两种扩增rDNA片段RFLP图谱用于快生型大豆根瘤菌的遗传多样性研究  

Genetic Diversity of Fast growing Soybean Nodulating Rhizobia Strains Based on RFLP of Amplified 16S rDNA Fragment and 16S～23S Intergenic Spacer Region rDNA Fragment

作　　者：覃筱婷[1] 周俊初[1] 李阜棣 

机构地区：[1]华中农业大学农业微生物重点实验室

出　　处：《农业生物技术学报》1998年第4期398-404,共7页Journal of Agricultural Biotechnology

基　　金：欧共体TS3-CT92-O127合作研究课题

摘　　要：根据Ｅ．ｃｏｌｉ基因组ｒＤＮＡ保守序列设计的两对引物，分别用于扩增３０株来源于我国不同地区的快生型大豆根瘤菌（Ｓｉｎｏｒｈｉｚｏｂｉｕｍｓｐｐ）菌株。扩增产物经几种较少识别序列的内切酶消化后，得出特征的限制性内切酶酶切图谱（ＲＦＬＰ）。该图谱可用于供试菌株种水平的鉴定及菌株ｒＤＮＡ分子的遗传多样性研究。选用来源于Ｅ．ｃｏｌｉ基因组１６ＳｒＤＮＡ保守序列的一对引物（ＰＨ和ＰＡ）将所有供试菌株都扩增出一条１．５ｋｂ的带，经四种内切酶消化后发现：新疆地区来源的菌株的酶切图谱显著不同于其它地区来源的菌，称其为群Ⅰ菌株。群Ⅰ菌株内部的酶切图谱还可以分为５种类型。其它地区来源菌株的酶切图谱基本一致（７５％），称其为群Ⅱ菌株。在群Ⅱ菌株内部，有少数菌株也表现有特殊性。选用来源于Ｅ．ｃｏｌｉ基因组的１６ＳｒＤＮＡ和２３ＳｒＤＮＡ保守序列的一对引物ＰＨＲ和Ｐ２３ＳＲＯ１将新疆来源的一群菌株扩增出一条１．５ｋｂ的带，而其它地区来源的菌株扩增产物为１．８ｋｂ。新疆来源的群Ｉ菌株扩增产物经３种内切酶消化后的ＲＦＬＰ图谱显著不同于群Ⅱ菌株。群Ⅰ菌株的ＲＦＬＰ图谱也不完全一致，表现为４种类型。群Ⅱ菌株ＲＦＬＰ图谱基本一致，但也发现有特?Thirty strains isolated from different areas of P.R.China were used to study the genetic diversity.A pair of primers derived from conserved 16S rDNA sequence of E.coli were used for amplifying tested strains. All strains produced a band of about 1500 bp. The amplification product were digested by four restriction endonucleases. Five to Seven distinct restriction patterns were detected with each endonuclease. The restriction patterns of the strains isolated from Xinjiang Area was obviously different from the patterns of that from the other Provinces in China.The strains from Xinjiang region were called “Group Ⅰ”while the strains from the other area were named “GroupⅡ”.Group I contained 5 genotypes (Ia,Ib,Ic,Id,Ie). Twenty two strains from the other areas contained 6 genotypes. Sixteen group Ⅱ strains(72.3%)showed an identical restriction pattern (ⅡA),but there still were several strain specific restriction patterns. Another pair of PHR and P23SRO1 primers was also used for amplifying tested strains.Group Ⅰ strains produced a band of 1500 bp while the group Ⅱstrains amplified a band of 1800 bp. Strains“NJ518”and“A602”failed to amplify.The amplified product was digested with three 4 base cutting restriction endonucleases too.Six to eleven restriction patterns were obtained with each enzyme.Ten genotypes of 16S～23S rDNA ISR were defined. GroupⅠstrains included 4 genotypes while group Ⅱproduced 6 genotypes. Fifteen groupⅡ strains showed an identical pattern and several strains showeds strain specific pattern.

关 键 词：快生型 大豆根瘤菌 RFLP 遗传多样性 

分 类 号：S154.381[农业科学—土壤学] Q939.114[农业科学—农业基础科学]