慢生型大豆根瘤菌GX201结瘤效率基因的分子遗传学研究
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摘要
本研究首先利用转座子Tn5gusA5对重组质粒pGXN201进行诱变，获得3.4kbEcoRI片段插入了Tn5gusA5的突变质粒pGXN215、pGXN216、pGXN217。对这些突变质粒进行亚克隆及测序分析并与基因库中已报道的慢生型大豆根瘤菌的DNA序列作比较，发现突变质粒pGXN217中Tn5gusA5恰好插入在一个未知功能的开放阅读框架内。将pGXN201的3.4kb EcoRI片段与M13作连接，获得亚克隆pGXN201C，对pGXN201C进行测序分析，发现与基因库中已报道的慢生型大豆根瘤菌的DNA序列有95％的同源性。随后，利用突变质粒pGXN217诱变慢生型大豆根瘤菌菌株GX201，获得了GX201的突变体菌株GX217；将pGXN201的3.4kbEcoRI片段与pLARF3连接获得亚克隆pGXN201cl，然后将pGXN201cl导入突变体菌株GX217，构建了GX217的功能互补菌株rGX217。
通过对野生型菌株GX201，突变体菌株GX217及其功能互补菌株rGX217进行植株试验，发现突变体菌株GX217在结瘤时间方面较野生型菌株GX201慢一天，其结瘤效率及竞争结瘤能力均比GX201明显降低。我们认为在突变体菌株GX217中Tn5gusA5所插入的开放阅读框架是一个新的与竞争结瘤有关的基因。
In this study, a recombinant plasmid pGXN201 was first mutated by using of transposon Tn5gusA5 mutagenesis, and three mutant plasmids , pGXN215, pGXN216 and pGXN217 were achieved. The 3.4kb EcoRI DNA fragment of the pGXN201 and the mutant EcoRI DNA fragments of the three mutant plasmids were subcloned into the EcoRI sites of the clone vectors Ml3 and the pGEM3Z-f(+) respectively.Through DNA sequencing and DNA sequence analysis by comparing with the Bradyrhizobium Japonicum DNA sequences reported, we found that there are four intact Open Reading Frames in the 3.4kb fragment and it was highly homologous to the Bradyrhizobium Japonicum DNA sequence reported, and the Tn5gusA5 containing in the pGXN217 was inserted in one of the four Open Reading Frames .This Open Reading Frame located on about 5.5kb upstream of the nfeC gene, which includes 207bp nucleotides, coding 68 putative amino acids and this is completely identical to that of the Bradyrhizobium Japonicum DNA sequence reported.
pGXN217 was transferred into Bradyrhizobium Japonicum strain GX201 by triparental mating using the helper plasmid pRK2073. Marker exchange was achieved by selection on YMA containing Sm, Km ,Gm, Spc et al four kinds of antibiotics. Mutants were confirmed by southern blotting and hybridization with the wild-type region and the Tn5 fragment respectively. The mutant strain GX217 was achieved. The 3.4kb EcoRI fragment of the plasmid pGXN201 was firstly subcloned into the EcoRI site of the plARF3, then the recombinant plasmid was introduced into the mutant strain GX217 by triparental mating using the helper pRK2073 and the mutant strain rGX217 was achieved.
Plant assay was performed with the wild-type strain GX201,the mutant strain GX217 and the function recovered strain rGX217.The results indicate that the mutant strain GX217 showed a day delay in nodulation time and its abilities of nodule formation efficiency and competitive nodulation were apparently decreased compared with the parental strain GX201 respectively. We conclude that the gene we studied is a new loci required for efficient nitrogen fixation and for competitive nodulation of soybeans by Bradyrhizobium Japonicum strain GX201.
