Construction Of Cucumber CDNA Microarray And ESTs And Their Application In The Study Of Stress Physiology

Environmental stress is one of the most important factors that reduce plant growth and limit crop quality and yields. But the stress tolerance mechanism involves the expression and interaction of a repertoire of genes rather than individual genes. Fortunately, recent approaches using microarray provide powerful tools to the global gene expression under a variety of developmental or environmental conditions. The superiority of microarray lies in miniaturization, automation and parallelism permitting large-scale acquisition of quantitative biological information. Cucumber (.Cucumis sativus L.) , a kind of vegetable originated from tropical zone, is very popular in the world. In order to gain a better understanding of stress tolerance mechanism of cucumber, we designed and constructed a cucumber cDNA microarray to study the changes of gene expression profiles of cucumber plants under the stress of magnesium starvation, heat, CMV infection. Also, we constructed a root cDNA library of cucumber and identified the ESTs related to salt stress from cDNA libraries. The results are as follows:1. Microarray technology plays increasing roles in the study of plant gene function. By using BLAST biosoftware and data from Genebank, the specific primers for key metabolism enzyme genes in cucumber were designed, and the specific fragments amplified by RT-PCR were cloned and sequenced. Then, a total of 432 cDNA sequences corresponding to genes involved in photosynthesis, carbon fixation, carbohydrate metabolism, water-water cycle, signal transduction, hormone metabolism, defense, photorespiration and amino acid metabolism were obtained and categorized. At the same time, a comprehensive comparison analysis and optimization of the amplification methods of clone DNA, the purification methods of PCR products, the combination of the spotting robot with printing substrates were carried out. At last, a high quality cDNA microarray with 423 cDNA fragments and 9 quality control cDNA fragments were built. The microarray was then used to study the changes of gene expression profiles of cucumber plants after exposure to magnesium starvation. The results showed that 10 genes were down-regulated, while 12 genes were up-regulated in magnesium deficient plants. The microarray will provide us a powerful tool for the study of gene function in response to a series of stresses.2. Heat stress is a common abiotic stress that leads to economic losses in agriculture. The effects of heat stress on the cucumber (Cucumis sativus L.) transcriptome were analyzed using a spotted microarray with 432 cDNAs. Significant changes in transcript abundance were observed for 77 genes, among them, 42 showed down-regulated expression and 35 showed up-regulated expression. Six representative clones induced or repressed from microarray analysis were selectedfor real-time PCR analysis. The real-time PCR results were consistent with the results from microarray data. Analysis of these heat-responsive genes showed that they belonged to a wide range of functional categories. Many genes for photosynthesis and nitrogen assimilation were strongly repressed. Some defense-related genes, on the other hand, were found to be up-regulated. In addition to these expression changes, some function unknown or novel unreported genes that respond to heat stress were also identified. A complex set of metabolic adaptations appears to occur during heat stress and some genes, such as Hsp70-l might play an important part in thermotolerance.3. CMV is one of main diseases that affect yields and quality of crops seriously. The effects of CMV stress on the cucumber (Cucumis sativus L.) were analyzed by microarray and physiological measurement. Significant changes in transcript abundance were observed for 67 genes, 42 of which down-regulated expression and 35 up-regulated expression. Five representative clones induced or repressed from microarray analysis were selected for real-time PCR analysis. The real-time PCR results validated the results from microarray data. Analysis of these CMV-responsive genes showed that they belonged to a wide range of functional categories. Many genes for photosynthesis and nitrogen assimilation were strongly repressed. Some defense-related genes, on the other hand, were found to be up-regulated. A complex set of metabolic adaptations appears to occur during CMV stress and some genes, such as PRl-la, PAL might play an important part in defense. Meanwhile, in parallel with changes in photosynthesis and some physiological parameters during CMV infection were analyzed to evaluate the relationship between transcriptional and physiological changes. The result showed that this gene expression pattern reflected well the physiological changes in CMV-stressed leaves.4. Cucumber (Cucumis sativus L.) is a widely used and important vegetable in the world and a sensitive plant to salinity stress. Up to now, the genomic information and the gene expression patterns of cucumber have not been studied in detail. We constructed a NaCl-treated cDNA library of cucumber roots. The titer of cDNA libraries was 6.8×105 pfu/mL, matching the requirement of high quality library. The recombinant rates were 98.05%, and the inserts were about 1.0-2.0kb. There were 3.5×10~5 clones in total cDNA library. The results indicated that the librarie was integrated and efficacious.5. We sequenced more than 2700 randomly selected clones from the cDNA library of cucumber roots. By sequence analysis, 1548 unique genes were identified, of which 971 were significantly similar to the sequences in public databases. These database-matched ESTs were further grouped into 14 function categories including: metabolism (21.63%), energy (4.63%), cell growth and division (2.27%), transcription (7.62%), protein synthesis (7.93%), protein destinationand storage (7.51%), transporter (7.00%), intracellular traffic (8.24%), cell structure (7.51%), signal transduction (11.02%), defence (9.27%), secondary metabolism (1.85%), and unclear classification (10.92%). Nearly 140 unique genes were significantly similar to known plant genes potentially implicated in plant salinity responses. These genes associated with osmoprotectants biosynthesis, transporter proteins, reactive oxygen scavengers, stress proteins, signaling components, metabolism and energy, transcriptional regulators, and so on. Our study provides an overview of mRNA expression profile and a diverse genetic resource for cucumber plant research.6. In order to sequence efficiently, the feature of MegaBACE 1000 DNA analysis system and the key parameter affecting the quality of DNA sequencing were discussed. It showed that the quality of plasmid DNA was the key parameter affecting sequencing, and the homogeneity of the sequencing reaction products was critical to improve efficiency of high-throughput DNA sequencing. It was significant and useful for reducing the cost and improving the success rate of cucumber EST sequencing.