Cloning And Characterization Of Transcription Factor CBF1 And Actin Gene From Larix Gmelinii

Low temperature is one of the key factors that greatly restricts growth and distribution of plants. Plant has specific mechanism to resist low-temperature stress, such as regulating the characteristic of biomembrane, osmoregulation, and so on. Cold-regulated (COR) genes play an important role for regulating metabolic activity under cold-stress conditions. Transcription factor CBF is a class of trans-acting factor, who is specifically induced by low temperature, and can control expression of COR genes whose promoter contain cis-acting elements, to activate a variety of cold-resistant mechanisms in plants. Therefore, the research on expression and regulation of CBF genes is significant for understanding cold-resistant mechanism in plants.In this study, a CBF1 ortholog was cloned from Larix gmelinii, referred to as LgCBFl, and its expression pattern was investigated. L. gmelinii is the major coniferous tree species in the plantation forests of northeast China, which has strong resistance to cold-stress. Therefore the research on LgCBF1 is very important for understanding the cold-resistant mechanism in L. gmelinii. To investigate the expression pattern of LgCBF1, a Actin gene was also isolated from L. gmelinii, referred to as LgAct, and the gene structure and phylogenetic evolution was analyzed. Actin gene has a stable transcription even under stress condition, therefore, it can be used as a control to investigate expression levels of functional genes in L. gmelinii.The main results are as follows:1. An ortholog of CBF1 transcription factor was isolated from L. gmelinii by degenerate-PCR. The result of blast search showed that the LgCBF1 gene has high sequence similarity to CBF1 genes of other plant species.2. Semi-quantitative PCR was used to investigate the expression level of LgCBF1 gene under low-temperature stress. The transcription of LgCBF1 was activated after treatment (4℃) for 10 min, and the maximum level of the Lg-CBF1expression was induced after 6 hours. When the cold-treated seedlings were transferred to 22℃, the expression of LgCBF1 significantly decreased, indicating that the transcription of LgCBF1 gene is rapidly regulated by cold-stress.3. Full-length sequences of L. gmelinii actin gene, referred to as LgAct, were isolated by using degenerate-PCR and RACE technique. The cloned LgAct cDNA was 1662 bp in length, encoding a protein of 377 amino acids, which shows more than of 77% nucleotide sequence similarity and more than of 93% amino acid sequence similarity to other plant actin genes. Genomic DNA of the LgAct contains 2564 bp, including 4 introns and 5 exons. The phylogenetic tree generated from the deduced amino acid sequences of plant actins revealed that the L. gmelinii and Picea abies actins is in a sister group to the clade of angiosperms, corresponding to plant evolutionary history.