| Larix gmelinii is a deciduous tree of Pinaceae, which presents a large proportion of coniferous forests in northeastern China, including Inner Mongolia, and alpine region of northwest and southwest China, as well as a major coniferous timber. As a typical tree in coniferous forests of temperate zone and frigid-temperate zone, the larch has a wide horizontal-distribution and vertical distribution, which reach to the upper limit of forest vertical distribution and can safely overwinter even in the low temperature of-50degree. L. gmelinii should has a particular antifreeze mechanism, as well as valuable resources of antifreeze gene.Therefore, it has an important significance to develop the antifreeze gene resources and to analyze the antifreeze mechanism of of the larch.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. The expression of plant chitinase is low under conventional condition, however, the expression of chitinase can be induce by low temperature-, drought-, salt stress and the other adverse environment.Using RT-PCR and rapid amplification of cDNA ends (RACE) method, a991bp of full-length cDNA sequence of Chitinase were isolated from L. gmelinii, referred to as Lgchia, which includes an ORF of813bp, a5’-untranslated region of36bp, a3’-untranslated region of143bp. The predicted ORF encods a protein of270amino acids, with a molecular weight of80.1kDa and the theoretical isoelectric point of5.07. The predicted Lgchia protein were analyzed using online software:the result of secondary structure prediction showed that random coil is the major secondary structure and extend strand and alpha helix are subordinate structure for the Lgchia. The prediction of subcellular localization showed that the Lgchia is a secretory protein which located in the secretory pathway and existed in the cytoplasm. The prediction of transmembrane domains showed that Lgchia contains a trans-membrane region in the N-terminal, indicating that Lgchia is a membrane-binding protein.The analysis of functional domains showed that the Lgchia belongs to chitinase.glyco.hydro.19family. Phylogenetic analysis revealed that the Lgchia shows close genetic relation with chitinase of Pseudotsuga menziesii.Semi-quantitative RT-PCR showed that Lgchia expresses in roots, stems and leave of seedlings of L.gmelinii, and higher expression was detected in stems, while the expression in leave and roots was very lower than that in stems. The expression of Lgchia was abviously induced by low-temperature, drought and salt stress. The transcript level of the Lgchia rapidly increased when grown at4℃for12h, while a relative lower increase in Lgchia expression was detected by treatment with15%PEG. Duringr the gradient of treatment with low-temperature (4℃) for1,6,12,24h, rapid increase in expression of Lgchia was observed in seedling grown in4℃for1-6h. The expression of Lgchia in L. gmelinii grown in4℃for1-24h was obviously higher than that in control plants, indicating that the Lgchia plays an important role in antifreeze of the larch.These results will provide theoretical and practical proof for analysing the function and regulational mechanism of the Lgchia and antifreeze mechanism of the larch, and provide a valuable candidate for cold-resistence improvement using antifreeze genes of L. gmelinii. |
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