通过对野生型菌株GX201，突变体菌株GX217及其功能互补菌株rGX217进行植株试验，发现突变体菌株GX217在结瘤时间方面较野生型菌株GX201慢一天，其结瘤效率及竞争结瘤能力均比GX201明显降低。我们认为在突变体菌株GX217中Tn5gusA5所插入的开放阅读框架是一个新的与竞争结瘤有关的基因。
In this study, a recombinant plasmid pGXN201 was first mutated by using of transposon Tn5gusA5 mutagenesis, and three mutant plasmids , pGXN215, pGXN216 and pGXN217 were achieved. The 3.4kb EcoRI DNA fragment of the pGXN201 and the mutant EcoRI DNA fragments of the three mutant plasmids were subcloned into the EcoRI sites of the clone vectors Ml3 and the pGEM3Z-f(+) respectively.Through DNA sequencing and DNA sequence analysis by comparing with the Bradyrhizobium Japonicum DNA sequences reported, we found that there are four intact Open Reading Frames in the 3.4kb fragment and it was highly homologous to the Bradyrhizobium Japonicum DNA sequence reported, and the Tn5gusA5 containing in the pGXN217 was inserted in one of the four Open Reading Frames .This Open Reading Frame located on about 5.5kb upstream of the nfeC gene, which includes 207bp nucleotides, coding 68 putative amino acids and this is completely identical to that of the Bradyrhizobium Japonicum DNA sequence reported.
pGXN217 was transferred into Bradyrhizobium Japonicum strain GX201 by triparental mating using the helper plasmid pRK2073. Marker exchange was achieved by selection on YMA containing Sm, Km ,Gm, Spc et al four kinds of antibiotics. Mutants were confirmed by southern blotting and hybridization with the wild-type region and the Tn5 fragment respectively. The mutant strain GX217 was achieved. The 3.4kb EcoRI fragment of the plasmid pGXN201 was firstly subcloned into the EcoRI site of the plARF3, then the recombinant plasmid was introduced into the mutant strain GX217 by triparental mating using the helper pRK2073 and the mutant strain rGX217 was achieved.
Plant assay was performed with the wild-type strain GX201,the mutant strain GX217 and the function recovered strain rGX217.The results indicate that the mutant strain GX217 showed a day delay in nodulation time and its abilities of nodule formation efficiency and competitive nodulation were apparently decreased compared with the parental strain GX201 respectively. We conclude that the gene we studied is a new loci required for efficient nitrogen fixation and for competitive nodulation of soybeans by Bradyrhizobium Japonicum strain GX201.
引文
1、曾定，《固氮生物学》，厦门大学出版社，1987。
2、樊庆笙，《固氮微生物学》，农业出版社，1993。
3、洪国藩、宋鸿遇，《固氮之光》，湖南科学技术出版社，1997。
4、陈因等，《生物固氮》，上海科学技术出版社，1985。
5、尤崇杓等，《生物固氮》，科学出版社，1987。
6、黄大昉、林敏主编，《农业微生物基因工程》，科学出版社，2001。
7、马庆生、武波、唐东阶等，外源 nfeC基因导入快生型大豆根瘤菌菌株HN01的行为分析，复旦学报(自然科学版)，1998，37(4)：445～448。
8、周刚林、武波、唐东阶等，慢生型大豆根瘤菌nfe基因的分子克隆，广西农业生物科学，1999，18(1)：29～32。
9、武波、周刚林、龙章德等，慢生型大豆根瘤菌nfeC基因的定位，广西农业生物科学，2001，20(2)：89～92。
10、武波、周刚林、龙章德等，慢生型大豆根瘤nfeC基因的克隆及序列分析，高技术通讯，2001，11(5)：6～9。
11、唐咸来，慢生型大豆根瘤菌GX201与结瘤固氮有关的lrp、putA基因的研究，中国科学院博士论文，2001。
12、Fischer H M,1994. Genetic regulation of nitrogen Fixation in Rhizobia. Microbiol. Rev, 58(3): 352～386.
13、Xavier Perret,Christian Staehelin, William J. Broughton et al. 2000. Molecular Basis of symbiotic promiscuity. Microbiol. Mol. Biol. Rev.,64(1): 180～201.
14、Herman P. Spaink, 2000, Root nodulation factors produced by Rhizobial bacteria. Annu. Rev. Microbiol,54: 257～288.
15、Spaink H P, 1995. The Molecular Basis of infection and nodulation by Rhizobia: the Ins and Outs of sympathogenesis. Annu. Rev. Phytopathal., 33: 345～368.
