大豆根瘤内生菌全细胞可溶性蛋白SDS-PAGE电泳图谱分析
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摘要
以大豆根瘤内生细菌为试验材料,分别采用单次点样、重复点样、不同上样量对其进行全细胞可溶性蛋白质十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)图谱分析,根据标准蛋白质各条带相对迁移率估算待测蛋白质亚基的分子量范围,结合16S r DNA测序结果区分大豆根瘤内生菌间亲缘关系,便于后续研究。结果表明:单次点样,不同菌株间蛋白质条带有差异,说明彼此间亲缘关系不同。经16S rDNA序列分析,菌株44最相似属种为Bacillus horikoshii、菌株52最相似属种为Sinorhizobium fredii、菌株70最相似属种为Ochrobactrum intermed;重复点样,菌株7与53的蛋白质图谱相同,经16S rDNA测序分析,两者最相似属种均为Bacillus subtilis,且最相似菌株均为HRA69,说明两者属于同一种内生菌。菌株123与125蛋白质图谱相近,经16S rDNA测序比对,两者最相似属种均为Bacillus cereus;电泳结果显示,对同一菌株不同上样量不影响蛋白质图谱,16S rDNA测序结果表明同一菌株最相似属种始终不变,即不影响亲缘关系。
Taking endophytic bacteria as experimental materials,electrophoresis analysis were performed by setting a single loading sample,repeated loading samples,different loading amount about sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) of whole cell soluble protein,depending on the relative motilities of each standard protein to estimate the molecular weight range of protein subunits,combinating with 16 S rDNA sequences to distinguish the relative molecular weight of different strains from soybean nodules to facilitate further research. The results showed that the protein bands of different strains were different,their genetic relationships were different,indicating more distant relatives. 16 S rDNA sequencing analyzed indicated strain 44 was the most similar genera Bacillus horikoshii,52 was the most similar to Sinorhizobium fredii,strain 70 was the most similar with Ochrobactrum intermedium;Repeated electrical samples suggested strains 7 and 53 had the same proteins profile,and the analysis of 16 S rDNA sequences showed the most similar genera was Bacillus subtilis,and the most similar strain was HRA69,indicating they were the same species. Protein profiles of 123 and 125 combinated with 16 S rDNA sequences and comparison,the most similar genera was Bacillus cereus. Electrophoresis results showed that different loading amount of same strain did not affect protein profiles,16 S rDNA sequences results furtherly confirmed the most similarity was invariant,did not affect the genetic relationship.
Taking endophytic bacteria as experimental materials,electrophoresis analysis were performed by setting a single loading sample,repeated loading samples,different loading amount about sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) of whole cell soluble protein,depending on the relative motilities of each standard protein to estimate the molecular weight range of protein subunits,combinating with 16 S rDNA sequences to distinguish the relative molecular weight of different strains from soybean nodules to facilitate further research. The results showed that the protein bands of different strains were different,their genetic relationships were different,indicating more distant relatives. 16 S rDNA sequencing analyzed indicated strain 44 was the most similar genera Bacillus horikoshii,52 was the most similar to Sinorhizobium fredii,strain 70 was the most similar with Ochrobactrum intermedium;Repeated electrical samples suggested strains 7 and 53 had the same proteins profile,and the analysis of 16 S rDNA sequences showed the most similar genera was Bacillus subtilis,and the most similar strain was HRA69,indicating they were the same species. Protein profiles of 123 and 125 combinated with 16 S rDNA sequences and comparison,the most similar genera was Bacillus cereus. Electrophoresis results showed that different loading amount of same strain did not affect protein profiles,16 S rDNA sequences results furtherly confirmed the most similarity was invariant,did not affect the genetic relationship.
