Cloning And Expression Analysis Of Chitinase Gene From Larix Gmelinii

Larix gmelinii is mainly distributed in northeastern China, which is the dominant species in the forest ecosystems of northeast region. The larch not only can survive in the extremely low temperature, but also can grow normally in the complex environment stresses, which is a good resource for the study of plant growth in the adversity. Chitinase is a pathogenesis-related protein in plants, which is an important portion of plant defense system and plays an important role in the resistance to low tempreture and pathogenesis. It has thermal hysteresis activity, which can reduce freezing point and inhibit the growth of ice crystal so as to protect the plants from frost damage.In this study, the cDNA sequence of Chitinase was isolated from L. gmelinii using RT-PCR, referred to as LgChi2. The ORF of LgChi2 is 831bp, which encodes 276 amino acid, with a predicted protein molecular weight of 28.4kDa and an isoelectric point of 7.83. The genomic DNA of the LgChi2 contains 1395bp including two introns and three exons. The result of phylogenetic tree suggests that the chitinase from larch, pseudotsuga menziesii and Picea abies had a relatively closer genetic relationship, which belong to ClassIV chitinase. Subcellular localization analysis revealed that the LgChi was localized in the cell periphery as a membrane-bound spot. Semi-quantitative RT-PCR results showed that LgChi2 was highly expressed in stem, which obviously induced by treatment of low temperature (4℃) in transcriptional level. The chitinase activity in L. gmelinii remained increase from 1 to 9d, and decreased at 11d after treatment with low temperature (4℃). The result suggested that the chitinase gene may play an important role in resisting to the low temperature stress in the larch.To detect the function and expression features of the LgChi, plasmid pEI101-LgChi and pET32a(+)-LgChi were constructed. Escherichia coli (DE3) cells harboring the recombinant plasmid were induced with IPTG and the fusion protein with an apparent molecular mass of 46 kDa was detected by denaturing polyacrylamide gel electrophoresis analysis. Overexpression of LgChi in E. coli enhanced cell growth under low-temperature. For investigating the expression feature of the LgChi in Arabidopsis, agrobacterium-mediated genetic transformation was performed Analysis of transgenic Arabidopsis indicated that overexpression of LgChi increased the activities of antioxidant enzymes under low temperature (4℃) stress, including ascorbate peroxidase (APX), catalase (CAT) and peroxidase (POD), that the activities of the antioxidant enzymes in the transgenic plants were higher than those in WT plants.Our findings may provide a theory foundation for elucidating the chitinase gene functions, gene expression regulation and antifreeze molecular mechanism of the larch, Meanwhile the study will provide a valuable candidate gene for genetic improvement of crops and forest.