利用原生质体融合构建竞争结瘤能力强且耐酸性固氮菌
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摘要
为获得固氮、竞争结瘤能力强且耐酸的功能菌株,促进新型多功能微生物肥料的研究与开发,以前期分离、鉴定获得的竞争结瘤能力强的大豆根瘤菌菌株YA-1和耐酸性菌株BQ-2为材料,按照功能互补的原则进行原生质体融合,针对融合子采用全氮比色法测定其固氮能力,利用BOX-PCR生物学技术评价其竞争结瘤能力,以GGE双标图的数学模型绘制出融合子和p H环境图标,进行融合子耐酸性状和稳定性的评价分析。结果表明:两供试菌株原生质体易于融合,连续传代10次后,共获得稳定融合子5株。固氮活性测定结果显示融合子YB-3生物固氮量最高,较出发菌株YA-1和BQ-2有显著的提高;融合子在竞争结瘤能力方面对两出发菌株进行了平衡,接种融合子YB-3菌株图谱与结瘤菌株图谱一致性较高,其占瘤率最高为95%,菌株竞争结瘤能力强于土著根瘤菌。GGE双标图分析结果表明,菌株耐酸性顺序为YB-2> YB-3> BQ-2> YB-4> YA-1> YB-5> YB-1,稳定性顺序为YB-5> YB-3> YB-1> YA-1> YB-2> YB-4> BQ-2,耐酸性强且稳定性较好的融合子菌株为YB-3。综合比较而言,获得了一株表现出双亲优良性状的集高固氮、竞争结瘤能力强且耐酸性的新型固氮菌YB-3。
In order to obtain the functional strains with strong nitrogen fixing,competitive nodules and acid resistance,promoting research and development of new multifunctional microbial fertilizer,this study used the YA-1 strain isolated and identificated with strong competitive nodules and BQ-2 strain that have acid resistance as materials. Protoplast fusion was performed according to the principle of complementary,we determined the nitrogen fixation capacity of the fusion hybrid by the total nitrogen colorimetry,evaluated its ability to compete tumor formation by BOX-PCR evaluate,drew the fusion child and p H environment icon with the mathematical model of GGE biplot,evaluate the acid resistance and stability of the fusion seeds. Results showed that: The protoplast of the two strains was liable to fuse,and after 10 consecutive generations,5 stable trains of fusion were obtained. The results of nitrogen fixation showed that the nitrogen fixation was the highest in the sub YB-3,which was significantly higher than that of the starting strain YA-1 and BQ-2. The fusion cells were balanced in terms of their ability to compete for nodule formation. The consistency between the bacterial map of the fusion strain YB-3 and the tumor-forming strain was higher,with the highest tumor rate 95%. The competition ability of the YB-3 strains was stronger than that of indigenous rhizobia. The result of GGE double plot analysis showed that the acid resistance sequence was YB-2 > YB-3 > BQ-2 > YB-4 >YA-1 > YB-5 > YB-1,and the stability sequence was YB-5 > YB-3 > YB-1 > YA-1 > YB-2 > YB-4 > BQ-2,and the fusion substrain with strong acid resistance and better stability was YB-3. In the comprehensive comparison,we obtained a new type of nitrogen fixing bacteria YB-3,with high nitrogen fixation,competitive nodule ability and acid resistance.
In order to obtain the functional strains with strong nitrogen fixing,competitive nodules and acid resistance,promoting research and development of new multifunctional microbial fertilizer,this study used the YA-1 strain isolated and identificated with strong competitive nodules and BQ-2 strain that have acid resistance as materials. Protoplast fusion was performed according to the principle of complementary,we determined the nitrogen fixation capacity of the fusion hybrid by the total nitrogen colorimetry,evaluated its ability to compete tumor formation by BOX-PCR evaluate,drew the fusion child and p H environment icon with the mathematical model of GGE biplot,evaluate the acid resistance and stability of the fusion seeds. Results showed that: The protoplast of the two strains was liable to fuse,and after 10 consecutive generations,5 stable trains of fusion were obtained. The results of nitrogen fixation showed that the nitrogen fixation was the highest in the sub YB-3,which was significantly higher than that of the starting strain YA-1 and BQ-2. The fusion cells were balanced in terms of their ability to compete for nodule formation. The consistency between the bacterial map of the fusion strain YB-3 and the tumor-forming strain was higher,with the highest tumor rate 95%. The competition ability of the YB-3 strains was stronger than that of indigenous rhizobia. The result of GGE double plot analysis showed that the acid resistance sequence was YB-2 > YB-3 > BQ-2 > YB-4 >YA-1 > YB-5 > YB-1,and the stability sequence was YB-5 > YB-3 > YB-1 > YA-1 > YB-2 > YB-4 > BQ-2,and the fusion substrain with strong acid resistance and better stability was YB-3. In the comprehensive comparison,we obtained a new type of nitrogen fixing bacteria YB-3,with high nitrogen fixation,competitive nodule ability and acid resistance.
