Mechanisms Of Leymus Chinensis To Adaptive Habitats And Cloning Of Gene Related To Betaine Synthesis On The Songnen Grassland

Leymus chinensis is a kind of perennial Gramineae plant of rhizome type, which mainly distributes in Northeast and Inner Mongolia of China. It is a constructive species of wide-ecological amplitude in arid grassland. Since Leymus chinensis is more tolerant to drought, leanness as well as salinity, it propagates very fast and has high level of biomass. Although it is not a typical halophyte, it has an ability to resist salt. It can grow normally in the soil containing total salinity 0.1～1.0%, pH8.3～10.1.Leymus chinensis came from different salinization grassland in the Songnen plain were the test material in the article, studying Dynamic changes of content of proline and betaine as the micromolecule osmoregulation substance and activities of superoxide dismutase (SOD), catalase (CAT) , peroxidase (POD) and betataine aldehyde dehydrogenase (BADH) in different seasons . The alkalization degree is major factor having an effect on changes of proline content. The betaine contents were positive correlation to electrical conductivity of soil.The pH of soil is major factor having an effect on change of SOD and POD activity and electrical conductivity of soil having an effect on change of CAT activity.From No.1 to No.5 sampling grassland, betaine and proline contents and activities of SOD,CAT,POD and BADH are all increased but MDA content is decreased with increasing of salinity degree of soil.Under the salinization stress, it synthesizes and accumulates quickly betaine to maintain normal function of cell, which is a defensive reaction adapting to environment. In higher plants, the synthesis of betaine begins from choline, which was catalyzed by choline monooxygenase (CMO) and BADH。Both the CMO and BADH were induced by salinity and drought. They were studied more in gene engineering of salt and drought resistance .The gene of CMO and BADH have already been separated and cloned from Suaeda salsa and Zea mays, ascribed to Chenopodiaceae and Gramineae plants respectively and other plants.The systematic study of homogenic gene of CMO and BADH in Leymus chinensis is the first time. The seedlings of Leymus chinensis were the test material which were treated with different concentrations of saline-alkali solutions. Using rapid amplification of cDNA ends (RACE), according to BADH and CMO gene sequences of Chenopodiaceae and Gramineae plants registered in the GenBank, a pair of arbitrary degenerate primers were designed in the conservative sequence to amplify by RT-PCR and obtained specific bands 905bp and 520bp respectively. PCR product were recovered, cloned to T-vector, transformed to EcoliDH-5α, obtained recombinant plasmid, which were sequenced after enzyme digestion and PCR identity. It showed that the homology was highest by comparing BADH gene sequence with barley and wheat of Gramineae plant and that the homology was highest comparing CMO sequence with Zea mays and Oryza sativa .which was improved that the middle fragment of CMO and BADH in Leymus chinensis had obtained. Then applied 3′RACE and 5′RACE, obtaining 3′-end and 5′-end sequence of BADH and obtaining 3′-end and 5′-end partial sequence before middle fragment of CMO. Splicing overlap sequences by DNAman and amplified the full-length ,the length of BADH and CMO were 1774bp and 1283bp,named LcBADH和LcCMO respectively.A pair of specific primers, named CS-1and CS-2, designed on the ends of LcBADH open reading frame(ORF) with BamHⅠand HindⅢenzyme site respectively. The sequence obtained from LcBADH in Leymus chinensis through sequencing analysis, the length of ORF was 1521bp, encoding 506 amino acid, molecular weight was 56.78kDa. The primary structure of LcBADH contained a ten- peptide (VSLELGGKSP), a highly conserved Cys aldehyde dehydrogenase sequence related to enzymatic function and a SKL residue at C terminus. Comparing nucleotide sequence of coding region in Leymus chinensis to BADH registered in the GenBank, the results that showed LcBADH had high homology with Gramineae plant, such as Hordeum vulgare (95%),Oryza sativa (91%)and Hordeum brevisubulatum (90%).A pair of specific primers, named PLc-1andPLc-2, designed on the ends of LcCMO open reading frame (ORF) with BamHⅠa nd EcoRⅠe nzyme site respectively. The sequence obtained from LcCMO in Leymus chinensis through sequencing analysis, the length of ORF was 1029bp encoding 343 amino acid, molecular weight was 37.73kDa. Using bioinformatics software DNAman to analysis, LcCMO had high homology with CMO from Zea mays,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,HindⅢand BamHI,EcoRI respectively, recovered, ligated, constructed expression vector named pET30a(+)-LcBADH and pET30a(+)-LcCMO, transformed E.coli BL21(DE3). when the temperature was 37℃and final concentration of IPTG was 1.0mmol/L inducing different time, the result showed that LcBADH and LcCMO were induced by agarose-electrophoresis analysis, the molecular weight of expressing protein were about 56.78kDa and 37.73kDa respectively.