慢生型大豆根瘤菌22-10春雷霉素敏感基因（ksgA）的研究
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摘要
本研究做了二方面的工作：
一方面利用Tn5gusA5随机诱变方法，对慢生型大豆根瘤菌22—10的染色体进行了插入诱变，筛选到抗春雷霉素(ksg)突变体6株。根据已知的Tn5gusA5序列，设计引物，经PCR检测，结果表明突变体染色体DNA上并无Tn5gusA5的插入，推测抗春雷霉素突变株为自发突变。
另一方面，从NCBl Genebank Blast中查到根癌农杆菌(Agrobacterium tumefaciens)、苜蓿中华根瘤菌(Sinorhizobium meliliti)、百脉根中慢生根瘤菌(Mesorhizobium loti)的春雷霉素敏感基因(ksgA)序列，经alignment(对齐)分析，选择高度保守区，设计引物。以慢生型大豆根瘤(B．japonicum)22-10的总DNA为模板，扩增出长度为520bp的DNA片段。通过NCBl Gene Bank Blast分析，表明它与已发表的Agrobacterium、Mesorhizobium和Sinorhizobium的ksgA基因具有相当高的一致性，说明扩增的520bp产物是慢生型大豆根瘤菌22-10的KsgA基因的部分片段。然后利用Tail-PCR方法扩增出520bp片段的两侧，获得一长度为1598bp的DNA片段，其内含一个完整的ORF(开放阅读框架)，该ORF长度为858bp，命名为B．jksgA。Blastx分析结果表明其氨基酸一级结构与已报道的苜蓿中华根瘤菌(Sinorhizobium meliloti)的ksgA基因的氨基酸同源性高达69％。初步认为已扩增的858bp的ORF是慢生型大豆根瘤菌22-10的ksgA基因。
根据已知的含有B．jksgA基因的1598bp DNA序列，设计引物POR扩增B．jkseA基因的ORF的两侧序列，左侧为395bp，右侧为345bp。同时从质粒载体上PCR扩增Km~r基因的ORF。将左侧395bp、Km~r基因的ORF、右侧345bp混合后，加入左侧片段的正向引物和右侧片段的反向引物进行PCR连接，获得B．jksgA基因缺失结构体(LksgA)。将LksgA结构体连接到plJ3200上，得到重组质粒plJ32LksgA，电脉冲转化法转化B．japonicum 22—10，以进行同源双交换，利用plJ3200(pLAFR系列)与pPH1Jl质粒的不相容性，赶走plJ32LkssA质粒，选择Rif抗性、Km抗性及春雷霉素抗性(Ksg~r)，获得B．jksgA基因置换突变体4株，PCR验证结果表明4株突变体的ksgA基因已被LksgA结构体定点置换。从而进一步证实了我们扩增到的B．jksgA基因是慢生型大豆根瘤菌22-10的春雷霉素敏感基因(ksgA)。
In this study ,there are two aspects:
On one hand, the TnSgusAS insertion mutagenesis of chromosome of Bradyrhizobium japonicum 22-10 were obtained by TnSgusAS arbitary mutagenesis, and six kasugamycin resistant mutants have been screened. And oligonucleotied primers were designed according to the sequence of Tn5, then used PCR to identify with TnSgusAS insertion of chromosome. The result shows that there is no TnS insertion in chromosome, and implicates that kasugamycin resistant mutant is autegenic mutation.
On the other hand, the sequence of Methyltransferase gene (ksgA) which dimethylates adjacent adenines near the 3 ' end of 16S rRNA about Agrobacterium tumefaciens; Sinorhizobium meliloti and Mesorhizobium loti were downloaded from NCBI Gene Bank Blast, and primers were designed in their conserved regions by alignment analysis to amplify fragment of ksgA gene from total DNA of B.japonicum22-10. DNA sequence shows that the segment consist of 520bp. NCBI Gene Blast analysis shows that this 520bp segment share high homology with known ksgA gene of Sinorhizobium meliloti. Then used Tail-PCR method to amplify sequence flanking the 520bp fragment, and obtained a 1598bp fragment which contain a complete open reading frame (ORF) and consist of 858bp named B.jksgA gene. Blastx analysis shows that B.jksgA gene share 69% % 69% and 65% homology at the amino acid level compared with reported ksgA gene of S.meliloti M.lito and A.tumefaciens.
