| The content and composition of soluble sugar in melon fruit play key roles in melon quality. Sugar accumulation and composition in melon fruit are controlled by sugar metabolism enzymes. The developments of plant biotechnology and molecular biology create a new path for studying on genes of melon metabolism enzymes and melon quality breeding.In this research, we constructed melon fruit cDNA library and cloned a cDNA fragment of melon fruit acid invertase gene. Melon fruit acid invertase (MAI) gene antisense expression vector was constructed and transferred into tobacco and melon especially mediated by agrobacterium tumefaciens strain LBA4404. Transgenic tobacco plants and kanamycin- resistance melon callus were obtained. The details were as follows:1. Construction of melon fruit cDNA libirayAt the beginning of 1990s, Clonetech in America invented SMART( the Switch Mechanism At the 5' end of RNA Transcript) technique. A modified oligo(dT) primer primes the first-strand synthesis reaction, and the SMARTIV Oligo serves as a short, extended template at the 5' end of the mRNA. When the RT reaches the 5' end, the enzyme' s terminal transferase activity adds a few additional nucleotides, primarily deoxycytidine, to the 3' end of the cDNA. The SMARTIV Oligo, which has an oligo(G) sequence at its 3' end base-pairs with the deoxycytidine stretch, creating an extended template. RT then switches templates and continues replicating to the end of the oligonucleotide. The resulting full-length ss cDNA contains the complete 5' end of the mRNA which then serves as a universal priming site in the subsequent amplification by LD PCR. However, at present this technique is seldomly applied to plants and the applicatin for melon hasn' t been reported. We firstly constructed a high quality cDNA library of yong melonfruit using SMART technique.Total RNA extracted from five days after pollination melon fruit was reverse transcripted into first strand cDNA using SMART technique. The double strand cDNA digested by sfi I (A and B) was ligated to SfiA and SfiB sites in the vector # TriplEx2. 8.54X105recombinants had been obtained after the mixture of ligation was packed in vitro and the host strain E. coli XLl-Blue was infected.100 clones was chosen randomly from the library and were tested cDNA inserts using PCR. The result indicated 81 percent of PCR products were over 500bp and the average length of cDNA inserts was 720bp. The recombinant rate of the library was 99. 53 percent by Blue/White Screening. These results showed that the cDNA library of melon fruit had been constructed successfully. This will provide theoretical basis for studying on the metabolism enzymes of melon fruit and other genes in the future.2. Cloning of a cDNA fragment of acid invertase gene from melon fruit and constructing of antisense expression vectorAt present, antisense RNA is used to inhibite some enzymes activities successfully. Antisense RNA can integrate into the genomic DNA of host and express stably. Antisense technology has been used in plant inprovement successfully for many species. The main measure for improving melon quality and increasing sugar content of melon fruit is to decrease the acid invertase activity in suitable period during the fruit development. Therefore, in this study we cloned a cDNA fragment of melon fruit acid invertase, constructed antisense expression vector and transformed it into tobacco and melon. These provides theoretical basis for transferring antisense acid invertase gene into melon and improving melon fruit quality at the level of molecular biology.PCR primers were designed based on the consensus domain of some acid invertase genes in GenBand. The aimed product with molecular weight of about 1kb was amplified from the total RNA isolated from 'Elizerbirth' melon fruit via RT-PCR.Then it was inserted into pMD18-T vector and sequenced. The result showed this cDNA fragment contained 1038bp and 346 amino acids determined by this fragment. The amino acid sequence shows 70-99 percent homo... |
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