Cloning And Expression Analysis In E.coli Of Choline Monooxygenase (CMO) Gene Of Leymus Chinese

Chinese leymus(Leymus Chinese),also named alkali grass,is ascribed to Gramineae plant of Leymus,speeding widely in temperate zone of our country.It is a wide ecological amplitude plant, adapting various environment strongly,such as salt,drought,leanness,cold and moist.Although it is not a typical halophyte, it has an ability to resist salt.It can grow normaly in the soil containing total salinity 0.1～0.14%, pH8.3～8.9, alkalifyl 3.5～40.9%.Synthesizing and accumulating micromolecule compounds to keep normal function is a defensive reaction when plants suffer from stress condition.Glycine betaine is one of important compatible micromolecule organics. Many vegetal cells accumulate a great deal of betaine under stress conditions, especially Chenopodiaceae and Gramineae, improving cell osmoregulation ability and stabilizing structure and function of intra-cellular macromolecule protein and biomembrane. Betaine was also detected in Chinese leymus. In high plants, betaine is synthesized by oxidation of choline, catalyzed reaction by two sequential steps:choline→(CMO)betaine aldehyde→(BADH) betaine. the first step is catalyzed by choline monooxygenase(CMO),a limiting enzyme in the betaine synthesis. CMO is one of many current investigations in genetic engineering of salt and drought resistance. CMO gene has already isolated from Zea mays and Oryza sativa, but it hasn't isolated from Chinese leymus.Chinese leymus was test material in the experiment, according to CMO gene sequences of Chenopodiaceae and Gramineae plants registered in the GenBank,designed a pair of primers in the conservative sesquence to amplify fragment,the unique band was obtained about 520bp. PCR product was recovered,cloned toT-vector,transformed to EcoliDH-5α, obtained recombinant plasmid, sequenced after enzyme digestion and PCR identity. Compared the result with other plants,it showed the homology was high with Zea mays and Oryza sativa,it was improved that the middle fragment of Chinese leymus CMO had obtained,then applied 3'RACE obtaining 3'-end sesquence and 5'RACE obtaining partial sesquence before middle fragment. Splicing overlap sequences by DNAman and amplified the full-lengthwhich was named LcCMO.A pair of specific primers designed on the ends of LcCMO open reading frame(ORF) to construct prokaryotic expression vector.The full-length sesquence of LcCMO was 1283bp including 217bp 3' untranslated region(UTR) and poly(A), the length of ORF was 1029bp encoding 343 amino acid, molecular weight was 37.73kDa.The sequence identities were 78.77% and 86.54% compared to CMO from Zea mays on the nucleotide and amino acids level respectively.Digested the recombinant plasmid and pET30a(+) by BamHI and EcoRI respectively,recovered, ligated,constructed expression vector named pET30a(+)-LcCMO,transformed E.coli BL21(DE3).when the temperature was 37 centi-degree and final concentration of IPTG was 1.0mmol/L inducing different time,the result showed that LcCMO was induced by SDS-PAGE electrophoretic analysis, protein molecular weight was about 37kDa.CMO cDNA sequence of Chinese leymus was cloned and expressed in E.coli firstly in this experiment,the aim was to research expression mechanism of salt resistant gene of plant, offered theory basis for sieving the salt resistant gene, explaining salt resistant molecule mechanism in Chinese leymus, reconstructing crops and cultivating new salt resistant species through genetic engineering.