Functional Characterization Of Genes Associated With Polyamine And Proline Biosynthesis And Metabolism In Apple

Polyamines not only regulate plant growth and development, but also are involved in the responses to various stresses, while proline is an important osmotic protective substance. The biosynthesis and metabolism of polyamines and proline are catalized by lines of enzymes, such as arginine decarboxylase (ADC) and spermine synthase (SPMS) for polyamines synthesis, while ?1-pyrroline-5-carboxylate dehydrogenase (P5CDH) for proline metabolism. To elucidate the molecular mechanism underlying polyamines and proline biosynthesis and metabolism, MdADC, MdACL5, MdSPMS and MdP5CDH were isolated from apple. Among them, MdADC encoding for ADC, MdACL5 and MdSPMS for SPMS, while MdP5CDH for P5CDH. The functions of those genes were characterized with gene expression analysis and transgenic strategy. The main results are shown as follows.1. The full-length cDNAs of MdADC, MdSPMS and MdACL5 genes were isolated from apple'Gala'tissue cultures. Their expression vectors pBI121-MdADC, pBI121-MdSPMS and pBI121-MdACL5 were constructed and introduced into tobacco Nc89, respectively, with Agrobacterium-mediated transformation. Transgenic tobacco lines harboring MdADC, MdSPMS and MdACL5, respectively, were obtained.2. Semi-quantitative RT-PCR and HPLC assay demonstrated that the overexpression of respective MdADC, MdSPMS and MdACL5 brought about increased polyamines content. As a result, all transgenic tobacco plants exhibited altered phenotypes such as reduced plant height, shortened internode and enlarged leaf area.3. Abiotic stresses resistance analysis showed that all transgenic tobaccos harboring MdADC, MdSPMS and MdACL5, respectively, were more resistant to PEG osmotic stress, low temperature and high salinity than the non-transgenic control. Transgenic tobaccos exhibited higher polyamine content, proline content, antioxidant enzyme activity, activity of root system, as well as lower MDA amount and relative conductivity. In addition, MdSPMS overexpression resulted in more significant transgenic phenotypes and enhanced resistance than MdACL5.4. The full length cDNA of MdP5CDH was isolated from apple tissue cultures with RT-PCR and RACE amplification. Expression analysis showed that MdP5CDH gene constitutively expressed in various tissues or organs. Also, its expression was induced by low temperature.5. The prokaryotic expression vectors pET-30a-MdP5CDH and pGEX-MdP5CDH were constructed. Specific proteins in 65.14 kDa and 88.44 kDa were induced by IPTG. Therefore, the molecular weight of MdP5CDH-encoded protein was 61.74 kDa.6. The ORF sense expression vector pJIT166-MdP5CDH and a specific fragment antisense expression vector pBI121-RevMdP5CDH of MdP5CDH gene were constructed and transformed into apple'Orin'callus, respectively. Semi-quantitative RT-PCR indicated that MdP5CDH overexpression occurred in ORF sense transformant, while suppression in fragment antisense transformant.7. MdP5CDH overexpression brought about decreased proline content in transgenic callus, while MdP5CDH suppression increased proline accumulation. The growth curves of apple callus lines showed that either MdP5CDH overexpression or suppression effected callus growth.