使vbp2的启动子控制rfp的表达,因此,可通过测定所表达的RFP的荧光强度来表征vbp2启动子的活性。结果表明:乙酰丁香酮（AS）和鼠李糖（Rha）均能诱导vbp2启动子表达RFP,AS和Rha的最佳诱导浓度分别为150 μmol/L和100 μmol/L。通过截短vbp2启动子DNA片段的方法,证明响应AS和Rha诱导的调控区域分别位于上游的165-260 bp和126-165 bp。

关键词: 根癌农杆菌, vbp2基因, 转录调控, 转录活性, 荧光蛋白

Abstract:

Agrobacterium tumefaciens is able to transfer DNA fragments（T-DNA）from its plasmid to plant cells in the form of T-complex and to integrate the T-DNA into the genome of host plants,thereby it has been used as an effective transgenic vector for plant. vbp2 is one of the three homologous genes encoding VBPs,and VBPs are involved in the transport of T-DNA by binding with the VirD2 in the T-complex. Bioinformatics analysis showed that there might be several transcriptional regulation elements in the promoter region of vpb2 gene. The vbp2 in the chromosome of A. tumefaciens was in situ replaced by the rfp encoding red fluoresce protein（RFP）using homologous recombination method,so that promoter of vbp2 controlled the expression of rfp. Therefore,the activity of vbp2 promoter can be characterized by the fluorescence intensity of the expressed RFP. The results showed that acetosyringone（AS）and rhamnose（Rha）both were able to induce the vbp2 promoter to expresse RFP. The optimal concentrations of AS and Rha for the induction were 150 μmol/L and 100 μmol/L,respectively. The test of the transcriptional activity of the truncated vbp2 promoter regions revealed that transcriptional regulation elements induced by AS and Rha were located on the regions of 165-260 bp and 126-165 bp upstream of the vbp2 gene,respectively.

Key words: Agrobacterium tumefaciens, vbp2 gene, transcriptional regulation, transcriptional activity, fluorescence protein

徐楠, 徐宇娟, 孙盼, 宗仁杰, 郭敏亮. 根癌农杆菌vbp2基因启动子转录调控的探析[J]. 生物技术通报, 2021, 37(12): 41-49.

XU Nan, XU Yu-juan, SUN Pan, ZONG Ren-jie, GUO Min-liang. Exploration of the Transcriptional Regulation of Agrobacterium tumefaciens vbp2 Promoter[J]. Biotechnology Bulletin, 2021, 37(12): 41-49.

图/表 7

表1 本研究所使用的引物

Table 1 Primers used in this study

引物 Primer	序列 Sequence（5' - 3'）	酶切位点 Restriction endonuclease site	用途 Purpose
Q1-F	TGTGGATCCAACGTCCGCTGCGCGA	BamH I	扩增vbp2上游526 bp片段 Amplifying upstream 526 bp segment of vbp2
Q1-R	AGCTCCTCGCCCTTGCTCACCATGGGAACGAGGTATCTGACTA	无None
Rfp-F	CGAAACGAAATAGGTGTACCCATCTGCCAC	无None	扩增rfp基因 Amplifying gene rfp
Rfp-R	CATGAGCGACGGGAATTCGCCTATCTGTGCCCCAGT	EcoR I	扩增rfp基因 Amplifying gene rfp
H-F	CTGGGGCACAGATAGCCGCGAGGTGTCAAAGCA	无None	扩增vbp2下游1 kb片段 Amplifying downstream 1 kb segment of vbp2
H-R	GAAGCTTATTGCGCTCGGGCCCAGTGGCACGG	Hind III
P-F	CCCAAGCTTGGGAGAGGCGGTTTGCGTATTGGGC	Hind III	扩增去除pUCA-19质粒的lacZ’启动子 Amplifying lacZ’ promoter with pUCA-19 plasmid removed
P-R	CCCAAGCTTACAGGAAACAGCTATGACCATGATT	Hind III
Egfp-F	TAGTCAGATACCTCGTTCCCATGGTGAGCAAGGGCGAGGA	无None	扩增gfp序列 Amplifying gfp sequence
Egfp-R	CCCAAGCTTTTACTTGTACAGCTCGTCCATGCC	Hind III	扩增gfp序列 Amplifying gfp sequence
Q2-F	CGCGGATCCCGATCCAGTTATAATGAATTTGC	Hind III	与Q1-R扩增vbp2上游463 bp序列 Amplifying upstream 463 bp sequence of vbp2 with Q1-R
Q3-F	CGCGGATCCAAGTAGTAGTAAGGTCGATCG	Hind III	与Q1-R扩增vbp2上游260 bp序列 Amplifying upstream 260 bp sequence of vbp2 with Q1-R
Q4-F	ATCAGGATCCGGCAGCGTGTTTATCAGG	Hind III	与Q1-R扩增vbp2上游165 bp序列 Amplifying upstream 165 bp sequence of vbp2 with Q1-R
Q5-F	CGCGGATCCGACCTACAAAATTCGACTAGCAC	Hind III	与Q1-R扩增vbp2上游126 bp序列 Amplifying upstream 126 bp sequence of vbp2 with Q1-R
Q6-F	CGCGGATCCGGCATTTGATCAACGTGTCG	Hind III	与Q1-R扩增vbp2上游97 bp序列 Amplifying upstream 97 bp sequence of vbp2 with Q1-R
Q7-F	CGCGGATCCATACCACCAGCGCGATTGC	Hind III	与Q1-R扩增vbp2上游60 bp序列 Amplifying upstream 60 bp sequence of vbp2 with Q1-R
Q8-F	CGCGGATCCTAGTCAGATACCTCGTTCCC	Hind III	与Q1-R扩增vbp2上游20 bp序列 Amplifying upstream 20 bp sequence of vbp2 with Q1-R