16、Denarie D, Debelle F., 1996. RhizobiumLipo-chitooligosaccharide nodulation factors: Signaling
moleculars mediating recognition and morphogenesis. Annu. Rev. Biochem., 65: 305-335．
17、 Kondorosi A , Kondorosi E , John M , 1996． The role of nodulation genes in bacterium-plant communication. Genetic Engineering vol. 13, shtlow J Keds , Plenum Press , New York , 115-136．
18、 Lerouge P , Roche P , Prome J C , et al 1991． Rhizobium meliloti nodulation genes specify the production of an Alfalfa-specific sulphated oligosaccharide signal. Nature , 344: 781-784．
19、 Denarie J , Cullimore J , 1993． Liop-oligosaccharide nodulation factors: a new class of signaling molecules mediating recognition and morphognesis. Cell, 74: 951-954．
20、 Heidstra R , Brsseling T , 1996． Nod factor-induced host responses and mechanisms of Nod Factor Preception. New Phytol. ,133: 25-43．
21、 Turlough M. Finan , Stefan Weidner, Kim Wong et al. 2001． The complete sequence of the 1683-kb pSym B megaplasmid from the N2 fixing endosymbiont sinorhizobium meliloti. Proc. Natl. Acad. Sci. USA.98(17) :9889-9894．
22、 Virginie Viprey , Andre Rosenthal William J Broughton et al. 2000． Genetic snapshots of the Rhizobium species NGR234 genome. Genome biology , 1(6) : research 0014． 1-0014． 17．
23、 Capela D. , Barloy-Hubler, F. et al. 2001． Analysis of the Chromosome sequence of the legume symbiont Sinorhizobium meliloti strain 1021． Proc. Natl. Acad. Sci. USA.98(7) : 9877-9882．
24、 Gottfert M , Sandr Rothlisberger, Christoph Kandig et al. 2001． Potential symbiosis-specific uncovered by sequencing a 410-kb DNA region of the Bradyrhizobium japonicum chromosome J. Bacteriol, 183(4) : 1405-1412．
25、 Francis Galibert , Turlough M. Finan, Sharon R. Long et al , 2001． The composite genome of the Legume symbiont Sinorhizobium meliloti. Science , 293: 668-672．
26、 Triplett E W , Sadowsky M J. 1992． Genetics of competition for nodulation of legumes. Annual Rev. Microbiol. , 46: 399-428．
27、 Toro N , and Olivares J. 1986． Characterization of a large plasmid of Rhizobium meliloti involved in enhancing nodulation. Mol Gen Genet, 202:331-335．
28、 Sanjuan J , and Olivares J., 1989． Implication of nifA in regulation of genes located on a Rhizobium
meliloti cryptic plasmid that affect nodulation efficiency. J. Bacteriol. ,171: 4154-4161．
29、 Sanjuan J , Olivares J. , 1991． NifA-NtrA regulatory system activates transcription of nfe , a gene locus involved in nodulation competitiveness of Rhizobium meliloti. Arch Microbiol , 155(6) : 543-548．
30、 Soto MJ , Zorzano A, Mercado-Blanco J et al. 1993． Nucleotide sequence and characterization of Rhizobium meliloti nodulation competitiveness genes of nfe. J Mol Biol, 229(2) : 570-576．
31、 Soto MJ , Zorzano A, Garcia-Rodiguez et al. 1994． Identification of a novel Rhizobium meliloti nodulation efficiency nfe gene homolog of Agrobacterium ornithine cyclodeaminase. Mol Plant Microbe Interact, 7(6) : 703-707．
32、 J Michiels , H. Pelemans .1995． Identification and characterization of a Rhizobium leguminosarum bv. Phaseoli Gene that is important for nodulation competitiveness and shows structural homology to a Rhizobium fredii host-induced gene. Mol. Plant-Microbe interat. 8(3) :468-472．
33、 J Y , Chun , and stacey G. 1994． A Bradyrhizobium japonicum gene essential for nodulation competitiveness is differentially regulated from two promoters. Mol. Plant-Microbe Interat . 7(2) : 248-255．
34、 Garcia-Rodriguez FM , Toro N. 2000． Sinorhizobium meliloti nfe (nodulation formation efficiency)genes exhibit temporal and spatial expression patterns similar to those of genes involved in symbiotic nitrogen fixation. Mol Plant Microbe Interact, 13(6) : 583-591．
35、 周俊初，刘孔鑫、杨润英等，广谱花生根瘤菌97-1菌株在花生和大豆根瘤中的分化和繁殖能力，微生物学报， 1992, 32 (4) : 278-284
36、 Eardly B D, Hannaway D B and Botfemley P J. 1985． Characterization of rhizobia from ineffective alfalfa nodules: ability to nodulate bean plants Phaseolus valgaris(L.). Appl. Environ. Microbil. , 50: 1422-1427
37、 Waelkens F, Voets T, Vlassak K et al. 1995． The nods gene of Rhizobia tropici strain CIAT899 is necessary for nodulation of Phaseolus Vulgaris and on Leucaena leucocephala. Mol. Plant-Microbe Interact.