引文
[1]俞艳春,文定良,罗心平,等.接种根瘤菌对豆科绿肥的固氮效果研究[J].云南农业科技,2006(2):21-22.(Yu H C,Wen D L,Luo X P,et al. Effect of rhizobium inoculation on nitrogen fixation of leguminous green manure[J]. Yunnan Nongye Keji,2006(2):21-22.)
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[4]赵宇枢,段玉玺,王媛媛,等.辽宁省大豆根瘤菌资源抗逆性及生防潜力研究[J].大豆科学,2009,28(1):113-117.(Zhao Y S,Duan Y X,Wang Y Y,et al. Stress resistance and biocontrol potential of soybean rhizobia resourses isolated from Liaoning province[J]. Soybean Science,2009,28(1):113-117.)
[5]江木兰,张学江,徐巧珍,等.大豆-根瘤菌的固氮作用[J].中国油料作物学报,2003,25(1):50-54.(Jiang M L,Zhang X J,Xu Q Z,et al. Nodulation and nitrogen-fixation in soybeanrhizobium[J]. Chinese Journal of Oil Crop Sciences,2003,25(1):50-54.)
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[7] Marinkovic J. Effect of bean inoculation with rhizobium phaseoli on soil microbial activity[J]. Soil and Plant,2006,55(1):61-66.
[8] Prasad J,Ram H. Effect of zinc,copper and Rhizobium inoculation on microbial population in soil and yield of greengram(Phaseolus radiatus)[J]. International Journal of Tropical Agriculture,1992,10(2):157-160.
[9]王静,马玉珍,史清亮.大豆根瘤菌与光合细菌混合接种效果[J].土壤肥料,1997(2):41-42.(Wang J,Ma Y Z,Shi Q L. Noculation effect of mixed inoculation of soybean rhizobia and photosynthetic bacteria[J]. Soil Fertilizer,1997(2):41-42.)
[10] Staehelin C,Charon C,Boller T,et al. Medicago truncatula plants overexpressing the early nodulin gene enod40 exhibit accelerated mycorrhizal colonization and enhanced formation of arbuscules[J]. Proceedings of the National Academy of Sciences of the United States of America,2001,98(26):15366-15371.
[11]谭娟.接种俄罗斯大豆根瘤菌对大豆生长和产量的影响[J].作物杂志,2007(4):36-37.(Tan J. Effects of inoculation with Russian soybean rhizobia on soybean growth and yield[J]. Crop Journal,2007(4):36-37.)
[12] Dudeja S S,Khurana A L. Persistence of Bradyrhizobium sp.(caianus)in a sandy loam[J]. Soil Biology&Biochemistry,1989,21(5):709-713.
[13]陈翠翠,马元武,冯永君,等. MADS-box家族蛋白在植物开花、结实及根瘤形成中的多功能调节作用[J].华北农学报,2008,23(S2):80-83.(Chen C C,Ma Y W,Feng Y J,et al.Multifunctional regulation of MADS-box family proteins in plant blooming,fruit mature and nodule formation[J]. Acta Agriculturae Boreali-Sinica,2008,23(S2):80-83.)
[14]单尹珮.微生物菌种选育技术的发展和研究[J].生物技术,2013,3(2):24-37.(Shan Y P. The development and research of microorganism breeding technology[J]. Biotechnology,2013,3(2):24-37.)
[15]霍乃蕊,韩克光.细胞融合技术的发展与应用[J].激光生物学报,2006,15(2):209-213.(Hong N X,Han K G. The development and application of cell fusion technology[J]. Journalof Laser Biology,2006,15(2):209-213.)
[16]张广志,杨合同,李纪顺,等.利用原生质体融合构建耐盐固氮菌[J].中国土壤与肥料,2010(2):86-89.(Zhang G Z,Yang H T,Li J S,et al. The application of protoplast fusion to build salt-tolerant nitrogen-resistant bacteria[J]. Chinese Soil and Fertilizer,2010(2):86-89.)
