Clone And Function Analysis Of Betaine Synthetase Gene Of Chromohalobacter Sp.ST307

Moderately halophilic bacteria is a kind of bacteria whose optimal NaClconcentration of growth is between 0.5 mol/L (3% or so) to 2.5 mol/L (15% or so).The bacteria exist widely in salt lakes, salterns, desert, high salt soil and saline foodand other surroundings. The moderately halophilic bacteria is different from extremehalophiles. The mechanism of salt tolerance is mainly to realize the balance byresistance to the outside high osmotic pressure by means of endocellular accumulationof compatible solute, Example glycine betaine. Glycine betaine, betaine for short, issynthesized under the guide of the choline dehydrogenase (betA) and betaine aldehydedehydrogenase (betB) in bacteria. Presently, the betaine and its hydrochloride havewide applications in chemical industry, medicine and industry, etc, and there is a goodapplied prospect of betaine synthetase in plant Gene engineering. The preliminarystudy found that the Chromohalobacter sp.ST307 belongs to the new population ofChromohalobacter sp and has optimum growth in the 0.8M NaCl concentration. Itssalt-tolerance mechanism and the research of betaine synthesis genes are of importantrole.The influence of cations and anions of endogenous moderately halophilicbacteria of suaeda salsa Chromohalobacter sp.ST307 on its growth are examined.And then we analyzed the accumulation of in vivo betaine of strain ST307. Finally,we focus on clone the betaine synthetase genes betA and betB, and analyze theirexpression. The concrete results are as follows:Among a variety of cations and anions examined in experiments, cations includeNa+、NH4+、Li+、K+、and Ca2+, anions include Cl-、SO42-、I-、Br-、NO3-. When theNa+(0.8M) in the medium is substituted for equimolar amounts of NH4+, Li+, K+,Ca2+, it turns out that strains ST307 never grow, indicating that the growth ofChromohalobacter sp.ST307 depends on Na+. When the Cl-(0.8M) in the medium issubstituted for equimolar amounts of SO42-, I-, Br-, NO3-, SO42-is growing very well, indicating that Cl-is non-essential of the growth of strain ST307. Further weinvestigate the growth of Chromohalobacter sp ST307 in media of various saltconcentration containing cation-anion compounds, and the result is that amongcations NH4+expedites its growth, and that in the NH4+existent medium, the strain’sdemand of Na+decreases, and that Ca2+inhibits the strain. Li+、K+are non-essentialof the growth. Anion SO42-expedites its growth. As the NO3-, Br-of the medium rampup, the biomass of the strains ramps down, indicating that NO3-and Br-inhibit itsgrowth. In the medium containing I-, Chromohalobacter sp ST307 never grow,indicating that I-has a strong inhibition effect.To endogenous moderately halophilic bacteria of Suaeda SalsaChromohalobacter sp.ST307, ethanol extraction method is used to extract betaine,and UV spectrophotometry to carry out the spectroscopy scan testing, and the resultsshow the endocellular accumulation of betaine in the Chromohalobacter sp.ST307. Atthe same time, from 3%, 5%, 7%, 9% salt concentration series etc., betaine isextracted and detected and the test data indicate that as the salt concentrationincreases, betaine accumulation also rises, and when the salt concentration of themedium increases from 3% to 9%, betaine accumulation increased by 20%,demonstrating that one of the mechanisms of salt tolerance of endogenous moderatelyhalophilic bacteria of Suaeda Salsa Chromohalobacter sp.ST307 is to accumulatebetaine .According to the conserved region of the betaine aldehyde dehydrogenase gene,we design a pair of degenerate primers, and use total DNA of endogenous moderatelyhalophilic bacteria of Suaeda Salsa Chromohalobacter sp.ST307 as templates toamplify and thus obtain the sequence of conserved region of betB gene of theChromohalobacter sp.ST307, and then through segment cloning method, obtain thenon-conservative unknown region of gene 5 ’and 3’ end of the known region, andfinally abtain the complete coding region of betB gene of Chromohalobactersp.ST307 by Splicing sequence. Result analysis shows that the gene has totally 1470nucleotides, encoding 489 amino acids. By BLAST sequence analysis, we find thatthis gene sequence has a high homology with many nucleotide sequences in GenBank database, up to 79%. Protein sequences comparison and the constructive result ofphylogeny show that the BADH sequence of Chromohalobacter sp.ST307 has closestevolutionary relationship with Chromohalobacter salexigens DSM3043 andHalomonas elongata DSM2581, possesses active sites (Asn170, Glu250, and Cys290),which are BADH-conservative and can combine NAD+, and possesses the active sitebinding the enzyme substrate.The cloned full-length gene of betaine aldehyde dehydrogenase is connected tothe expression vector pET-27b, and then detected the expression effect by measuringthe biomass. We found that, by adding IPTG induction, the biomass at differentsalinities has all increased with the rate of increase up to 36%. This illustrates that theintroduction of BADH synthase gene of endogenous moderately halophilic bacteria ofSuaeda Salsa Chromohalobacter sp.ST307 boosts the salt tolerance of E.coli, and wefurther validate the osmotic adjustment function of betaine aldehyde dehydrogenasegene of Chromohalobacter sp.ST307 cloned in this experiment.