Physiological Responses Under Cold Stress And Expression Analysis And Cloning Of ELI Gene Of Winter Rapeseed(Brassica Rapa L.)

To study the cold-resistance mechanism of winter rape during cold-resistance provides more basis for winter rapeseed breeding.Experiments were conducted on winter rapeseed with different cold-resistance levels.The photosynthetic characteristics,physical and chemical properties,and cold-resistant gene expression levels were studied.The results are as follows:1.During the cold stress process,the photosynthetic characteristics of different cold-resistant winter rapeseed.seedlings showed the same change trend,but the degree of change was different.After low temperature stress,the chlorophyll content of functional leaves of winter rapeseed.showed a decreasing trend,and the chlorophyll content of functional leaves of winter rapeseed.with stronger cold resistance was decomposed faster.Photosynthesis gas exchangs parameters were easily affected by temperature.Photosynthetic rate（Pn）,Stomatal conductance（Gs）and Transpiration rate（Tr）of the leaves were all decreased after treatment at low temperature,and the content of leaves Intercellular CO₂concentration（Ci）was increased,which relieved the consumption of nutrients at low temperatures,and provides sufficient organic compounds for the cold resistance of winter rapeseed.2.In the process of low temperature stress,the physicochemical properties of winter rapeseed were changed.At 4°C,the contents of Malondialdehyde（MDA）,Soluble sugar（SS）,Soluble Protein（SP）,Superoxide dismutase（SOD）,Peroxidase（POD）,and Catalase（CAT）in different cold-resistant winter rapeseed increased,and Soluble sugar（SS）,Soluble Protein（SP）,Superoxide dismutase（SOD）,Peroxidase（POD）,and Catalase（CAT）could effectively protect the leaf membrane structure and prevent the loss of water in the leaf cells.which also increased the cell fluidity of the plant leaves.Superoxide dismutase（SOD）,Peroxidase（POD）,and Catalase（CAT）can effectively reduced the Malondialdehyde（MDA）content and alleviate the damage caused to the leaves.Therefore,these physical and chemical indicators reflect the difference in cold resistance.3.Transcriptome of 11 genes were differentially screened to quantitative fluorescence analysis showed,When the temperature dropped to 4°C,the relative expression level of KIN₂in winter rape with relatively weak cold resistance was increased,and the relative expression level of this gene in winter rapa with relatively low resistance to cold was decreased.The relative expression levels of OLEO1 and ELI were completely different with KIN₂.In contrast, relative expression of RS281 was up-regulated,and varieties with strong cold resistance were up-regulated,and relative expression levels of LEAD8,MAM1,CYT1,KIN₂,CHSY,unknown genes,and APRR9 genes were down-regulated.The relative expression level of RS281 was up-regulated,and the varieties with strong cold resistance were up-regulated.The relative expression levels of LEAD8,MAM1,CYT1,KIN₂,CHSY,unknown genes and APRR9 genes were down-regulated.In the course of temperature reduction,these genes act simultaneously to increase the plant’s ability to resist cold,mitigate the damage caused by the low temperature environment,and increase its resistance to low temperature stress.4.The cDNA sequences of Lenox,Neib,Tianyou2（T2）,Tianyou4（T4）,Longyou6（L6）,Longyou7（L7）,Longyou8（L8）,and Longyou9（L9）ELI genes in winter rapeseed were amplified by PCR.The sequence identity of the ELI gene in the transcriptome was as high as99.06%with Lenox,Neib,Tianyou2（T2）,Tianyou4（T4）,Longyou6（L6）,Longyou7（L7）,Longyou8（L8）,and Longyou9（L9）clones.The gene protein encodes 195 amino acids with an isoelectric point of 9.24;the relative molecular mass is 20.4728 kD;and the encoded protein is mainly composed of a-helix and random-curbed hydrophobic stable protein.The alignment analysis of 8 rape varieties revealed that there were 21 mutations in the ELI gene.The amino acid sequence comparison revealed that there were 6 mutations,of which the conserved region Longyou8（L8）had 3 mutation points,and Tianyou2（T2）,Tianyou4（T4）,Longyou6（L6）,Longyou7（L7）,and Longyou9（L9）had 1 mutation point.