According to 1598bp DNA sequence which contain B.jksgA gene, primers were designed to amplify sequence flanking B.jksgA ORF, which left flanking sequence is 395bp, and right is 345bp. At same time used ORF of
Km resistance gene to replace the ORF of ksgA gene, then B.jksgA gene deletion constructs (LksgA) have been obtained. The LksgA construct was ligated into multipurpose broad host rang cloning vector pIJ3200, and recombinant plasmid pIJ32LksgA was constructed. Recombinant plasmid was transformed Bradyrhizobium japonicum 22-10 with electroporation method, then double homogenous exchange occurred in with incompatible plasmid pPHUI. And obtained four B.jksgA gene replacement mutants through selecting Rif resistance, Km resistance and Kasugamycin resistance. PCR result shows that the ksgA gene of four mutant is just replaced by LksgA construct. It has proved that B.jksgA gene is the kasugamycin sensitivity gene(ksgA) of B.japonicum22-10.
一方面利用Tn5gusA5随机诱变方法，对慢生型大豆根瘤菌22—10的染色体进行了插入诱变，筛选到抗春雷霉素(ksg)突变体6株。根据已知的Tn5gusA5序列，设计引物，经PCR检测，结果表明突变体染色体DNA上并无Tn5gusA5的插入，推测抗春雷霉素突变株为自发突变。
另一方面，从NCBl Genebank Blast中查到根癌农杆菌(Agrobacterium tumefaciens)、苜蓿中华根瘤菌(Sinorhizobium meliliti)、百脉根中慢生根瘤菌(Mesorhizobium loti)的春雷霉素敏感基因(ksgA)序列，经alignment(对齐)分析，选择高度保守区，设计引物。以慢生型大豆根瘤(B．japonicum)22-10的总DNA为模板，扩增出长度为520bp的DNA片段。通过NCBl Gene Bank Blast分析，表明它与已发表的Agrobacterium、Mesorhizobium和Sinorhizobium的ksgA基因具有相当高的一致性，说明扩增的520bp产物是慢生型大豆根瘤菌22-10的KsgA基因的部分片段。然后利用Tail-PCR方法扩增出520bp片段的两侧，获得一长度为1598bp的DNA片段，其内含一个完整的ORF(开放阅读框架)，该ORF长度为858bp，命名为B．jksgA。Blastx分析结果表明其氨基酸一级结构与已报道的苜蓿中华根瘤菌(Sinorhizobium meliloti)的ksgA基因的氨基酸同源性高达69％。初步认为已扩增的858bp的ORF是慢生型大豆根瘤菌22-10的ksgA基因。
根据已知的含有B．jksgA基因的1598bp DNA序列，设计引物POR扩增B．jkseA基因的ORF的两侧序列，左侧为395bp，右侧为345bp。同时从质粒载体上PCR扩增Km~r基因的ORF。将左侧395bp、Km~r基因的ORF、右侧345bp混合后，加入左侧片段的正向引物和右侧片段的反向引物进行PCR连接，获得B．jksgA基因缺失结构体(LksgA)。将LksgA结构体连接到plJ3200上，得到重组质粒plJ32LksgA，电脉冲转化法转化B．japonicum 22—10，以进行同源双交换，利用plJ3200(pLAFR系列)与pPH1Jl质粒的不相容性，赶走plJ32LkssA质粒，选择Rif抗性、Km抗性及春雷霉素抗性(Ksg~r)，获得B．jksgA基因置换突变体4株，PCR验证结果表明4株突变体的ksgA基因已被LksgA结构体定点置换。从而进一步证实了我们扩增到的B．jksgA基因是慢生型大豆根瘤菌22-10的春雷霉素敏感基因(ksgA)。
In this study ,there are two aspects:
On one hand, the TnSgusAS insertion mutagenesis of chromosome of Bradyrhizobium japonicum 22-10 were obtained by TnSgusAS arbitary mutagenesis, and six kasugamycin resistant mutants have been screened. And oligonucleotied primers were designed according to the sequence of Tn5, then used PCR to identify with TnSgusAS insertion of chromosome. The result shows that there is no TnS insertion in chromosome, and implicates that kasugamycin resistant mutant is autegenic mutation.
