| Cold resistance in plants is a complex quantitative characteristic involving genes in large quantities.Isolated cold resistance-related genes exert limited effects on cold resistance.The lack of major genes with high contribution rates is the major limiting factor for molecular breeding of cold-resistant crops.Research on AFPs is an important entry point for this area.Antifreeze proteins were found to directly bind to the surface of ice crystals,reduce the freezing point of solutions and eliminate ice crystal hazards,playing a critical role in cold resistance.’Longyou 7’(Brassica rapa)can resist an extremely low temperature of-30°C.It is the most cold-tolerant winter rapeseed variety in China.The effects of biochemical characteristics and photosynthetic characteristics were studied.The antifreeze protein BrAFP1 of winter rape was isolated and identified from the seedlings of Longyou 7 after low temperature treatment.The BrAFP1 gene was cloned and the spatial and temporal characteristics of BrAFP1 gene expression at low temperature were analyzed.The gene function was preliminarily verified,and the main results are as follows:The effects of low temperature stress on physiological and biochemical characteristics and photosynthetic characteristics of winter rape seedlings were studied.The results showed that(1)at room temperature,the chloroplasts of winter rape.seedlings were spindle or elliptical,and the buttress structure of grana lamellae was intact and clear.The stroma lamellas were arranged neatly and orderly,and the chloroplast contains starch granules.After treatment at-4°C for 24 h,the chlorophyll content in leaves of winter rape.seedlings decreased significantly.The number of chloroplasts in Tianyou 4 variety of winter rape.with weak cold-resistant variety decreased,part of chloroplast outer membrane ruptured,matrix slice fractured with only leaving lamellar fragments.There was no starch granule accumulation in the chloroplast,the vacuolar membrane contracted obviously,and the vacuole volume became significantly smaller;the stomata were in the closed and semi-closed state;the Longyou7 granule could still maintain the integrity in its buttress structure,and most of the stroma lamellas were completely continuous.A small number of stroma lamellas were damaged,and a small amount of starch granules accumulated in the chloroplast,and the vacuolar membrane was intact;the stomata were in a semi-closed state.(2)Antioxidant enzyme activities(SOD,CAT,POD,APX),osmotic adjustment substances(SS,SPr,Pro)and malondialdehyde content(MDA)and relative electrical conductivity in seedlings’ leaves of different cultivars of winter rape.under low temperature stress all showed the increasing trend.The increase range of electrical conductivity was greatly different due to the different cold resistance of the variety.The increase range of strong cold-resistant variety was less than that of weak cold-resistant variety,indicating that the strong cold-resistant variety can better maintain the integrity of the plasma membrane.The anti-freeze protein BrAFP1 of winter rape.was isolated and identified,and its sub-cellular localization was carried out.The BrAFP1 gene was cloned,and the main results were as follows:(1)The whole protein extract in seedling leaves of Longyou 7 variety after low temperature treatment isolated the protein with ice crystal morphology modification and recrystallization inhibition activity.It was identified by mass spectrometry as β-1,3-glucanase,which was the antifreeze protein of winter rape,named BrAFP1,and BrAFP1 was localized in the nucleus and cytoplasm.(2)ORF sequence length of winter rape.BrAFP1 gene was 1032 bp,encoding 343 amino acids,and containing signal peptide,with 38.130 kDa at molecular weight.The expression of BrAFP1 gene was up-regulated under low temperature stress,indicating that this gene played an important role in the low temperature response of winter rape.The BRAFP1 overexpression transgenic lines and Arabidopsis afp1 mutants were used to initially analyze the function of BrAFP1 gene.The results showed that compared with wild-type plants,the expression level of BrAFP1 gene in over-expressed transgenic plants under low temperature increased by 3.76 times.And its electrical conductivity and MDA content were significantly reduced;while the expression level of the mutant afp1 gene was maintained at a very low level,the osmotic adjustment substance content was significantly reduced,and the electrical conductivity and MDA content were significantly increased.It indicated that overexpression of BrAFP1 gene at low temperature could increase the cold adaptability of plants to the environment,while mutant afp1 plants were more sensitive to low temperature.The BrAFP1 gene in 14 varieties of winter rape.with different cold resistance having been cloned encoded the hydrophobin with 343 amino acids.There were 5 mutations in its base,including 3 synonymous mutations(488,749 and 992 sites)and 2 sense mutations(52 and 607 sites),resulting in amino acid substitution at 2 sites.The typical representative varieties of different cold-resistant winter rapes includes Longyou 7,Longyou 8 and Tianyou 4 varieties were selected as materials to analyze the temporal and spatial characteristics of BrAFP1 gene expression.The results showed that:(1)the expression level of BrAFP1 gene in leaves increased significantly after 3 h of low temperature treatment,and reached the highest value at 18 h-24 h.Then,with the increase of cold stress time,the expression level began to decrease rapidly;While,the expression level of BrAFP1 gene in leaves was significantly higher than that in petiole and root tissues.In addition,the expression of BrAFP1 gene in leaves of winter rape.seedlings was significantly induced after cold stress at 4°C and-4°C;While the expression of BrAFP1 gene was significantly decreased after cold stress at-8°C,and the leaves of plants showed obvious symptoms of freezing damage.(2)Longyou7 variety of super-strong cold-resistant winter rapes and Tianyou4 variety of weak cold-resistant winter rapes at both ends of the varieties were selected as the materials.After the seedlings were treated at – 4°C for 0 h,12 h,24 h and 48 h,the results showed that: After 24 h of treatment,the expression level of CNGC and ICE1 genes in seedling leaves of Longyou7 variety increased significantly,while the expression level of OST1 and CBF2 genes was cold induced after 12 h treatment. |
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