Screening and Functional Evaluation of Four Larix kaempferi Promoters

Promoters are powerful tools for breeding new varieties using transgenic technology. However, the low and unstable expression of target genes is still a limiting factor in <i>Larix kaempferi</i> (Lamb.) Carr (Japanese larch) genetic transformation. In this study, we analyzed <i>L. kaempferi</i> transcriptome data, screened out highly expressed genes, cloned their promoters, and constructed plant expression vectors containing the <i>β-glucuronidase</i> (<i>GUS</i>) reporter gene driven by these promoters. Recombinant vectors were introduced into the <i>L. kaempferi</i> embryogenic callus by means of the <i>Agrobacterium</i>-mediated transient or stable genetic transformation method, and the promoter activity was then determined by measuring <i>GUS</i> expression and its enzyme activity in the transformed materials. Four highly expressed genes were identified: <i>L. kaempferi Zhang Chen Yi-1</i> (<i>LaZCY-1</i>), <i>Zhang Chen Yi-2</i> (<i>LaZCY-2</i>), <i>Translationally Controlled Tumor Protein (LaTCTP)</i>, and <i>ubiquitin</i> (<i>LaUBQ</i>). The 2000 bp fragments upstream of ATG in these sequences were cloned as promoters and named <i>pLaZCY-1</i>, <i>pLaZCY-2</i>, <i>pLaTCTP</i>, and <i>pLaUBQ</i>. Semi-quantitative and quantitative RT-PCR analyses of transient genetic transformation materials showed that all four promoters could drive <i>GUS</i> expression, indicating that they have promoter activities. Semi-quantitative and quantitative RT-PCR analyses and the histochemical staining of stable genetic transformation materials showed that the <i>pLaUBQ</i> promoter had higher activity than the other three <i>L. kaempferi</i> promoters and the <i>CaMV35S</i> promoter. Thus, the <i>pLaUBQ</i> promoter was suggested to be used in larch genetic transformation.