On the other hand, the sequence of Methyltransferase gene (ksgA) which dimethylates adjacent adenines near the 3 ' end of 16S rRNA about Agrobacterium tumefaciens; Sinorhizobium meliloti and Mesorhizobium loti were downloaded from NCBI Gene Bank Blast, and primers were designed in their conserved regions by alignment analysis to amplify fragment of ksgA gene from total DNA of B.japonicum22-10. DNA sequence shows that the segment consist of 520bp. NCBI Gene Blast analysis shows that this 520bp segment share high homology with known ksgA gene of Sinorhizobium meliloti. Then used Tail-PCR method to amplify sequence flanking the 520bp fragment, and obtained a 1598bp fragment which contain a complete open reading frame (ORF) and consist of 858bp named B.jksgA gene. Blastx analysis shows that B.jksgA gene share 69% % 69% and 65% homology at the amino acid level compared with reported ksgA gene of S.meliloti M.lito and A.tumefaciens.
According to 1598bp DNA sequence which contain B.jksgA gene, primers were designed to amplify sequence flanking B.jksgA ORF, which left flanking sequence is 395bp, and right is 345bp. At same time used ORF of
Km resistance gene to replace the ORF of ksgA gene, then B.jksgA gene deletion constructs (LksgA) have been obtained. The LksgA construct was ligated into multipurpose broad host rang cloning vector pIJ3200, and recombinant plasmid pIJ32LksgA was constructed. Recombinant plasmid was transformed Bradyrhizobium japonicum 22-10 with electroporation method, then double homogenous exchange occurred in with incompatible plasmid pPHUI. And obtained four B.jksgA gene replacement mutants through selecting Rif resistance, Km resistance and Kasugamycin resistance. PCR result shows that the ksgA gene of four mutant is just replaced by LksgA construct. It has proved that B.jksgA gene is the kasugamycin sensitivity gene(ksgA) of B.japonicum22-10.
引文
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[2] Benjamin Lewin, Genes Ⅱ. 1985, John Wiley & Sons.
[3] Cundliffe E. Antibiotic inhibitors of ribosome. The molecular basis of antibiotic action. 1990． znd edit, John Wiley and Sons, New York
[4] Sigmund.C.D, Ettayebi. M, Borden.A, et al. Antibiotic resistance mutations in ribosomal RNA genes of Escherichia coli.Methods Enzymel. 1988, 164:673-690．
[5] Cuudliffe. E, Recoglution sies for antibiotics within rRNA. The ribosome:structure function evolution. 1990． American society for microbiolog, Washington DC.
