Expression of a Novel Potato Patatin ClassⅠ cDNA in Transgenic Tobacco and Its Lipid Acylhydrolase Activity
Si Huaijun1 Liu Jun2 Xie Conghua1**
(1.College of Horticulture and Forestry, Huazhong Agricultural University, Wuhan 430070, China；2.College of Life Science and Technology，Huazhong Agricultural University, Wuhan 430070, China)
Abstract：Abstract: Patatin is the trivial name for a family of glycoproteins that accounts for up to 40% of the soluble protein in potato (Solanum tuberosum L.) tubers. Unlike most other storage proteins, patatin has a lipid acylhydrolase (LAH) activity. It is proposed that patatin may be involved in particular biological processes, especially tuber formation. Despite a detailed molecular and biochemical analysis of patatin, its physiological role and function, especially in potato tuber formation and regulation have not been elucidated. In order to approach this question, we have isolated a classⅠpatatin cDNA (GenBank accession No. AF498099) from a cDNA library constructed from wild diploid potato species Solanum chaconse under tuber inducing conditions. We constructed the expression vector pBSSP by fusing the patatin class I cDNA with CaMV 35S promoter and introduced it into tobacco without patatin gene to prove its expression and LAH activity. The expression vector pBSSP was introduced into Agrobacterium tumefaciens strain LBA4404 by electroporation and then transferred into tobacco (Nicotiana tabacum ) via Agrobacterium tumefaciens system. Leaves from 4-week-old plant grown in vitro of tobacco line T1 were used for transformation. Transformants were selected on MS medium containing 2.25 mg/L BA, 0.3 mg/L NAA, 100 mg/L kanamycin and 400 mg/L carbenicillin. When green shoots reached 1~1.5 cm, transferred them to the selective rooting MS medium supplemented with 50 mg/L kanamycin and 200 mg/L carbenicillin. A total of 45 kanamycin resistance plants were obtained. In order to prove patatin class Ⅰ cDNA was integrated into genome of tobacco, DNA was isolated from putative transformed and control tobacco plants. PCR analysis using patatin class Ⅰ cDNA specific primers was performed on 45 putatively transformed plants. There were 40 transformed plants showed a 500 bp amplification product which was missing in non-transformed control plants. The PCR amplification results were conformed by PCR-Southern blot analysis. The PCR products were electrophoresed on 1% agarose gel and transferred to positively charged nylon membranes (Boehringer Mannheim, Germany). Labeling, hybridization and detection were carried out using a DIG High Prime DNA Labeling and Detection Starter KitⅠ(Roche, Germany) following the manufacturer’s instructions. The results showed that the PCR product was really patatin cDNA fragment and patatin cDNA has been integrated intogenome of tobacco. Expression of the class I patatin cDNA in the transformed tobacco plants was also verified by Northern hybridization analysis using DIG RNA Labeling Kit (Roche, Germany). The results showed the predicted transcript of class I patatin cDNA in the transformed plants while no transcript in the control ones. This indicated that the class I patatin cDNA was well transcribed in the transgenic tobacco plants.The total soluble protein content of transformed plant leaves was measured by Bradford method. The highest protein content of transformed plant increased up to 6.4 mg/g FW that was 64.1% higher than that of the control plant. Lipid acyl hydrolase (LAH) analysis demonstrated that LAH activity was increased obviously in leaves of the transformed tobacco plants. The average LAH activity of 6 different transformed plants was increased by 67.7% compared to the control plant.The present experiment proved that this new classⅠpatatin cDNA expresses accurately and has normal function with LAH activity, and provides a foundation for a further study on physiological role and function of patatin to look into its relationship with tuber formation and regulation.
