The Comparative Studies On The Characterization Of Chromosomal And PSym Borne Genes Of The Isolates From Astragalus Sinicus

208 isolates from Astragalus sinicus were collected from 51 locations, 7 provinces in southern China. 16S rRNA gene and 16S-23S rDNA intergenic spacer are the representation of chromosomal information and common nod genes (nodD,B,C) as the marks of pSym information.All the strains produced only one 1.5kb fragment of 16S rDNA by PCR-amplification with the primes pA and pH. With the respective disposition of restriction endonucleases Hinfl. Mspl. Haelll and TaqI, four types of band pattens were presented. The 16S rDNA of four strains representing three types of strains were partially sequenenced. By comparing with the related sequeane of other rhizobia, the results from clustering analysis indicated that the four strains are all belonged to Mesorizobium but in two groups, HB5A4-1 and HN14A16-2 choser to M. loti, ZJ15B7-1 and HN8A1-1 more related to M. huakuii, CCBAU2609. The great discrepancy among the strains is present in M. huakuii.Using the primes, PHr and p23SR01, for amplifying 16S-23S intergenic spacer (IGS), 202 isolates produced one fragment, 1.8-2.0 kb, and 5 isolates produced unique land, 540bp, except one isolate with both the fragments, The result suggests that the recombination of rDNA operon occurred in evolution. By the digestion with the same restriction endonucleases as above, 14 IGS-types were produced, which can by placed into 5 groups (90% similarity level). In contrast to 16S rDNA types, these IGS-types are predominantly in conformity with them.101 strains isolated from various locations were examined by the Echardt's method and produced 16 types of plasmid profiles, 1-4 plasmids and 36-360 MDa in size. The hybridization of plasmid DNA, with the probe, nodDBC (4.2kb) cloned from M.huakuii 7653R, indicated that the nodulation genes are all located on the largest or the second plasmids.EcoRl-digested genomic DNAs of all the isolates are hybridized with nodDBC probe and nine types of positive band patterns were discovered. There are strong correspondences between the chromosomal genotypes and the hybriding types, indicatingthat coadaptation between them may occur. By the hybridization of Pst-I digested genomic DNA with the same probe, 28 strains including all the EcoRI-digested hybriding types of isolates created 10 types of hybridization patterns and their dendrogram was constructed. In comparison of the chromosomal genotypes. There is no closer relationship among their plasmid genotypes in certain chromosomal background and vice versa. It implies that the origin of common nod genes may be different from that of chromosomal genes.We noticed that a conservative fragment of 2.0kb occurs in almost all the strains by analyzing the positive bands in the Pstl-hybriding pattern. It should be the sequence including nodB and partial nodD and C according to corresponding sequence of M. huakuii 7653R. In order to examine the variation of the fragment in the isolates. A pair of primes, pG1 and pG2 was designed for its PCR-amplification. 25 strains in various chromosomal and plasmid genotypes all produced the expecting fragment. By the digestion with HindIIL MspK HinfI and HaeIII, only the disposition with HaeIII gave different types, one type involved in 20 strains and two mutational types in other strains. It is very interesting that there is the same mutational type in different chromosomal background. It confirms that gene transfer had taken place in its evolution and furthermore, the mutation happened before gene transfer.Finally, we profound one hypothesis: (1) the loss, recombination of symbiotic genes and gene transfer exist in the evolutional process of rhizobia. (2)there is the coadaptation of not only between symbiotic sequences of rhizobia and its host plant but also between the nonsymbiotic sequences and chromosomal genes and chromosomal genes.