Cloning And Characterization Of The Sucrose Synthase-encoding Gene From Muskmelon Fruit And Transformation

Muskmelon (Cucumis melon L.) is one of the major horticultural crops. The content and composition of soluble sugars in fruit play key roles in muskmelon quality. Sucrose, glucose and fructose are the main soluble sugars。In melon fruit, the accumulation and composition of the soluble sugars in melon fruit are controlled by sucrose metabolism enzymes including sucrose synthase (SS), sucrose phosphate synthase (SPS) and acid invertase (AI). The previous studies showed that the SS played a key role in sucrose metabolism in muskmelon fruit. Therefore, cloning and characterization of SS encoding gene from muskmelon is necessary to toward the genetic improvement of the quality of muskmelon fruit.In this study, we cloned the SS full-length cDNA from muskmelon fruit by RT-PCR and 3', 5' RACE and this clone was named CmSS1. The expression levels of CmSS1 in the tissue-organ and during fruit development in muskmelon were analyzed by Real-time RT-PCR. We constructed the antisense expression vector of CmSS1 and transformed it to muskmelon by ovary injection method and Agrobacterium-mediated method . Five transgenic plants were obtained by PCR screening. The main results are as follows:1. The full-length cDNA of SS gene from muskmelon fruit were cloned by RT-PCR and 3′, 5′RACE. The sequence analysis showed that SS gene contained 2585 bp and coded 804 amino acid residues.2. Real Time PCR analysis indicated that CmSS1 transcripts are easily detected in the leaves, stems, mature fruit, roots and flowers. The highest expression level existed in root, However, in mature fruit showed the lowest level. With the development of fruit, the expression level of CmSS1 showed a decreased trendy. CmSS1 mRNA strongly expressed in 5 DAP fruit, the lowest level existed in 30 DAP fruit.3. A 1.4 Kb fragment of the CmSS1 was removed from pMD18-T vector by BamHⅠand SalⅠdigested and the fragment was ligated into the plant expression vector pBI121 in the antisense orientation. We constructed the antisense expression vector of CmSS1 successfully by PCR and digested with restricted enzymes.4. Anti-CmSS1 was transformed to muskmelon by ovary injection, at present we got the injected seeds.5. Anti-CmSS1 was transformed to muskmelon by Agrabactria-mediated and five transgenic plants were obtained by PCR screening.