Genetic Diversity And Proteomic Analysis On Medium And Low Temperature-adapting Strains Of Soybean Rhizobia Isolated From Northeast Regions

In this stuy, soybean rhizobia were isolated and verified from soybean root nodules collected fromnortheast regions of China. Genetic diversity and phylogeny of these rhizobia and reference strains wereanalysed. Certain representive strains were set to medium and low temperature screening andvermiculite pot experiment. At last, proteomics technology was used to study mechanisms of strainsgrowing under medium temperature. This paper had important meanings to reveal species distributionand genetic diversity of soybean rhizobia from northeast regions, and gave an opportunity to explore themechanism of growing and capacity difference in nodulation and nitrogen fixing of rhizobia strainsunder medium and low temperature.312soybean rhizobia strains were isolated and verified from347soybean nodules which wereselected form18soybean cultivars in14sites in the northeast regions of China. In order to reveal thegenetic diversity and phylogeny of soybean rhizobia, all312soybean rhizobia strains were analysed.The results indicated that，all the tested rhizobia belonged to Bradyrhizobium, of which B. japonicumwas the dominant species. Under IGS PCR-RFLP72%and16S rDNA PCR-RFLP76%similarity level,tested rhizobia were divided into6and4groups respectively. The BOX-PCR fingerprint showed that,the tested rhizobia were divided into31groups under90%similarity level, which suggested theabundance genetic diversity of Bradyrhizobium in this areas. Based on above comprehensive analysis, itwould appear that, the genetic diversity of tested strains was closely related to the geographicalenvironment, rather than their soybean cultivar hosts.According to genetic diversity, hosts and geographical origins,29soybean bradyrhizobia stemedfrom Heilongjiang province and B.japonicum USDA110were selected to undergo medium and lowtemperature growth screening. The results showed that tested strains had various growing capacityunder medium and low temperatures and21strains were obtained which could grow better undermedium and low temperature(6~17℃). Seven strains having different growing capacity under mediumand low temperatures were set to proceed medium and low temperature vermiculite pot experiment. Bymeasuring plant over ground dry weight and total nitrogen content, root nodule number and dry weight,one strain（sB.japonicum5438）were excluded having superior nodulation and nitrogen fixing efficiency.Two soybean bradyrhizobia (B. japonicum5411and B. japonicum5438), gorwing well but havingdifferent nodulation and nitrogen fixing efficiency under medium and low temperatures, were chose tomeasure their growth curve under15℃and28℃, which subsequently showed that tested strains hadvery low growth rate in15℃. Then the two strains were cultured12d and72h under15℃and28℃respectively to make sure in logarithmic phase. After that, cells were collected and proteomicstechnology was used to analyse extracted total proteins. The results indicated that, compared to28℃,when growing under15℃,5411had15up-regulated or induced protein spots and27down-regulated orrepressed protein spots;5438had16up-regulated or induced protein spots and30down-regulated orrepressed protein spots. Different expressed protein spots was identified by MOLDI-TOF massspectrum, and33proteins of5411,30proteins of5438were identified. After analyzing different expressed proteins, we can find that, proteins participating in energy andsubstrate synthesis metabolism pathways were down regulated when strains growing under15℃, forexample, malate dehydrogenase, biotin carboxyl carrier protein subunit of acetyl-CoA carboxylase andoxidoreductase of5411; electron transfer flavoprotein large subunit, malate dehydrogenase anddeoxyuridine5’-triphosphate nucleotidohydrolase of5438. When strains growing under15℃, proteinsprotecting cell against oxygen injury were down-regulated, while proteins helping preprotein foldingand translocation were up-regulated, which indicated that strains confronted lower oxygen pressure butfaced more uncertain factors in preprotein folding.Other than similar regulated proteins above, the two strains also adopted other strategies to adaptmedium and low temperatures，which may eventually lead5438had higher nodulation and nitrogenfixing efficiency than5411under medium and low temperatures. The different strategies referred to theregulations that， aldehyde dehydrogenase and nitrogen regulatory protein PIIof5411weredown-regulated, while acetyl-CoA acetyltransferase and RNA polymerase sigma-E factor (Sigma-24)protein of5438were up-regulated.