[17]黄熬梅,安韶山,曲东,等.两种测定土壤微生物量氮方法的比较初探[J].植物营养与肥料学报,2005,11(6):830-835.(Huang A M,An S S,Qu D,et al. Comparison between two methods of determination soil microbial biomass nitrogen[J]. Soil Science and Plant Nutrition,2005,11(6):830-835.)
[18]王金生,丁宁,吴俊江,等.大豆根瘤菌接种效应及竞争结瘤能力分析[J].大豆科学,2017,36(5):761-767.(Wang J S,Ding N,Wu J J,et al. Analysis of the inoculation effect of soybean rhizobia and the competitive nodulation ability[J]. Soybean Science,2017,36(5):761-767.)
[19]王金生,王君,吴俊江,等.基于GGE-biplot的大豆根瘤菌抗逆性资源筛选[J].大豆科学,2017,36(6):894-899.(Wang J S,Wang J,Wu J J,et al. Screening the resistance resources of Rhizobium japonicum based on GGE-biplot[J]. Soybean Science,2017,36(6):894-899.)
[20] Janice E T. Inlluense of the size of indigenous Rhizobial populations on establishment and symbiotiec; performance of introduced Rhizobia on field-grown legumes[J]. Environ Microbiol,1991,57(1):19-28.
[21] Streit W,Kosch K,Werner D. Nodulation competitiveness of Rhizohium leguminosarum by phaseoli and Rhizohium tropic strains measured by glucuronidase(GUS)gene fusigns[J]. Biology and Fertility of Soils,1992,14:140-144.
[22]肖猛,刘晓云,刘桂霞,等. BOX-PCR分子标记对补播紫花苜蓿共生根瘤菌田间竞争结瘤能力的研究[J].华北农学报,2011,26(1):187-190.(Xiao M,Liu X Y,Liu G X,et al.Study on competitive nodulation ability of rhizobia in symbiosis with reseeding Medicago sativa in field test by using BOX-PCR molecular marker method[J]. Acta Agriculturae Boreali-Sinica,2011,26(1):187-190.)
[23] Gabriel K R. The biplot graphic display of matrices with applicalion to principal component analysis[J]. Biometrika,1971,58:453-467.
[2]陈文新.建议将豆科植物根瘤菌共生固氮体系的应用纳入西部大开发规划[N].科学时报,2000-04-17(1).(Chen W X.It is suggested that the application of symbiotic nitrogen fixing system of legume rhizobia be included in the western development plan[N]. Science Times,2000-04-17(1).)
[3]陈文新,汪恩涛,陈文峰.根瘤菌一豆科植物共生多样性与地理环境的关系[J].中国农业科学,2004,37(1):81-86.(Chen W X,Wang E T,Chen W F. The relationship between the symbiotic promiscuity of rhizobia and legumes and their geographical environments[J]. Scientia Agricultura Sinica,2004,37(1):81-86.)
[4]赵宇枢,段玉玺,王媛媛,等.辽宁省大豆根瘤菌资源抗逆性及生防潜力研究[J].大豆科学,2009,28(1):113-117.(Zhao Y S,Duan Y X,Wang Y Y,et al. Stress resistance and biocontrol potential of soybean rhizobia resourses isolated from Liaoning province[J]. Soybean Science,2009,28(1):113-117.)
[5]江木兰,张学江,徐巧珍,等.大豆-根瘤菌的固氮作用[J].中国油料作物学报,2003,25(1):50-54.(Jiang M L,Zhang X J,Xu Q Z,et al. Nodulation and nitrogen-fixation in soybeanrhizobium[J]. Chinese Journal of Oil Crop Sciences,2003,25(1):50-54.)
[6]马中雨,李俊,张永芳,等.大豆根瘤菌与大豆品种共生匹配性研究[J].大豆科学,2008,27(2):221-227.(Ma Z Y,Li J,Zhang Y F,et al. Symbiotic matching between soybean rhizobium and soybean cultivars[J]. Soybean Science,2008,27(2):221-227.)
[7] Marinkovic J. Effect of bean inoculation with rhizobium phaseoli on soil microbial activity[J]. Soil and Plant,2006,55(1):61-66.
[8] Prasad J,Ram H. Effect of zinc,copper and Rhizobium inoculation on microbial population in soil and yield of greengram(Phaseolus radiatus)[J]. International Journal of Tropical Agriculture,1992,10(2):157-160.