38、 Lewin A, Rosenberg C, Meyer Z A H et al. 1987． Multiple host-specificity loci of the broad-host-range Rhizobium sp. NGR234 selected using the widely compatible legume Vigna unguiculata. Plant. Mol. Biol., 8: 447-459
39、 Dusha I, Olah B, Szegletes Z et al. 1999． Syrm is involved in the determination of the amount and ratio of the two forms of acidic exopolysaccharide EPSI in Rhizobium melilotii. Mol. Plant-Microbe Interact., 12: 755-765
40、 Marie C, Plaskitt K A and Downie J A .1994． Abnormal bacteroid development in nodules induced by a glucosamine synthase mutant of Rhizobium leguminosarum. Mol. Plant-Microbe Interact. 7:482-487
41、 Tate R, Riccio A, Caputo E et al. 1999． The Rhizobium etli metZ gene is essential for methionine biosynthesis and nodulation of Phaseolus Vulgris. Mol. Plant-Microbe Interact. 12:24-34
42、 Sequence accession numbers: Chromosome AL591688(EMBL, CON entry); pSymA accession AE006469(GenBank); pSymB accession number AL591985(EMBL).
43、 Leong S, Ditta G, helinski D.Heme. 1982． biosynthesis in Rhizobium. J Biol Chem, 257: 8724-8730
44、 Hahahan D. 1983． Studies on transformation of Escherichia coli with plasmid. J. Mol. Biol, 166:557-580
45、 Beringer J E, Beynon J L, Buchanan-wollaston A V, et al. 1978． Transfer of the drug-resistance transposon Tn5 to Rhizobium. Nature. 276:633-634
46、 Friedman A M, Long S R, Brown S E, et al. 1982． Construction of a broad host range cosmid cloning Vector and its use in the genetic analysis of Rhizobium mutants. Gene, 18:289-296
47、 Wen PC, Tsong TK. 1993． A simple and rapid method for the preparation of gram-negative bacterial genomic DNA.Nucleic Acids Res. 21(9) :2260
48、 唐东阶，大豆品种-根瘤菌株最佳组合筛选试验。广西农学院学报, 1989, 8 (1) : 49-55
49、 Kundig,C.,H.Hennecke,and M. Gottfert. 1993． Correlated physical and genetic map of the Bradyrhizobium japonicum 110 genome J.Bacteriol. 175:613-622
50、 Wang CL. 1986． Host plant effects on hybrids of Rhizobium leguminosarum Biovars viceae and trifolii. J.Gen.Microbiol., 132: 2063-2070
51、 Birnboim HC, Doly J. 1979． A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7: 1513
52、 Jefferson R A, Burgess S M, Hisrsh d. 1986． P-Glucuronidase from Escherichia coli as a gene-fusion marker. Proc.Natl Acid Sci USA. 83: 8447-8451
53、 Jefferson R A. 1987． Assaing chimeric genes in plants, the GUS gene fusion system. Plant Mol Biol Report. 5: 387-405
2、樊庆笙，《固氮微生物学》，农业出版社，1993。
3、洪国藩、宋鸿遇，《固氮之光》，湖南科学技术出版社，1997。
4、陈因等，《生物固氮》，上海科学技术出版社，1985。
5、尤崇杓等，《生物固氮》，科学出版社，1987。
6、黄大昉、林敏主编，《农业微生物基因工程》，科学出版社，2001。
7、马庆生、武波、唐东阶等，外源 nfeC基因导入快生型大豆根瘤菌菌株HN01的行为分析，复旦学报(自然科学版)，1998，37(4)：445～448。
8、周刚林、武波、唐东阶等，慢生型大豆根瘤菌nfe基因的分子克隆，广西农业生物科学，1999，18(1)：29～32。
9、武波、周刚林、龙章德等，慢生型大豆根瘤菌nfeC基因的定位，广西农业生物科学，2001，20(2)：89～92。
10、武波、周刚林、龙章德等，慢生型大豆根瘤nfeC基因的克隆及序列分析，高技术通讯，2001，11(5)：6～9。
11、唐咸来，慢生型大豆根瘤菌GX201与结瘤固氮有关的lrp、putA基因的研究，中国科学院博士论文，2001。
12、Fischer H M,1994. Genetic regulation of nitrogen Fixation in Rhizobia. Microbiol. Rev, 58(3): 352～386.
