| Huanghemi muskmelon (Cucumis melo cv. Huanghemi ) plant were wildly accepted by the farmers in Gansu province, because of its delicious taste, rich nutrition and sweat aroma. But the production was seriously affect by postharvest diseases during storing and transportation. During ripening and senescence of respiratory climacteric fruits, Ethylene has an important role. In order to reduce the losses caused by postharvest diseases, it is necessary to control the production of ethylene during the fruit ripening. However, it has not much improvement in dealing with this problem by using traditional methods. We sought to solve this problem by biotechnology. To obtain transgenic muskmelon plant that resists diseases, antisense gene encoding an amino-cyclopropane-1-carboxylie acid synthase (ACS) was introduced into muskmelon plant by antisense RNA technology. Antisense RNA, a kind of posttranscriptional gene silencing (PTGS),which presumptive mechanism is that sense and antisense transcripts from genes were brought together by crossing to form double-stranded RNAs, and then these double-stranded RNAs were incised into 21-25nt interferential small RNA(siRNA).The siRNA is involved in triggering homology-dependent gene silencing(HDGS). In the pathway of Ethylene biosynthesis, ACC is the direct pre-cursor of Ethylene. The conversion of SAM to ACC was recognized as the rate-limiting step which was catalysed by ACS. ACS is encoded by multigene families, and its expression is tissue- and cell-specific. If the expression of ACS gene was silenced by antisense technology, production of Ethylene can be inhibited, and the ripening of fruit can also be retarded. So the fruit will have a long storing life, and posthavest diseases will strongly reduce. In this study, Total RNA, isolated from wounded mesocarp tissue of mature muskmelon (Cucumis melo cv. Huanghemi) fruit, was successfully amplified by Reverse Transcription-Polymerase Chain Reaction ( RT-PCR ). Two specific target sequence of ACS cDNA segments (706bp and 974bp) were obtained. The 974bp cDNA fragment then was inserted into a cloning vector pGEM-3zf digested with Sma I and the recombinant was named pACS44, while the 706bp cDNA fragment was inserted into a cloning vector pBluscript IISK(+) digested with EccRV and the recombinant was named pACC. The recombinants were confirmed by sequencing. The two cDNA sequences shared highly homologous to that of the ACC synthase cDNA from other Cucubitae plants, especially ripening-specific muskmelon ACS(ME-ACSA)gene(99%). This indicate that the cloned ACS cDNAs are relate to ripening process of fruit.The 974bp length cDNA sequence was submitted to Genbank database, and the Genbank accession number is AY320515. The 974bp fragment which was digested with Sad and Xba I was inserted into a binaryvector pBI121 digested with the same enzymes in a reverse orientation drived by cauliflower mosaic virus 35S ( CaMV 35S) promoter and nopaline synthase (NOS) terminator, giving a construct containing an antisense ACC synthase gene. PCR analysis demonstrated that the foreign antisense cDNA fragment has been seccesfully introduced into the binary vector pBI121. The construct was named pBACS. The cotyledons explants, embryonic callus tissue explants and leaf disc explants transformation/ regeneration system was respectively modified for miskmelon(Cucumis melo cv. Huanghe). Both embryonic callus tissue explants and cotyledon sections explants can be used to regenerate transformed plants. But the cotyledon section explants more easily give rise to shoots than the embryonic callus tissue explants, and the frequency of regeneration is very high(91%). Disarmed Agrobacterium tumefaciens strain EHA105(rif)containing antisense vector pBACS was used for transformation. In this way .antisense ACS gene was introduced into muskmelon plants. By tissue culture, the efficient regeneration systemfor muskmelon using cotyledons as explants was established, and the elongated transgenic muskmelon shoots were obtained.
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