| Cucumber (Cucumis sativus L.) is one of the ten top cultivate vegetable crops in the world, and the total planting area and total yield of cucumber in China have taken the first place in the world. However, cucumber downy mildew, which is one of the most destructive diseases of cucumber leaves in cucumber production area, has great threat to cucumber production. In view of little is known about the molecular mechanisms of this disease and ESTs analysis has obvious advantages on studying functional genomics, the construction of cDNA Library from cucumber leaves infected with Pseudoperonospora cubensis and analysis on relevent ESTs have great meaning on studying the gene expressions in the process of cucumber leaves interacting with P. cubensis, cloning resistance-related genes and revealing resistance mechanism.In this research,10 different methods of extraction total RNA were evaluated about the availabilities for isolating total RNA from the different tissues of cucumber, such as roots, stems, leaves, and young fruit. As a result, the improved SDS method were chosen to extract total RNA from leaves of the disease-resistant cucumber cultivar '649' at 4 h,8 h,16 h,24 h,48 h and 72 h after infected by P. cubensis respectively, and then, equal quantity mixed as reverse transcription template, the full-length cDNA library was constructed used CreatorTM SMARTTM cDNA Library Construction Kit. Following, the positive clones were chosen randomly from the constructed cDNA library to sequencing, and finally the high quality ESTs were analysised through bioinformatics. The research has led to the following results:1. The results showed that cucumber belongs to the plant that RNA exaction easily; total RNA in four different tissues of cucumber was all extracted successfully with 10 different methods, but where was some discrepancy among the extraction effect. Overall, the extraction effect of total RNA in leaves and young fruit extracted by 10 different methods better than that of extracted from stems and roots. The RNAplant Reagent method and Improved SDS method have better extraction effect of total RNA from leaves; the Trizol Reagent method, the SDS method and the improved SDS method have better extraction effect of total RNA from young fruit; the purity quotient of total RNA in stems, roots extracted by 10 different methods was all not high, but the integrity are all better.2. In the prosess of constructing cDNA library, the cycle number of LD-PCR was determined to be 21, and a better ligation effect was obtained when the proportion of cDNA and pDNR-LIB vector was 1.5:1. The results showed that the primary titer of the constructed cDNA library was 5.5×106 pfu·mL-1, and 6.5×109 pfu·mL-1 for amplified library. The recombination rate was about 99%. The size of inserted cDNA fragment ranged from 0.5 kb to 2 kb, majority at about 1 kb.3.3360 positive clones were chosen randomly from the constructed cDNA primary library of Cucumber leaves to sequencing and a total of 3091 ESTs were obtained successfully. At last,2903 high quality ESTs were acquired after removing vector sequences, repeat sequences and short sequences (<100 bp) with SeqClean software. The ESTs length ranged from 101bp to 604bp and the average length was 414.6bp. The average content of GC was 42.44%.4.2507 unigenes (included 211 contigs and 2296 singlets) were clustered obtained with CAP3 program from 2903 ESTs and the proportion of the novelty was 86.36%. The length of unigenes ranged from 101 bp to 1426bp and the average length was 422.73bp. The average content of GC was 38.21%. Besides, there were 53 unigenes with length above 600bp.5.The analysis of genes expression abundance results showed that the number of high abundance expression genes (expression frequency≥5) was 18, which took 0.72% of the total. The number of middle abundance expression genes (5>expression frequency≥2) was 139, which took 7.69% of the total. The rest were low abundance expression genes, its number took 91.60% of the total. This distribution indicated that most gene in the cucumber leaves were low abundance expression genes.6. Homology origin analysis showed that 1653 unigenes (65.94%) had matching sequences in the NCBI nr database. There had comprehensive sequence homology origin and the origin more than 110 species, which mainly were Cucumis sativus (23.29%), Vitis vinifera (13.43%), Populus (13.19%),Glycine max(10.41%) and so on.7. A total of more than 500 function-known proteins were obtained by blastx analysis used 2507 assembled unigenes in the NCBI and Swiss-Prot database and these proteins could be divided into four categories based on their function annotation. The protein function of 1547 unigenes with function known or function putative were determined by UniProt database and the results were then used to construct gene expression map. A total of 2231 functional genes were given (including one-gene with multi-effect), including 427 (19.16%) plant disease resistance/defense related genes and 56 (2.51%) plant signal transduction related genes.8. The 2507 assembled unigenes were analyzed by blastp in the COG database, and the results showed that there were only 662 unigengs in the COG database obtained annotation information, referring to 23 protein functions, which including 351 (53.01%) information storage and processing related genes,207 (31.27%) metabolism related genes,44 (6.65%) cell processing related genes,5 (0.76%) disease resistance/defense related genes and 55 (8.31%) function indefinite genes.9. The 2507 assembled unigenes were analyzed in the KEGG database, and the results showed that a total of 1851 unigenes had related annotation which involved 44 non-repeat metabolic pathway. Pathway analysis showed that these metabolic pathway mainly focused on various types of amino acids synthesis and metabolism, organics synthesis and metabolism, it also included 3 kinds of signal transduction pathway,3 kinds of disease occurrence pathway,1 antigen processing pathway and 1 cholera bacillus infection pathway. 10. A total of 468 unigenes had no any information through blast in NCBI, Swiss-Prot, KEGG and COG database, and finally they were identified as new genes.
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