13、Xavier Perret,Christian Staehelin, William J. Broughton et al. 2000. Molecular Basis of symbiotic promiscuity. Microbiol. Mol. Biol. Rev.,64(1): 180～201.
14、Herman P. Spaink, 2000, Root nodulation factors produced by Rhizobial bacteria. Annu. Rev. Microbiol,54: 257～288.
15、Spaink H P, 1995. The Molecular Basis of infection and nodulation by Rhizobia: the Ins and Outs of sympathogenesis. Annu. Rev. Phytopathal., 33: 345～368.
16、Denarie D, Debelle F., 1996. RhizobiumLipo-chitooligosaccharide nodulation factors: Signaling
moleculars mediating recognition and morphogenesis. Annu. Rev. Biochem., 65: 305-335．
17、 Kondorosi A , Kondorosi E , John M , 1996． The role of nodulation genes in bacterium-plant communication. Genetic Engineering vol. 13, shtlow J Keds , Plenum Press , New York , 115-136．
18、 Lerouge P , Roche P , Prome J C , et al 1991． Rhizobium meliloti nodulation genes specify the production of an Alfalfa-specific sulphated oligosaccharide signal. Nature , 344: 781-784．
19、 Denarie J , Cullimore J , 1993． Liop-oligosaccharide nodulation factors: a new class of signaling molecules mediating recognition and morphognesis. Cell, 74: 951-954．
20、 Heidstra R , Brsseling T , 1996． Nod factor-induced host responses and mechanisms of Nod Factor Preception. New Phytol. ,133: 25-43．
21、 Turlough M. Finan , Stefan Weidner, Kim Wong et al. 2001． The complete sequence of the 1683-kb pSym B megaplasmid from the N2 fixing endosymbiont sinorhizobium meliloti. Proc. Natl. Acad. Sci. USA.98(17) :9889-9894．
22、 Virginie Viprey , Andre Rosenthal William J Broughton et al. 2000． Genetic snapshots of the Rhizobium species NGR234 genome. Genome biology , 1(6) : research 0014． 1-0014． 17．
23、 Capela D. , Barloy-Hubler, F. et al. 2001． Analysis of the Chromosome sequence of the legume symbiont Sinorhizobium meliloti strain 1021． Proc. Natl. Acad. Sci. USA.98(7) : 9877-9882．
24、 Gottfert M , Sandr Rothlisberger, Christoph Kandig et al. 2001． Potential symbiosis-specific uncovered by sequencing a 410-kb DNA region of the Bradyrhizobium japonicum chromosome J. Bacteriol, 183(4) : 1405-1412．
25、 Francis Galibert , Turlough M. Finan, Sharon R. Long et al , 2001． The composite genome of the Legume symbiont Sinorhizobium meliloti. Science , 293: 668-672．
26、 Triplett E W , Sadowsky M J. 1992． Genetics of competition for nodulation of legumes. Annual Rev. Microbiol. , 46: 399-428．
27、 Toro N , and Olivares J. 1986． Characterization of a large plasmid of Rhizobium meliloti involved in enhancing nodulation. Mol Gen Genet, 202:331-335．
28、 Sanjuan J , and Olivares J., 1989． Implication of nifA in regulation of genes located on a Rhizobium
meliloti cryptic plasmid that affect nodulation efficiency. J. Bacteriol. ,171: 4154-4161．
29、 Sanjuan J , Olivares J. , 1991． NifA-NtrA regulatory system activates transcription of nfe , a gene locus involved in nodulation competitiveness of Rhizobium meliloti. Arch Microbiol , 155(6) : 543-548．
30、 Soto MJ , Zorzano A, Mercado-Blanco J et al. 1993． Nucleotide sequence and characterization of Rhizobium meliloti nodulation competitiveness genes of nfe. J Mol Biol, 229(2) : 570-576．
31、 Soto MJ , Zorzano A, Garcia-Rodiguez et al. 1994． Identification of a novel Rhizobium meliloti nodulation efficiency nfe gene homolog of Agrobacterium ornithine cyclodeaminase. Mol Plant Microbe Interact, 7(6) : 703-707．
