Study On The Function Of Stress Resistance Gene PcHsf And PcHsp70-90 In Pistacia Chinensis

Pistacia chinensis Bunge.is one of the most important bioenergy species in China,it has long fruiting period,but the seed setting rate and the single fruit yield are both low,dioecism is considered to be the main cause of low yield.In the investigation,our team found that the resources of monoecious Pistacia can be self-fertilizated,breaking the bottleneck of low yield of dioecious plants.In order to find out the sex determination mechanism of Pistacia chinensis,and put the monoecious plants into the actual production,the key stages of sex phenotype differentiation were selected for correlation analysis of transcriptome,small RNA and proteome,some genes and proteins affecting sex determination were screened.Among them,the HSF and HSP families related to stress resistance are closely related to the flower development of Pistacia chinensis.In order to further understanding the function of PcHsf and PcHsp70-90 genes in Pistacia chinensis,with a view to revealing the mechanism of response to adversity during the floral development of Pistacia chinensis.1.Pchsf and PcHsp70-90 genes were successfully cloned,and the full-length sequence of the genes was analyzed by bioinformatics.The amino acid multiple sequence alignment and phylogenetic tree analysis of Pchsf and PcHsp70-90 showed that Pchsf and PcHsp70-90 were closely related to citrus.2.Four plant expression vectors 35S::PcHsf,35S::PcHsf-EGFP,35S::PcHsp70-90 and 35S::PcHsp70-90-EGFP were constructed.Agrobacterium transformation method was used to transfer them into wild type Arabidopsis,and T1 generation transgenic positive seedlings were obtained by resistance screening,and further verified by PCR.and T3 transgenic homozygous lines were obtained by PCR verification,T3 transgenic homozygous lines were obtained by continuous screening.3.The subcellular localization analysis of transgenic Arabidopsis thaliana by laser confocal microscopy showed that PcHsf gene expression was located in the nucleus.4.Overexpression of PcHsf and PcHsp70-90 both promoted flowering and growth of Arabidopsis.The phenotypic observation of transgenic PcHsf and PcHsp70-90 Arabidopsis and wild type Arabidopsis showed that transgenic Arabidopsis bolting and flowering earlier than wild type,and the number of rosette leaves was more when flowering,the growth rate was also significantly higher than wild type.In addition,the bolting height of PcHsp70-90 transgenic plants was 1/3 higher than wild type at the 5th week,and the number of small flowers was twice as high as wild type.5.Overexpression of PcHsf and PcHsp70-90 both enhance the drought resistance and heat resistance of the plant.Drought was simulated on 300 m M and 350 m M mannitol medium.The germination rates of the two transgenic Arabidopsis thaliana seeds were 14%,26%,22%,and 34% higher than those of the wild type,respectively.After 3 days of rehydration,the survival rate was 6 times and 7 times that of wildtype respectively;with 50 ? high temperature heat shock PcHsf,PcHsp70-90 and wild-type Arabidopsis thaliana seeds,their survival rates were 4%,11%,27.5%,All the genotype plants were withered and wilted after 7 days under high temperature stress at 35 ?,bolting and drooping showed dying symptoms,and then cultured for one week after returning to normal environment,most wild-type seedlings died,the survival rate was 16.6%,and The survival rates of PcHsf and PcHsp70-90 were 88.9% and 97.6%,respectively.6.The POD activity of Pchsp70-90 transgenic Arabidopsis under normal and drought conditions is significantly higher than that of other genotypes.When subjected to drought and high temperature stress,the MDA content and proline content of all plants increased significantly.The increase in MDA content of Arabidopsis was lower than that of wild-type Arabidopsis,and the average value of proline content was 1.5 times of that of wild-type.The effect of high temperature stress on the physiological indexes of Arabidopsis is basically similar to that of drought stress,but it is more severe,which may be related to the high temperature itself accompanied by the evaporation of plant water.The measurement results of these indicators proved from a physiological level that overexpression of PcHsf and PcHsp70-90 is beneficial to plants' ability to resist drought and high temperature stress,and the drought tolerance and heat resistance of PcHsp70-90 transgenic seedlings are stronger than that of PcHsf transgenic seedlings.