[9]王静,马玉珍,史清亮.大豆根瘤菌与光合细菌混合接种效果[J].土壤肥料,1997(2):41-42.(Wang J,Ma Y Z,Shi Q L. Noculation effect of mixed inoculation of soybean rhizobia and photosynthetic bacteria[J]. Soil Fertilizer,1997(2):41-42.)
[10] Staehelin C,Charon C,Boller T,et al. Medicago truncatula plants overexpressing the early nodulin gene enod40 exhibit accelerated mycorrhizal colonization and enhanced formation of arbuscules[J]. Proceedings of the National Academy of Sciences of the United States of America,2001,98(26):15366-15371.
[11]谭娟.接种俄罗斯大豆根瘤菌对大豆生长和产量的影响[J].作物杂志,2007(4):36-37.(Tan J. Effects of inoculation with Russian soybean rhizobia on soybean growth and yield[J]. Crop Journal,2007(4):36-37.)
[12] Dudeja S S,Khurana A L. Persistence of Bradyrhizobium sp.(caianus)in a sandy loam[J]. Soil Biology&Biochemistry,1989,21(5):709-713.
[13]陈翠翠,马元武,冯永君,等. MADS-box家族蛋白在植物开花、结实及根瘤形成中的多功能调节作用[J].华北农学报,2008,23(S2):80-83.(Chen C C,Ma Y W,Feng Y J,et al.Multifunctional regulation of MADS-box family proteins in plant blooming,fruit mature and nodule formation[J]. Acta Agriculturae Boreali-Sinica,2008,23(S2):80-83.)
[14]单尹珮.微生物菌种选育技术的发展和研究[J].生物技术,2013,3(2):24-37.(Shan Y P. The development and research of microorganism breeding technology[J]. Biotechnology,2013,3(2):24-37.)
[15]霍乃蕊,韩克光.细胞融合技术的发展与应用[J].激光生物学报,2006,15(2):209-213.(Hong N X,Han K G. The development and application of cell fusion technology[J]. Journalof Laser Biology,2006,15(2):209-213.)
[16]张广志,杨合同,李纪顺,等.利用原生质体融合构建耐盐固氮菌[J].中国土壤与肥料,2010(2):86-89.(Zhang G Z,Yang H T,Li J S,et al. The application of protoplast fusion to build salt-tolerant nitrogen-resistant bacteria[J]. Chinese Soil and Fertilizer,2010(2):86-89.)
[17]黄熬梅,安韶山,曲东,等.两种测定土壤微生物量氮方法的比较初探[J].植物营养与肥料学报,2005,11(6):830-835.(Huang A M,An S S,Qu D,et al. Comparison between two methods of determination soil microbial biomass nitrogen[J]. Soil Science and Plant Nutrition,2005,11(6):830-835.)
[18]王金生,丁宁,吴俊江,等.大豆根瘤菌接种效应及竞争结瘤能力分析[J].大豆科学,2017,36(5):761-767.(Wang J S,Ding N,Wu J J,et al. Analysis of the inoculation effect of soybean rhizobia and the competitive nodulation ability[J]. Soybean Science,2017,36(5):761-767.)
[19]王金生,王君,吴俊江,等.基于GGE-biplot的大豆根瘤菌抗逆性资源筛选[J].大豆科学,2017,36(6):894-899.(Wang J S,Wang J,Wu J J,et al. Screening the resistance resources of Rhizobium japonicum based on GGE-biplot[J]. Soybean Science,2017,36(6):894-899.)
[20] Janice E T. Inlluense of the size of indigenous Rhizobial populations on establishment and symbiotiec; performance of introduced Rhizobia on field-grown legumes[J]. Environ Microbiol,1991,57(1):19-28.
[21] Streit W,Kosch K,Werner D. Nodulation competitiveness of Rhizohium leguminosarum by phaseoli and Rhizohium tropic strains measured by glucuronidase(GUS)gene fusigns[J]. Biology and Fertility of Soils,1992,14:140-144.
[22]肖猛,刘晓云,刘桂霞,等. BOX-PCR分子标记对补播紫花苜蓿共生根瘤菌田间竞争结瘤能力的研究[J].华北农学报,2011,26(1):187-190.(Xiao M,Liu X Y,Liu G X,et al.Study on competitive nodulation ability of rhizobia in symbiosis with reseeding Medicago sativa in field test by using BOX-PCR molecular marker method[J]. Acta Agriculturae Boreali-Sinica,2011,26(1):187-190.)
[23] Gabriel K R. The biplot graphic display of matrices with applicalion to principal component analysis[J]. Biometrika,1971,58:453-467.
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