| Melon is one of the best ten fruits in the world. And the melon variety Hetao, which is the major economic crop in the west Inner Mongolia, a typical respiratory climacteric fruit, exhibit extremely rapid decrease in flesh firmness after fruit ripening so that their production is seriously compromised.The full-length cDNAs encoding 1-aminocyclopropane-l-carboxylic acid synthase (ACSl),1-aminocyclopropane-l-carboxylic acid oxidase (ACOl), ethylene receptor ETR2 and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) were amplified by reverse transcription polymerase chain reaction (RT-PCR) from ripening fruit of melon (Cucumis melo L. cv. Hetao). The analysis of nucleotide sequences showed that the length of cDNAs were 1531bp (ACS1),1035bp (ACOl), 2304bp (ETR2) and 1939bp (HMGR) respectively. And these cDNAs encode 493aa (ACS1),318aa (ACO1),767aa (ETR2) and 588aa (HMGR) respectively. These nucleotide sequences shared high similarity to those of the melon ACS1, ACO1, ETR2 and HMGR reported previously. And the deduced amino acid sequences are consistent with those of the melon reported previously.The expression characterization of these genes during melon fruit development and ripening was analyzed by real-time quantitative RT-PCR. The results indicated that the expression levels of ACS1 and ACO1 began to increase at 30 day after pollination (DAP), then decreased after the highest expression levels were found at 40 DAP. The expression levels of the both genes increased by 60 times and 300000 times compared with those of 15 DAP, respectively. There were no changes of the expression level of ETR2 from 15 DAP to 30 DAP. The expression level began to increase at 35 DAP and the increasing continued until 45 DAP. The highest level was 9 times than 15 DAP. There was a peak of the expression level of HMGR at 25 DAP, that was 3 times than 15 DAP, and then decreased at 30 DAP, reincreased at 35 DAP. The expression level declined after reaching the maximum at 40 DAP that was 16 times than 15 DAP.After digestion with Aseâ… and Pmeâ… , the 35S-MCS-nos fragment was cloned into the plant binary vector pPZP221 (GenBank accession No.:U10491) digested with Aseâ… and Pmeâ… to form vector pPZP201 (35S-nos). Consequently, pPZP201 (35S-nos) no longer contained the selection marker aacC1 compared to pPZP221. The fragments of about 588bp and 710bp in the coding region of the ACS1 and ACO1 gene were constructed reversely into pPZP201 (35S-nos) respectively. The resulting plasmids pPZP201-ACS1 and pPZP201-ACO1 were digested with restriction enzymes Psyâ… and Paeâ… to produce vector-free and marker-free linear cassettes about 2.3kb. The melon cultivar Hetao was transformed with these linear gene cassettes via pollen-tube pathway. A large number of transformed seeds were obtained. The PCR data showed that the transgenes were inserted into the recipient's genome. Based on phenotype analysis, the T2 transgenic strains with antisense genes showed improved storage capacity. The RT-PCR analysis showed that the antisense ACO1 gene was expressed in the transgenic fruits. The T2 transgenic fruits with antisense ACS1 and ACOl showed endogenous ethylene production level at approximately 7% and 5% of those of wild-type fruits, respectively. These fruits demonstrated improved storage capacity without soft flesh and fungal infection, and exhibited extended shelf-life of 30 days compared to shorter than 12 days for the wild type fruits. The flowers of T2 transgenic melons with the antisense ACO1 gene were investigated. The results showed that there were not significantly differences between the transgenic flowers and the non-transgenic ones in pollen morphology and pollen germination. The nodes of bisexual flowers of the transgenic plants (11th node) were higher than the non-transgenic ones (8th node). Pedicel abscission of the transgenic plants was delayed in vitro condition compared with that of the non-transgenic plants. And the pedicels of the transgenic plants abscised an average of 9.6 hours later than the non-transgenic ones.
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