Molecular Diversity And Phylogenetic Analysis Of Culturable Halophiles In Qinghai Lake

Objective Halophiles are microorganisms that thrive in environments with highconcentrations of salt and plays important role on maintaining ecological balance.Qinghai Lake, which is located in the northeastern of Qinghai-Tibet Plateau, is thelargest saltlake in China. The salinity of the water is approximately6%and contains alot of halophiles. The environment of Qinghai Lake is high-salt, low-pressure, andhypoxia. Until now, few researches were reported about the halophilic characteristics,physiological and biochemical characteristics, diversity of the population, molecularevolution and salt tolerance mechanisms of the halophiles in Qinghai Lake.Our goal is to isolate Halophiles from Qinghai Lake and study their salt-tolerantproperties. The phylogenetic tree will be constructed by cloning and sequencingtechnology, and the evolutionary relationship of halophile molecular species of QinghaiLake will be investigated. At first,16S rDNA and16S-23S internal transcribed spacer（ITS） fragments from10Halomonas strains in Qinghai Lake were amplified by PCR,then Sequenced, and analyzed by Blast and RFLP. Moreover, the protein differencesamong species were analyzed by SDS-PAGE. Based on the data obtained from ourresearch, we could understand the physiological and genetic characteristics of halophileresources in Qinghai Lake.Methods20Water samples were collected from different locations aroundQinghai Lake. The halophilic strains were isolated using RM medium, at37℃. Thecolonies of obtained strains were Gram stained and observed on the plates with RMmedium. The purified strains were cultured with different concentration of NaCl toanalyze their salt tolerant ability. One-step tests were carried out to study the effect oftemperature, pH and concentration of Mg²⁺to the bacterial growth. Different carbonand nitrogen sources were also analyzed with these strains to give foundations for their classification.The genomic DNA of35halophiles in Qinghai Lake were extracted. The16SrDNA gene was amplified by PCR using bacterial universal primers. The PCR productswere were purified and sequenced. Based on the16S rDNA sequences, phylogenetictree was constructed by using MAGE4.0software. The result showed that10strainsbelongs to Halomonas pp. RFLP method,16S-23S rDNA the ISR gene analysis andprotein SDS-PAGE were also used to analyze these10strains bacteria.Results By screening analysis of20different halophilic microbian,35strains ofhalophiles were isolated,34strains belongs to bacteria and1strain belongs to fungi.The morphology characteristics of bacterian colonies were detected. round or ovalcolonies accounted for88.57%, and the major color is white or milky white. Due toexistence of natural pigment, approximately40%of the bacterial colonies were red,orange-red, yellow or light blue, respectively. Microscopic observation showed thatGram-negative bacteria accounted for about74.28%. In addition, long rod or shortrod-shaped was approximately80%, spherical or curved shape was about14.28%,and5.7%belonged to filamentous shape.Based on the salt gradient experiments, these35strains halophiles in QinghaiLake belongs to the moderate halophile which accounted for62.85%, slight halophileaccounted for about22.85%, non-halophilic bacteria for14.28%. Results indicated thatthe type of moderate halophiles were the dominant populations in Qinghai Lake.15strains moderately halophiles were chosen for growth characteristic study, theresult showed that moderate halophiles in Qinghai Lake could grow at10⁴5°C. Somestrains could even grow at5°C, and this results were related with the plateauenvironment of Qinghai Lake. The isolated moderate halophiles could grow at the pHrange of5.5¹1, and the optimal pH was greater than7.5. This result was closelyrelated to the saline-alkali environment. With the difference of halophilic properties, themoderate halophile showed different need of Mg²⁺, and also showed different tolerancelevels on Mg²⁺, the highest tolerant concentration was1.6mol/L.The utilization of carbon and nitrogen sources in the separated15typical moderatehalophiles were studied. The results showed that the majority of the moderatelyhalophiles could utilize fructose, sucrose, glucose, maltose, lactose, galactose, xylose,rhamnose-sugar, raffinose, trehalose, andcellobiose, as a carbon source. The utilizationof other carbon sources was different among strains. For the utilization of nitrogensources, most bacteria strains could grow with yeast extract, peptone, ammonium sulfate, ammonium nitrate, ammonium chloride, urea, potassium nitrate, sodium nitrateand potassium nitrate, but could not grow with sodium nitrite.Through the extraction of bacterial genomic DNA and the amplification of16SrDNA by PCR, the phylogenetic tree was constructed and analyzed. It indicated that thecultivable halophilic bacteria population in Qinghai Lake belonged to four mainevolutionary branch unit, named Gammaproteobacteria, Firmicutes, Actinomycetalesand Alphaproteobacteria, containing14different affiliated groups.Based on16S rDNA analysis, it showed that10strains in Qinghai Lake werebelonged to the Halomonas of Oceanospirillales. They are Gram-negative bacteria andmoderate halophiles （salinity range is0.4³.5mol/L, and the optimal salinity is0.5².0mol/L）, but the morphological features and growth characteristics showedsignificant differences among them. By16S-23S rDNA ITS polymorphism analysis,whole protein analysis through SDS-PAGE and16S rDNA sequnces from the10Halomonas strains, it initially revealed that these10strains could be divided into sevenevolutionary branch units. It also confirmed that the sequnce difference among thespecies was high consistent in10Halomonas strains. Meanwhile, this result verifiedthat the significance of molecular characteristics of multiphase study was veryimportant for the unit classification at the beginning period.Conclusion Based on the characteristic of the plateau environment, our wereported the16S rDNA sequence homology of35halophiles from Qinghai Lake, andthe preliminary analysis for the population diversity of cultivable halophiles. This studyprovided the theoretical basis for the latter cloning and expression of salt-tolerant genesfrom halophiles in Qinghai Lake. It also provided theoretical support for the agricultureengineering resisting to salinization, high-salt wastewater treatment systems andengineering bacteria application development.