32、 J Michiels , H. Pelemans .1995． Identification and characterization of a Rhizobium leguminosarum bv. Phaseoli Gene that is important for nodulation competitiveness and shows structural homology to a Rhizobium fredii host-induced gene. Mol. Plant-Microbe interat. 8(3) :468-472．
33、 J Y , Chun , and stacey G. 1994． A Bradyrhizobium japonicum gene essential for nodulation competitiveness is differentially regulated from two promoters. Mol. Plant-Microbe Interat . 7(2) : 248-255．
34、 Garcia-Rodriguez FM , Toro N. 2000． Sinorhizobium meliloti nfe (nodulation formation efficiency)genes exhibit temporal and spatial expression patterns similar to those of genes involved in symbiotic nitrogen fixation. Mol Plant Microbe Interact, 13(6) : 583-591．
35、 周俊初，刘孔鑫、杨润英等，广谱花生根瘤菌97-1菌株在花生和大豆根瘤中的分化和繁殖能力，微生物学报， 1992, 32 (4) : 278-284
36、 Eardly B D, Hannaway D B and Botfemley P J. 1985． Characterization of rhizobia from ineffective alfalfa nodules: ability to nodulate bean plants Phaseolus valgaris(L.). Appl. Environ. Microbil. , 50: 1422-1427
37、 Waelkens F, Voets T, Vlassak K et al. 1995． The nods gene of Rhizobia tropici strain CIAT899 is necessary for nodulation of Phaseolus Vulgaris and on Leucaena leucocephala. Mol. Plant-Microbe Interact.
38、 Lewin A, Rosenberg C, Meyer Z A H et al. 1987． Multiple host-specificity loci of the broad-host-range Rhizobium sp. NGR234 selected using the widely compatible legume Vigna unguiculata. Plant. Mol. Biol., 8: 447-459
39、 Dusha I, Olah B, Szegletes Z et al. 1999． Syrm is involved in the determination of the amount and ratio of the two forms of acidic exopolysaccharide EPSI in Rhizobium melilotii. Mol. Plant-Microbe Interact., 12: 755-765
40、 Marie C, Plaskitt K A and Downie J A .1994． Abnormal bacteroid development in nodules induced by a glucosamine synthase mutant of Rhizobium leguminosarum. Mol. Plant-Microbe Interact. 7:482-487
41、 Tate R, Riccio A, Caputo E et al. 1999． The Rhizobium etli metZ gene is essential for methionine biosynthesis and nodulation of Phaseolus Vulgris. Mol. Plant-Microbe Interact. 12:24-34
42、 Sequence accession numbers: Chromosome AL591688(EMBL, CON entry); pSymA accession AE006469(GenBank); pSymB accession number AL591985(EMBL).
43、 Leong S, Ditta G, helinski D.Heme. 1982． biosynthesis in Rhizobium. J Biol Chem, 257: 8724-8730
44、 Hahahan D. 1983． Studies on transformation of Escherichia coli with plasmid. J. Mol. Biol, 166:557-580
45、 Beringer J E, Beynon J L, Buchanan-wollaston A V, et al. 1978． Transfer of the drug-resistance transposon Tn5 to Rhizobium. Nature. 276:633-634
46、 Friedman A M, Long S R, Brown S E, et al. 1982． Construction of a broad host range cosmid cloning Vector and its use in the genetic analysis of Rhizobium mutants. Gene, 18:289-296
47、 Wen PC, Tsong TK. 1993． A simple and rapid method for the preparation of gram-negative bacterial genomic DNA.Nucleic Acids Res. 21(9) :2260
48、 唐东阶，大豆品种-根瘤菌株最佳组合筛选试验。广西农学院学报, 1989, 8 (1) : 49-55
49、 Kundig,C.,H.Hennecke,and M. Gottfert. 1993． Correlated physical and genetic map of the Bradyrhizobium japonicum 110 genome J.Bacteriol. 175:613-622
50、 Wang CL. 1986． Host plant effects on hybrids of Rhizobium leguminosarum Biovars viceae and trifolii. J.Gen.Microbiol., 132: 2063-2070
51、 Birnboim HC, Doly J. 1979． A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7: 1513
52、 Jefferson R A, Burgess S M, Hisrsh d. 1986． P-Glucuronidase from Escherichia coli as a gene-fusion marker. Proc.Natl Acid Sci USA. 83: 8447-8451
53、 Jefferson R A. 1987． Assaing chimeric genes in plants, the GUS gene fusion system. Plant Mol Biol Report. 5: 387-405