The Physiologycal Changes Rule Of Cold Tolerance And Transcriptome And Expression Analysis Of Related Genes In Cucumber

Temperature is the main limiting factors of natural plant geographic distribution and yield formation, also one of the necessary conditions for crop growth and development. Cucumber(Cucumis sativus. L.), the thermophilic plant that is sensitive to temperature and weak to cold resistance, was one of the important type vegetable in the production of facility and open field cultivation in our country. But in the period of actual agricultural production, the chilling injury will happen often, deceased crop production and caused huge economic losses to the broad masses of farmers, that was a serious natural disaster of the agricultural production and this phenomenon performed more outstanding in Heilongjiang province. The study used cucumber seedling as the object under low temperature stress of 6 ℃, adapted chilling index, physiological and biochemical index determination, transcriptome, GO analysis of gene different expression, pathway analysis, real-time fluorescence quantitative PCR and RNAi technology et al, trying to figure out the plant’s physiological mechanism of low temperature stress reaction from physiological and molecular sides, and aiming to lay a foundation for cucumber cold tolerance cultivation、promoting the progress of cold tolerance genetic engineering、researching the regulation of key gene and improving the cold resistance by means of molecular, meanwhile providing a certain physiological and biochemical and molecular theory basis for cucumber breeding and cold tolerance cultivation.Through the survey of chilling injury index that had got 6 cold resistance strains, 5 cold sensitive strains and 9 intermediate type strains from 20 collected cucumber inbred lines, and identified that the cold resistance has no obvious relation with cucumber variety type. Cucumber had changed physiological and biochemical metabolism after low temperature stress, the relative electrical conductivity and osmotic regulation substance of soluble sugar、soluble protein、proline and MDA content were higher, cholophyll content was lower, SOD、POD and CAT activity were promoted. According to the chilling injury index, that had identified the related physiological and biochemical index of cold tolerance in cucumber. The relative conductivity used the increment as cold tolerance index, lower than 14.36 was cold resistance cucumber and higher than 25.67 was cold sensitive cucumber. The MDA content used increment or increase percentage as cold tolerance index, lower than 4.17 or 166.14% was cold resistance cucumber and higher than 5.98 or 223.97% was cold sensitive cucumber. The proline content used increment as cold tolerance index, higher than 54.00 was cold resistance cucumber and lower than 45.00 was cold sensitive cucumber. The soluble sugar content used increment as cold tolerance index, higher than 1.09 was cold resistance cucumber and lower than 0.63 was cold sensitive cucumber. The soluble protein content used increment as cold tolerance index, higher than 20.38 was cold resistance cucumber and lower than 14.90 was cold sensitive cucumber. The chlorophyll content used decrease amount or decrease percentage as cold tolerance index, lower than 0.48 or 36.36% was cold resistance cucumber and higher than 0.87 or 69.23% was cold sensitive cucumber. The SOD activity used increment or increase percentage as cold tolerance index, higher than 21.56 or 141.75% was cold resistance cucumber and lower than 13.92 or 59.05% was cold sensitive cucumber. The POD activity used increment or increase percentage as cold tolerance index, higher than 73 or 73.74% was cold resistance cucumber and lower than 16 or 16.84% was cold sensitive cucumber. The CAT activity used increment as cold tolerance index, higher than 12.85 was cold resistance cucumber and lower than 9.66 was cold sensitive cucumber. At the same time, the study had obtained the correlation of physiological and biochemical indexes, the chilling injury index, was significant positive correlated with relative conductivity and MDA content, and significant negative correlated with proline content, soluble sugar content, soluble protein content, chlorophyll content, POD activity, SOD activity and CAT activity.The study used the identified cold tolerance cucumber inbred line A, cold sensitive cucumber inbred line D, and they were disposed by 6℃ low temperature, named A1 and D1 as materials, so as to gain 14302 genes from A, 15302 genes from A1, 14843 genes from D, 14993 genes from D1 through the transcriptome sequencing. Through different expressed gene analysis, that had got 3353 up-regulated genes and 1629 down-regulated genes from A-VS-A1, and 886 up-regulated genes and 262 down-regulated genes from D-VS-D1; and discovered 1300 new transcript of material A, 1253 new transcript of material A1, 1264 new transcript of material D, 1213 new transcript of material D1. The alternative splicing had been obtained from experiment materials, 43716 from A, 49134 from A1, 38223 from D and 48501 from D1. By comparing consistency sequences with the reference sequence, thus had found SNPs, 36539 from A, 37004 from A1, 32967 from D, and 39189 from D1.Through GO function significant enrichment analysis, the experiment had identified: In A-VS-A1, the most enrichment were: non-membrane-bounded organelle of cellular component, total of 288 genes; hydrolase activity of molecular function, total of 107 genes; DNA replication initiation of biological process, total of 20 genes. In D-VS-D1, the most enrichment were: external encapsulating structure of cellular, total of 20 genes; oxidoreductase activity of molecular, total of 8 genes; response to chemical of biological process, total of 133 genes. Meanwhile, 376 different expression genes had been found in cold tolerance material and none in cold sensitive material, 167 genes up-regulated and 209 genes down-regulated.Through metabolic pathways analysis, cold resistance material had 278 differentially expressed genes and cold sensitive material had 83 differentially expressed genes in plant hormone signal transduction pathway; cold resistance material had 241 differentially expressed genes and cold sensitive material had 58 differentially expressed genes in plant pathogen interaction pathway; cold resistance material had 17 differentially expressed genes and cold sensitive material had 3 differentially expressed genes in porphyrin and chlorophyll metabolism; cold resistance material had 20 differentially expressed genes and cold sensitive material had 2 differentially expressed genes in peroxisome; cold resistance material had 6 differentially expressed genes and cold sensitive material don’t have the pathway in fatty acid metabolism; cold resistance material had 7 differentially expressed genes and cold sensitive material had none differentially expressed genes in photosynthesis; cold resistance material had 28 differentially expressed genes and cold sensitive material had 4 differentially expressed genes in arginine and proline metabolism; cold resistance material had 15 differentially expressed genes and cold sensitive material had 9 differentially expressed genes in regulation of autophagy. Not only located the differentially expressed genes in the protein and enzyme, and got the full length sequence of the differentially expressed genes.By using the real-time fluorescent quantitative PCR of up-regulated gene Cucsa.038100.1 and down-regulated gene Cucsa.178120.1, that had identified gene Cucsa.038100.1 expressed best in material A, and the expression was a little higher at disposed hour 0, reduced then reached to the highest expression time disposed hour 4, reduced then reached to another peak time. Gene Cucsa.178120.1 expressed best in material D at disposed hour 0, reduced then reached to the highest expression time disposed hour 4 in material A, reduced then reached to another peak time at disposed hour 12. Up-regulated gene had been identified to play a major role thorough comparative analysis.