Cloning Of Polygalacturonase (PG) Gene In Melon (Cucumis Melo L.) And Construction Of The Antisense Sequence Plant Expressing Vector

Most of Cucumis melo are typically climacteric fruits. When the fruits are physiologically mature, a climacteric peak appears soon during the post harvest period. That leads to softening and rot of fruit flesh and greatly decreases quality of the storage and transportation. According to statistics, the loss rate of Xinjiang Hami melon stored in short-term at the sale area was up to 5%~15%, and that of Gansu Huanghemi melon was about 29.3%. Therefore, the effective way to solve the difficult problem is to breed varieties that can endure store transportation.The softening and mature of fruits related to degradation of a certain number of pectin, which mainly due to the role of Polygalacturonase (PG). Many experiments have proved that the content of mRNA of PG1 is the most amounts and it can degrade gelatin in the cell wall during the fruit ripening period. The application of constructing a plant expression vector containing PG gene by antisence gene technology and transfer it into tomato plants has been succeed, and it had a good prospect for commercialization. In melon crops, Kristen had successfully cloned a cDNA complete sequence of melon PG1 gene,which laid the foundation for the use of antisense PG1 gene to regulate the storage and transportation period.The study was a part of the project that aimed to keep fresh and prolong mature period by molecular breeding method. The general goal of the study was to obtained new varieties of melon through genetic transformation technology. The objective of the study was to obtain the complete sequence of PG1 gene and construct the plant expression vector transferred into tobacco to show the plant expression vector was successfully construed. The studies laid a solid base for further transformation of Cucumis melo varieties with the PG1 gene to improve the quality of melon fruitsThe results showed as follow:1. One special primerfor was designed according to the MPG1 cDNA.Special amplification reaction was carried out using the plasmid DNA extracted from the pCMPG/E.coli as template. The amplification production was tested by electrophoresis and the target gene fragment the size of which was about 1200bp was recovered. Then ligated it with pGEM-T Easy Vector and the clone vector was obtained.2. Plasmid pGEM-PG and pBI121 was double digested with restriction endonucleases SacI and XbaI to retrieve the PG1 gene and the linear vector without gus gene. Thus the linear pBI121 and the PG1 had reversely matching ends. After ligating the two fragments DNA,transformed into E.coli cells. Indentification of restriction map by SacI/XbaI digestion indicated that 1200bp fragment was obtained. The inserted directions were identified by EcoRâ…¤digested.Thus,the...