Cloning Of TPI And Analysis Of Related Genes In Response To Stress In Leymus Chinensis

Leymus chinensis, which has good tolerance to cold, drought, salt-alkali and leanness, plays important roles in ecological and environmental protection. Yellow-green and gray-green are two ecotypes of Leymus chinensis, which display significant differences in phenotypes, physiology of photosynthesis, and stress resistance. However, the molecular mechanism of such differences has not been elucidated. In this work, we analyzed the expression of LcGAPDH and LcTPI, which are related to carbon metabolism and show different expression level in the above-mentioned ecotypes. Meanwhile, the gene of LcTPI was cloned, the construct of p1304-LcTPI was made and transformed into Arabidopsis, and a primary functional analysis of LcTPI was also conducted.Triose phosphate isomerase(TPI) is a key enzyme in carbon metabolism, which is involved in stress response and is closely related to the metabolism of methylglyoxal(MG) in plants. By utilizing the RACE cloning technology, we got two full-length LcTPI cDNA, which have 1069 bp and 1053 bp, from yellow-green Leymus chinensis. There are 762 bp in the open reading frame(ORF), encoding a protein of 253 amino acids. The LcTPI full length DNA of yellow-green was 2787 bp, and that of gray-green was 2776 bp.Bioinformatic analysis showed that the molecular weight of LcTPI is 26.94 kDa, and the isoelectric point is 5.57. It has 4 glycosylation sites and 15 phosphorylation sites. Lc TPI is a member of the TIM family and has a highly conserved structural motif. The percentages of ?-helix, random coil, ?-strand, and ?-turn in the protein are 47.83%, 28.85%, 16.21%, and 7.11%, respectively. Based on the analysis of phylogenetic tree, this protein is more evolutionarily close to the homologous protein in monocotyledon species.LcTPI coding sequence was cloned into pCAMBIA1304 plant expression vector. The p1304-LcTPI construct was introduced into Agrobacterium tumefaciens GV3101, and then transformed into Arabidopsis thaliana plants by floral-dipping method. Transformed plants were selected by growth on hygromycin-containing medium and identified by PCR. Compared with the wild type, the transgenic plants showed increased tolerance to methylglyoxal.The plants of yellow-green and gray-green of Leymus chinensis were treated with 200 mM salt, 200 mM alkaline and 20% PEG respectively, and the expression of LcTPI and LcGAPDH in response to abiotic stress was analyzed by qRT-PCR. The expression of LcTPI and LcGAPDH was altered responses to salt-alkali and drought. The expression of LcTPI and LcGAPDH between two ecotypes was differences under salt-alkali and drought, which may be associated with the various resistance to abiotic stress between two ecotypes of Leymus chinensis.