华癸中慢生根瘤菌焦磷酸硫胺素结合蛋白基因tpp的功能研究
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摘要
在构建M.huakuii tpp基因突变株的基础上,通过自生生长和植物盆栽试验,研究焦磷酸硫胺素结合蛋白tpp基因在根瘤菌的生长及与紫云英宿主共生固氮过程中的功能。通过同源重组获得华癸中慢生根瘤菌tpp突变菌株HKtpp,并进一步研究tpp基因突变对菌株生长及共生固氮功能的影响。结果显示:tpp基因突变会减缓菌株生长,突变菌株胞内的谷胱甘肽还原酶活性显著降低。植物盆栽试验表明,突变菌株感染紫云英宿主形成有效的红色根瘤,但是其固氮酶活降低了26.4%。结果表明:tpp基因在根瘤菌的生长和共生固氮中发挥着重要作用。
Mesorhizobium huakuii tpp mutant HKtpp was obtained by homologous recombination,and the effects of mutant HKtpp on the growth and symbiotic nitrogen fixation function of the strain were studied by autogenous growth and plant pot experiment. The results showed that the mutant HKtpp reduced the strain growth,and the activity of glutathione reductase in the mutant strain was significantly decreased. The mutant HKtpp formed effective red nodules,but its nitrogenase activity was decreased by 26.4%. It is indicated that tpp gene played an important role in the growth and symbiotic nitrogen fixation of Rhizobium.
Mesorhizobium huakuii tpp mutant HKtpp was obtained by homologous recombination,and the effects of mutant HKtpp on the growth and symbiotic nitrogen fixation function of the strain were studied by autogenous growth and plant pot experiment. The results showed that the mutant HKtpp reduced the strain growth,and the activity of glutathione reductase in the mutant strain was significantly decreased. The mutant HKtpp formed effective red nodules,but its nitrogenase activity was decreased by 26.4%. It is indicated that tpp gene played an important role in the growth and symbiotic nitrogen fixation of Rhizobium.
引文
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[2] HOSIE A H,ALLAWAY D,POOLE P S. A monocarboxylate permease of Rhizobium leguminosarum is the first member of a new subfamily of transporters[J]. Bacteriol,2002,184(19):5436-5448.
[3] 周俊初,曹燕珍.快生型根瘤菌营养生理的研究[J].华中农学院学报,1981(3):44-57.
[4] JOHNSTON A W B,BERINGER J E. Identification of the rhizobium strains in pea root nodules using genetic markers[J]. Journal of general microbiology,1975,87(2):343-350.
[5] KARUNAKARAN R,EBERT K,HARVEY S,et al. Thiamine is synthesized by a salvage pathway in Rhizobium leguminosarum bv. viciae strain 3841[J].J Bacteriol,2006,188(18):6661-6668.
[6] CHENG G J,LI Y G,ZHOU J C. Cloning and identification of opa22,a new gene involved in nodule formation by Mesorhizobium huakuii[J]. FEMS Microbiol Lett,2006,257: 152-157.
[7] SEMPRINI S,COLLINS C C,GONCZ K C,et al. Construction and purification of pSABR 01 a pUC19-derived vector optimizedfor cloning full-length cDNA[J].Biotechnology letters,2003,25: 1275-1280.
[8] FLGURSKI D H,HELINSKI D R.Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans[J].Biochemistry,1979,76(4):1648-1652.
[9] 周艳琳,何冬兰,李晓华,等. katG基因在豌豆根瘤菌抗氧化中的功能[J].微生物学报,2015,55(7):843-850.
[10] 田梦洋,何冬兰,李晓华,等.豌豆根瘤菌ohrB基因的抗氧化和共生固氮表型[J].华中农业大学学报,2016,35(3):54-60.
[11] 刘娟,王雅琴,刘刚,等.发酵液中还原型谷胱甘肽三种测定方法的改进及其比较[J].北京化工大学学报(自然科学版),2004(3):35-38.
[12] 王群,刘朝巍,徐文娟.紫外分光光度法测定玉米过氧化氢酶活性新进展[J].中国农学通报,2016,32(15):159-165.
[13] 张腾国,聂亭亭,孙万仓,等.逆境胁迫对油菜谷胱甘肽还原酶基因表达及其酶活性的影响[J].应用生态学报,2018,29(1):213-222.
[14] TATé R,CERMOLA M,RICCIO A,et al. Glutathione is required by Rhizobium etli for glutamine utilization and symbiotic effectiveness[J]. Mol Plant Microbe Interact,2012,25 (3):331-340.
[15] 彭杨,殷杰,田梦洋,等. OsmC基因在豌豆根瘤菌抗氧化和共生中的功能[J].中南民族大学学报(自然科学版),2017,36(2):25-29.
[16] 温永煌,汤健民.用乙炔还原法测定满江红的固氮活性[J].Journal of integrative plant biology,1978(3):273-275.
[17] RAMAKRISHNAN K,ANDREAS F H,ALISON K E,et al. BacA is essential for bacteroid development in nodules of galegoid,but not phaseoloid legumes[J].Bacteriol,2010,192(11):2920-2928.
[18] 戴少波. 硫胺素焦磷酸依赖酶催化机理研究[D].西安:西北大学,2012.
[19] PENNINCKX M J.An overview on glutathione in Saccharomyces versus non-conventional yeasts[J].FEMS yeast research,2002,2:295-305.
[20] DUNN M F. Rhizobial amino acid metabolism:polyamine biosynthesis and functions[M]//D’MELLO J P F.The handbook of microbial metabolism of amino acids.Wallingford:CAB International,2017:352-370.
[21] LI Y,WEI H,LI F,et al. Supplementation of branched-chain amino acids in protein-restricted diets modulates the expression levels of amino acid transporters and energy metabolism associated regulators in the adipose tissue of growing pigs[J]. Animal nutrition,2016,2(1):24-32.
[22] LODWIG E M,LEONARD M,MARROQUI S,et al. Role of polyhydroxybutyrate and glycogen as carbon storage compounds in pea and bean bacteroids[J]. Mol Plant Microbe Interact,2005,18: 67-74.
[23] 周俊,孙爱珍,曾礼漳,等. 硫胺素通过提高线粒体氧化状态促进植物快速响应外界胁迫的研究[J].激光生物学报,2012,21(4):340-345.
[24] 陈大松.紫云英共生固氮相关基因在铵胁迫及盐胁迫下的表达研究[D].武汉:华中农业大学,2007.