| Cucumber (Cucumis sativus L.) is a kind of important vegetable in the world. It is highly nutrient and popular with people. It originated from the northern part of India, south of the Hymalays. Cucumber had been cultivated in India 3000 years ago. With the migration of the south Asians, cucumber spreaded to the south of China, southeastern Asia and Japan etc., and has been cultivated in China for 2000 years. Cucumber is a kind of annual climber plant that likes to grow in warm condition. The interval between cucumber's seeding and harvest is so short that it is adaptable for all-year cultivation. The ovary is the edible part, crisp and alimental. The common theories and methods of plant genetic breeding had lost their advantage in improving physiological character. It means that the genetic improvement of character became smaller because of long-time evaluating the breeding value by phenotypic value. With the development of molecular genetics and biotechnology, there comes to possibility to improve the plant physiological character on the molecule level. Owing to the limitation of inner-specific resource, gene project will be the effective way to recombine the useful genes in the improvement of cucumber breeding. The research of the cucumber genes can provide the foundation for further molecular breeding. It is easy to sample and analyse young fruit of cucumber that possess all genes in mature fruit. Using cucumber fruit as material and TriplEx2 as vector, Cucumber (Cucumis sativus L.) cDNA library has been constructed in our study. We selected and sequenced partial colones, compared the homology and analyzed the gene function by bioinformation method. 1. Construction of Cucumber (Cucumis sativus L.) cDNA libraryThe total RNA of Cucumber (Cucumis sativus L.) was extracted with Trizol kit, and the mRNA was extracted with PolyATtract (?)) mRNA Isolation SystemIII, the agar gel electrophoresis showedthe total RNA has three bands and the degree of purity could fit for the following experiments.Using SMART IV Oligonucleotide and CDSHI/3' PCR Primer as primer, PowerScriptTMReverse Transcriptase as polymerase, first-strand cDNA has synthesized and following dscDNAsynthesized by primer LD-PCR. After PCR purification and sfi I digestion, small cDNA fragmentand some rRNA removed by CHROMA SPIN-400 column. Then cDNA fraction was ligated to TriplEx2 vector and packaged , cDNA library was gotten.Packaging product can be stored at -80℃ for at least one year in 7% DMSO.2. Identifying the quality of cDNA library1:10 dilution of the packaging extract was made and mixed with XL 1-Blue overnight culture, in which, 0.7% top agar, IPTG and x-gal were added. The mixture was poured onto 90mm LB/MgSO4 plates prewarmed to 37℃ and the plates were inverted and incubated at 37℃ overnight. By counting the plagues and calculating the ratio of white (recombinant) to blue (nonrecombinant) plaques, the titer of the phage was 1.165×106pfu/ml, and the recombinant percentage was 94.17%. Using EcoR I and HindIII digestion, the results show the length of inserting fragments were beyond 500bp. The titer of amplified library was about 1010 pfu/ml.3. EST sequencingPartial clones were selected from cDNA library, after EcoR I and HindIII. digestion and 1% agar gel electrophoresis, the result showed most of the clones had inserting fragment. In the experiment, 141 positive clones were sequenced with pTriP5 ' Sequencing Primer (CTCCGAGATCTGGAC) and the success ratio was 98.58%. 36 full-length sequences were obtained and the longest fragment was 706bp. 79.43% fragments gathered in the zone of 500~700bp.4. Analysis of sequencing resultThe analysis showed that some sequences encoded the same gene. After assembling by clustal W, we obtained 116 unigenes. Comparing the homology and analyzing the gene function by bioinformation method, we found 97 unigenes had significant similarities with the known EST, and the other 42 unigenes had low or no similarities with the known genes or EST sequences.
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