| Sorbus pohuashanensis(Hance)Hedl.is a deciduous tree belonging to Sorbus L.Rosaceae.It has high ornamental,ecological and medicinal value.S.pohuashanensis grows naturally in slopes and valley forests at 800-2200 m.When it is introduced vertically to low altitude areas,the leaf "sunburn" phenomenon often occurs in summer due to high temperature stress,which seriously affects the ornamental value of Sorbus Chinensis and the development and utilization of germplasm resources.Based on the transcriptome and genome data,this study explored the key gene of S.pohuashanensis in response to high temperature stress,heat shock transcription factor A3(SpHsfA3a/b),and used molecular biology and bioinformatics technology to analyze its function and regulatory mechanism.The main conclusions are as follows:(1)The CDS length of SpHsfA3 a gene was 1602 bp,encoding 533 amino acids,and the CDS length of SpHsfA3 b gene was 1455 bp,encoding 484 amino acids.SpHsfA3a/b contained 60 and49 phosphorylation sites,respectively.SpHsfA3a/b had no signal peptide.The transmembrane structure analysis results showed that SpHsfA3 a had one spiral from outside to inside at 450-470,which belonged to the transmembrane protein.SpHsfA3 b did not have the transmembrane structure.Subcellular localization prediction indicated that SpHsfA3a/b was located in the nucleus.Multiple sequence alignment showed that SpHsfA3a/b had conserved domains of heat shock transcription factors.Phylogenetic tree analysis showed that SpHsfA3a/b is closely related to Pyrus bretschneideri and Malus domestica,which belong to the Rosaceae.(2)The endogenous expression analysis results showed that SpHsfA3a/b was expressed in different organs of S.pohuashanensis,and the expression level was the highest in fruits and leaves,followed by roots.The expression level was lower in stem,bud,flower(blooming stage)and other organs,the lowest in flower buds,indicating that SpHsfA3a/b was involved in the growth and development process of plants.The expression of SpHsfA3a/b was up-regulated under 42℃ and reached its peak at 4 h of stress,with the expression levels 230 times and 1000 times of the control,respectively.Indicate that SpHsfA3a/b can be rapidly induced in response to high temperature stress.Under salt stress,the expression of SpHsfA3a/b showed an increasing trend,and reached the highest level at the 15 th day of stress,which was 12 times and 7 times of that in the control group,respectively.Indicated that SpHsfA3a/b was induced to be expressed under salt stress and participated in the process of salt stress response.Under drought stress,the expression of SpHsfA3a/b was up-regulated,and the expression level increased first and then decreased.The expression level reached the highest at the 9th day of drought stress,which was 7 times and 49 times of the control group,respectively.Indicated that SpHsfA3a/b played a certain role in drought stress.(3)The subcellular localization of tobacco leaves was observed and SpHsfA3a/b was all located in the nucleus.SpHsfA3a/b was transferred into Escherichia coli and treated with high temperature stress at 55 ℃,NaCl salt stress and mannitol drought stress.The results showed that SpHsfA3a/b could improve the tolerance of Escherichia coli to high temperature stress,salt stress and drought stress to different degrees.The effects of SpHsfA3a/b on high temperature,salt and drought stress were preliminarily verified.The physiological function of SpHsfA3a/b was further verified by allogeneic transformation of Arabidopsis.The results showed that SpHsfA3a/b was upregulated under high temperature stress,and the survival rate of SpHsfA3a/b overexpressed transgenic lines and hsfa3 mutant complementary lines were higher than the control lines.SpHsfA3a/b enhanced the heat resistance of transgenic Arabidopsis by increasing POD,SOD,CAT activities and Pro,soluble sugar content.Further studies showed that overexpression of SpHsfA3a/b promoted the expression of positive regulatory genes such as At HSP70,At HSP90,At HsfA1 e,At HsfA2,At APX2,At Gol S1,At HSP20 s,and inhibited the expression of negative regulatory factors of At Hsf B2 B.The results indicated that SpHsfA3a/b are positive regulatory factors under high temperature stress.However,there was no significant difference transgenic Arabidopsis lines between Arabidopsis thaliana and the control under NaCl salt stress and mannitol stress.(4)The molecular regulatory mechanism of SpHsfA3a/b in response to high temperature stress was explored by double luciferase assay.It was found that the expression of SpHSP26.5-1/-2 was regulated by SpHsfA3a/b.Further,it was proved that SpHsfA3a/b regulates the expression of SpHSP26.5-1 mainly by binding to SpHSP26.5-1pro-2 fragment(TCTGAGAAGA),and SpHsfA3a/b regulates the expression of SpHSP26.5-2 by binding to SpHSP26.5-2pro-1 fragment(TTTTTAGAATATG)through yeast one-hybrid assay,so as to respond to high temperature stress.These results indicate that SpHsfA3a/b is localized in the nucleus and can be induced to express under high temperature,salt and drought stress.Under high temperature stress,SpHsfA3a/b can directly regulate the expression of SpHSP26.5-1/-2 to activate the heat stress response,and then improve the thermotolerance of transgenic Arabidopsis by promoting the expression of heat-resistant genes,increasing the contents of soluble sugar and proline,and the activities of POD,SOD and CAT. |