引文
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[25]张露露,王昕陟.非连续型垂直版SDS-PAGE电泳技术分析蛋白质的方法[J].黑龙江畜牧兽医, 2015(11):91-93
[2]王志伟,纪燕玲,陈永敢.植物内生菌研究及其科学意义[J].微生物学通报, 2015, 42(2):349-363
[3]杨波,陈晏,李霞,等.植物内生菌促进宿主氮吸收与代谢研究进展[J].生态学报, 2013, 33(9):2656-2664
[4]丁健,陈贝,袁建军,等.植物修复重金属污染及内生细菌效应[J].微生物学通报, 2011, 38(6):921-927
[5]饶小莉,沈德龙,李俊,等.甘草内生细菌的分离及拮抗菌株鉴定[J].微生物学通报, 2007, 34(4):700-704
[6] Ji HL, Wang ET, Chen WF, et al. Genetic diversity and potential for promotion of plant growth detected in nodule endophytic bacteria of soybean grown in Heilongjiang province of China[J]. Soil Biology and Biochemisitry,2008, 40(1):238-246
[7] Sheng XF, Xia JJ, Jiang CY, et al. Characterization of heavy metalresistant endophytic bacteria from rape roots and their potential in promoting the growth and lead accumulation of rape[J]. Environmental Pollution,2008, 156(3):1164-1170
[8]王志伟,陈永敢,王庆璨,等.中国植物内生微生物研究的发展和展望[J].微生物学通报, 2014, 41(3):482-496
[9]杨欣,曾宪军,丁仁芳,等.一株红景天内生细菌的筛选及初步研究[J].微生物学通报, 2015, 42(10):1962-1970
[10]姜晓宇,高菊生,徐凤花,等.水稻种子内生细菌多样性及其分泌植物生长素能力的鉴定[J].微生物学报, 2013, 53(3):269-275
[11]李淑彬,黄娟,周仁超,等.南药植物高良姜内生细菌多样性及其促生潜力[J].生态学报, 2015, 35(10):3204-3213
[12]王宇光,顾文亮,张欣,等.香蕉内生拮抗细菌KKWB-10的分离鉴定及其内生性证明[J].中国农学通报, 2012, 28(24):183-187
[13]王爱娜,王灏,李殿荣,等.琼脂糖与聚丙烯酰胺凝胶电泳在SSR鉴定杂交油菜种子纯度中的比较[J].西北农业学报, 2012, 21(8):101-106
[14] Schagger H, Jagow GV. Tricine-sodium dodecyl sulfate-polyacrylamide gelelectrophoresis for the separation of proteins in the range from 1 to 100 KDa[J]. Analytical Biochemisity, 1987:166(2):368-379
[15]石继红,赵永同,王俊楼,等. SDS-聚丙烯酰胺凝胶电泳分析小分子多肽[J].第四军医大学学报, 2000, 21(6):761-763
[16]牛红军,李建文,张栋,等.两种光合细菌生物转化槲寄生培养液菌体中几种同工酶的变化[J].微生物学通报, 2011, 38(7):1043-1050
[17]袁燕,王红斌,杨文荣,等. SDS-PAGE电泳法测定蒜头果蛋白的相对分子质量[J].云南民族大学学报, 2015, 24(2):123-125
[18] Zakhia F, Jeder H, Willems A, et al. Diverse bacteria associated with root nodules of spontaneous legumes in Tunisia and first report for nifH-like gene within the genera Microbacterium and Starkeya[J].Microbial Ecology, 2006, 51(3):375-393
[19] Zhao LF, Xu YJ, Sun R, et al. Identification and characterization of the endophytic plant growth prompter Bacillus cereus strain MQ23isolated from Sophora alopecuroides root nodules[J]. Brazilian Journal of Microbiology, 2011, 42(2):567-575
[20]何亮,陈群,曾忠铭,等.通过特异PCR扩增和16S rDNA序列分析检测动弯杆菌[J].微生物学报, 2005, 45(1):27-30
[21]汤旭,姜海,赵鸿雁,等. 16S rDNA序列分析在鉴定布鲁氏菌中的应用[J].微生物学通报, 2013, 40(7):1290-1296
[22]周阳薇,刘正坪,魏艳敏,等.植物根系土壤细菌的分离与鉴定[J].中国农学通报, 2015, 31(7):212-217
[23]陈亚飞,孙宇,高丰衣,等. SDS-聚丙烯酰胺凝胶电泳法测定蛇毒抗瘤蛋白的相对分子质量[J].中国生化药物杂志, 2004, 24(5):300-303
[24]姜玮瑜,梁革梅,林毅,等.对Bt蛋白抗性和敏感的棉铃虫中肠细菌群落的比较[J].微生物学报, 2010, 50(6):828-834
[25]张露露,王昕陟.非连续型垂直版SDS-PAGE电泳技术分析蛋白质的方法[J].黑龙江畜牧兽医, 2015(11):91-93