[6] Ishiyama,T.et al. 1965, J.Antibiotics ser.A. 18:116-119．
[7] Tanaka .N, et al. 1965, J.Antibiotics ser. 18:139-144．
[8] Masukawa.H,et al. 1968, J.Mol.Biol.28:161-167
[9] Okugama A,.et al. Biochem.Biophys.Res.commun. 1971, 43:196-199
[10] Okuyama A,et al. J.Antibiotics, 1972, 25:212-218
[11] Helser T.L, et al.1971, Natare New Biol 12:233
[12] Helser T.L,Davies JE, Dahlberg.JE, Mechanism of kasugamgcin resistanle in Escherichia coli. Natare New Biology. 1972, 235(5) :6-9
[13] Okugama A, et al.1975． The binding of kasugamycin to the Eschierichia coli ribosomes. 1975, J.Antibiotics,28:903-905．
[14] Fouts KE, Barbour SD, Transductional mapping of ksg B and a new Tn5-induced kasugamycin resistance gene ksgD in Escherichia coli K-12 . J. Bacterio.1981, 145(2) :914-919
[15] Van Buul CPJJ, Van Knippenberg PH. Nucleotide sequence of the ksgA gene of Escherichia coli comparison of methyltransferases. Effecting dimethylation of adenosine in ribosomal RNA. Gene. 1985, 38:65-72．
[16] Van Buul CP, Hamersma M, Visser W, et al. Partial methylationn of two adjacent adenosines in ribosome from Euglena gracilis chloroplasts suggests evolutionary loss of an intermediate stage in the methlytranfer reaction.Nucleic Acids Res. 1984, 12(23) :9205-9208
[17] RoaBB, Connolly DM, Winkler ME. Overlap between pdxA and kgA in the complex pdxA-ksgA-apaG-apaH operon of Escherichia coli K-12． J.Bacteriol, 1989, 171(9) : 4767-4777．
[18] Leveque F.S, Blanchin R, Fagat G, et al. Design and characterization of Escherichia coli mutants devoid of Ap4N-hydrolase activity. J. Mol. Biol. 1990(212) :319-329．
[19] Hokko-Chem, patent. 1993, JP, 5023187．
[20] 樊妙姬，马庆生．根瘤菌竞争结瘤的研究进展．微生物学通报. 1996． 23(6) : 360-363．
[21] Anton V.S, Catherine L.S and Albert E. D. Isolation of kasugamycin Resistant Mutants in the 16S Ribosomal RNA of Escherichia coli. J. Mol. Biol. 1999, 293:1-8
[22] Anton V.S, Alber E.D. Mutational analysis of the conserved bases C1402 and A1500 in the center of the decoding domain of Escherichia coli 16SrRNA reveals an important tertiary interaction. J.Mol.Biol. 2001, 308:457-463．
[23] Pease A.J, Roa BB, Luo W. Positive growth rate-dependent regulation of the pdxA. kgA, and pdxB genes of Escherchia coli K-12． J. Bacterial. 2002,184(5) : 1359-1369．
[24] Capcla D, Barloy-Hubbler F, Gouzy J. Analysis of the chromosome sequence of the legume symbiont. Sinorhizobium meloti. strain 1021. Proc. Natl. Acad. Sci. U.S.A. 2001, 98(17): 9877-9882.
[25] Galibert F, Finan T.M, Long S.R. The composite genome of the legume symbiot. Sinorhizobium meloti. Sience. 2001, 293(5530): 668～672.
[26] Wood D.W, Setubal J.C, Kcul R. The Genome of the Nature Genetic Engineer Agro bacterium tumefaciens C58. Science, 2001, 294(5550), 2317-2323.
[27] Kaneko T, Nakamura Y, Sato S. Complete genome structure of the nitrogen-fixing symbiotic bacterium Mesorhizobium loti. DNA Res. 2000, 7(6):331-338.
[28] 樊妙姬，刘涛，陈荣基等．抗春雷霉素大豆根瘤菌突变株诱变及其生物固氮特性的分析．广西农业大学学报．1996，15(4)：285—290．
[29] Isoi T, Yoshida S. Growth nodulation and nitrogen fixation of soybean plants with seeds treated with Kasugamycin. Soil Sci. Plant Nutr. 1990, 36(2): 283-288.
[30] Hanahan D. Studies on tranformation of Escherichia coli with plasmid. J. Mol. Biol. 1983, 166:557-580.
[31] Friednan A M, Long SR, Brow SE, et al. Construction of broad host rangecosmid cloning vector and its use in the genetic analysis of Rhizobium mutants.Gene, 1982, 18:289-296.
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