表1 本研究所使用的引物

Table 1 Primers used in this study

引物 Primer	序列 Sequence（5' - 3'）	酶切位点 Restriction endonuclease site	用途 Purpose
Q1-F	TGTGGATCCAACGTCCGCTGCGCGA	BamH I	扩增vbp2上游526 bp片段 Amplifying upstream 526 bp segment of vbp2
Q1-R	AGCTCCTCGCCCTTGCTCACCATGGGAACGAGGTATCTGACTA	无None
Rfp-F	CGAAACGAAATAGGTGTACCCATCTGCCAC	无None	扩增rfp基因 Amplifying gene rfp
Rfp-R	CATGAGCGACGGGAATTCGCCTATCTGTGCCCCAGT	EcoR I	扩增rfp基因 Amplifying gene rfp
H-F	CTGGGGCACAGATAGCCGCGAGGTGTCAAAGCA	无None	扩增vbp2下游1 kb片段 Amplifying downstream 1 kb segment of vbp2
H-R	GAAGCTTATTGCGCTCGGGCCCAGTGGCACGG	Hind III
P-F	CCCAAGCTTGGGAGAGGCGGTTTGCGTATTGGGC	Hind III	扩增去除pUCA-19质粒的lacZ’启动子 Amplifying lacZ’ promoter with pUCA-19 plasmid removed
P-R	CCCAAGCTTACAGGAAACAGCTATGACCATGATT	Hind III
Egfp-F	TAGTCAGATACCTCGTTCCCATGGTGAGCAAGGGCGAGGA	无None	扩增gfp序列 Amplifying gfp sequence
Egfp-R	CCCAAGCTTTTACTTGTACAGCTCGTCCATGCC	Hind III	扩增gfp序列 Amplifying gfp sequence
Q2-F	CGCGGATCCCGATCCAGTTATAATGAATTTGC	Hind III	与Q1-R扩增vbp2上游463 bp序列 Amplifying upstream 463 bp sequence of vbp2 with Q1-R
Q3-F	CGCGGATCCAAGTAGTAGTAAGGTCGATCG	Hind III	与Q1-R扩增vbp2上游260 bp序列 Amplifying upstream 260 bp sequence of vbp2 with Q1-R
Q4-F	ATCAGGATCCGGCAGCGTGTTTATCAGG	Hind III	与Q1-R扩增vbp2上游165 bp序列 Amplifying upstream 165 bp sequence of vbp2 with Q1-R
Q5-F	CGCGGATCCGACCTACAAAATTCGACTAGCAC	Hind III	与Q1-R扩增vbp2上游126 bp序列 Amplifying upstream 126 bp sequence of vbp2 with Q1-R
Q6-F	CGCGGATCCGGCATTTGATCAACGTGTCG	Hind III	与Q1-R扩增vbp2上游97 bp序列 Amplifying upstream 97 bp sequence of vbp2 with Q1-R
Q7-F	CGCGGATCCATACCACCAGCGCGATTGC	Hind III	与Q1-R扩增vbp2上游60 bp序列 Amplifying upstream 60 bp sequence of vbp2 with Q1-R
Q8-F	CGCGGATCCTAGTCAGATACCTCGTTCCC	Hind III	与Q1-R扩增vbp2上游20 bp序列 Amplifying upstream 20 bp sequence of vbp2 with Q1-R

图1 生物信息学预测的vbp2基因启动子区可能存在的转录调控元件

Fig.1 Feasible transcriptional regulator in the promoter region of vbp2 gene predicted by bioinformatics

图2 用于进行同源重组构建根癌农杆菌突变体的质粒pEX18Km-rfp的鉴定 A:用构建质粒时所有的限制性内切酶水解质粒所得片段大小;B:用插入片段两端的引物从细菌中扩增得到的PCR产物

Fig.2 Identification of plasmid pEX18Km-rfp used for the construction of A. tumefaciens mutant via homologous recombination A:DNA fragments of the plasmid cleaved by the restriction endonuclease. B:The PCR products amplified from bacteria by primers inserted into the 2 ends of the fragment

图3 根癌农杆菌粗提液中RFP的含量与粗提液的荧光强度的关系

Fig.3 Relationship between fluorescent intensity and RFP content in A. tumefaciens crude extract

图4 不同浓度的乙酰丁香酮（A）和L-鼠李糖（B）诱导vbp2启动子表达RFP的情况

Fig. 4 vbp2 promotor-promoting RFP expressions induced by different concentrations of AS（A）or Rha（B）

图5 受不同长度的vbp2启动子片段控制的egfp表达质粒的构建和鉴定。 A:被截短的vbp2启动子片段的长度、位置及可能存在的调控元件;B:用PCR鉴定这些携带受不同长度的vbp2启动子片段控制的egfp表达质粒的根癌农杆菌菌株

Fig.5 Construction and identification of the egfp expression plasmids controlled by different lengths of vbp2 promotor A:The length,region and possible regulatory elements of the truncated vbp2 promotor fragment. B:PCR-identified A. tumefaciens strains that carry plasmids expressing egfp under the control of different lengths of vbp2 promotor

图6 分别以150 μmol/L AS（A）和100 μmol/L Rha（B）诱导不同长度vbp2启动子表达GFP的情况

Fig. 6 GFP expression promoted by different lengths of vbp2 promotor under the induction of 150 μmol/L AS（A）or 100 μmol/L Rha（B）

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