| Chrysanthemum(Chrysanthemum morifolium)is known as one of the top ten traditional flowers in China and one of the four major cut flowers in the world.It has high ornamental value and economic value,and the market demand is strong.In the actual production process,heat stress and salt stress seriously affect the yield and quality of chrysanthemum.Excavating excellent resistance genes and conducting relevant gene function research,which is the premise of genetic engineering breeding for chrysanthemum resistance.Plant HSP90 protein plays an important role in response to abiotic stresses.At present,there are few reports on gene cloning and functional analysis of HSP90 in chrysanthemum.In this study,the CmHSP90.5 gene was cloned from the chrysanthemum’Jinba’,and its sequence structure,expression characteristics,and subcellular localization characteristics were analyzed.Than CmHSP90.5 gene was transformed into Arabidopsis thaliana by Agrobacterium-mediated inflorescence infection,and a preliminary study was conducted to determine whether the gene mediates the plant’s resistance to heat and salt stress.The main findings are as follows:1.We cloned a HSP90 gene from the chrysanthemum ’Jinba’.The complete open reading frame is 2,376 bp,which is highly homologous to the Arabidopsis HSP90 family gene AtHSP90.5,and is named CmHSP90.5.Sequence analysis showed that it encodes 791 amno acids;the predicted molecular weight of the CmHSP90.5 protein is 89.80 kD,and the theoretical isoelectric point is 4.90;the prediction analysis of subcellular localization shows that the protein is mainly located in the chloroplast;tissue quantitative analysis founds that CmHSP90.5 expressed in various tissues,and the expression level of the leaf tissues was the highest.Analysis of expression profiles under stress revealed that CmHSP90.5 was rapidly induced by heat and salt stress.2.We constructed pORE-R4-CmHSP90.5 plant expression vector and transformed CmHSP90.5 into Arabidopsis Col,and obtained CmHSP90.5 overexpression transgenic Arabidopsis.Transgenic plants and wild-type plants were treated with heat stress and salt stress.The results showed that the survival rate of transgenic Arabidopsis seedlings was significantly lower than that of wild-type Arabidopsis after treatment at 42 □ for 2 hours,the survical rate of the two transgenic lines were 21.7%and 14.7%,and that in the wild-type plants was 39.0%;after 24 h treatment at 42□,the relative electrolyte leakage rate of transgenic plants was significantly higher than that of wild-type plants,those in the two transgenic lines were 43.9%and 39.9%,and that in the wild-type plants was 25.1%;the total chlorophyll content of transgenic plants under heat stress was significantly lower than wild-type plants,the chlorophyll contents in the two transgenic lines are 17.3 mg·g-1 and 15.6 mg·g-1,respectively,and the wild-type control is 20.9 mg·g-1;The activities of SOD,CAT and APX enzymes in transgenic plants were significantly lower than those of wild-type plants before and after heat treatment.Under heat stress,the expression levels of AtHSFA7a,AtHSFB2b,AtHSP101,AtHSP15.7 and AtHSP17.6C genes in transgenic plants decreased significantly compared to wild type,indicating that CmHSP90.5 might affect the active oxygen pathway and regulate the expression of HSFs and HSPs thereby affects heat resistance.3.On 1/2MS medium containing 100 mM NaCl,the germination rate of CmHSP90.5 overexpressing transgenic Arabidopsis seeds was significantly lower than that of the wild-type plants.The germination rate of two transgenic lines were 66.7%and 56.7%,while that in the wild-type control was 84.3%;the green seedling rate of transgenic Arabidopsis seedlings was significantly lower than the wild-type plants,the green seedling rate in two transgenic lines were 20.7%and 10.0%,and that in the wild-type control was 35.3%.After 14 days of irrigation treatment with 250 mM NaCl,the fresh and dry weights of the transgenic plants were significantly lower than those of the wild-type plants.The fresh weights of the two transgenic plants were 0.20 g and 0.16 g,and the wild-type plants was 0.36 g.The dry weight was 0.04 g and 0.03 g,and the wild-type plants was 0.07 g;the soluble sugar content was significantly lower than the wild-type plants before and after salt treatment,and the proline content was significantly lower than the wild-type plants after salt treatment.It is speculated that CmHSP90.5 may weaken the osmotic regulation of plants in response to salt stress,thereby reducing the tolerance of plants to salt stress. |
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