Cloning And Characterization Of Sucrose Metabolism Enzymes Encoding Genes From Muskmelon And Transformating Anti-CmSPS1 To Muskmelon

Muskmelon (Cucumis melo L.) is one of the major horticultural crops. The content and composition of soluble sugar in fruit play key roles in muskmelon quality. Sucrose, glucose and fructose are the main soluble sugars, and the content and composition of sugars play an important role in fruit quality. Soluble sugar accumulation and composition in melon fruit are controlled by sugar metabolism enzymes including sucrose synthase (SS), sucrose phosphate synthase (SPS) and acid invertase (AI). Many researchers showed that the increase of SPS activity and the decrease of AI activity are necessary for sucrose accumulation in muskmelon fruit. Therefore, using modern molecular biology method to change the activities of sugar metabolism enzymes is great significance to quality breeding of muskmelon.In this study, we cloned the SPS and AI full-length cDNAs from muskmelon fruit by RT-PCR and 3', 5' RACE and analyzed the time-specific expressions of them. We constructed the antisense expression vector of SPS gene and transformed it to muskmelon by ovary injection method. Two transgenic plants were obtained by PCR and PCR southern blot. The main results are as follows:1. The full-length cDNAs of SPS and AI gene from muskmelon fruit were cloned by RT-PCR and 3', 5' RACE. The sequence analysis showed that SPS gene contained 3373 bp and coded 1054 amino acid residues. This gene has been registered in GenBank (Accession DQ521271) and named CmSPS1. AI gene contained 2178 bp and coded 636 amino acid residues (GeneBank accession EU260044), and named CmS-AIV1.2. Northern Blot analysis indicated that CmSPS1 transcripts are easily detected in the leaves, stems, and mature fruit, but cannot be detected at all in roots and flowers, suggesting that CmSPS1 may play a role in leaf, stem, and fruit development in muskmelon. CmS-AIV1 transcripts are easily detected in the flowers and mature fruit, but cannot be detected at all in leaves, stems and roots. To further investigate the developmental regulation of CmSPS1 and CmS-AIV1 in fruit mesocarp tissues, the RNA blot analysis were carried out using different mesocarp tissues from 5 DAP to ripening. The result showed that CmSPS1 mRNA was not detected in the mesocarp tissues before 20 DAP. However, the CmSPS1 strongly expressed in the mesocarp tissues after 25 DAP. In contrast to CmSPS1, CmS-AIV1 expression can be detected during fruit development, but the expression levels of CmS-AIV1 declined as fruit mature.3. A 830 bp fragment of the CmSPS1 was removed from pMD18-T vector by Hincâ…¡and Kpnâ… and the fragment was ligated into the plant expression vector pROK2 in the antisense orientation. We constructed the antisense expression vector of CmSPS1 successfully by PCR and digested with restricted enzymes.4. Anti-CmSPS1 was transformed to muskmelon by ovary injection and two transgenic plants were obtained by PCR and PCR Southern Blot. The transformation rate was approximately 0.25 %.