Expression Characteristics And Preliminary Functional Identification Analysis Of PwUSP1 In Picea Wilsonii

Picea willsoni is a representative gymnosperm in pine,and has strong adaptability to the environment.Many genes related to stress resistance have been isolated from Picea willsoni.The acquisition of these important genes provides a potential for breeding new varieties of high quality,disease and insect pests resistance by transgenic technology.Universal stress protein(USPs)is a tolerance-related gene which plays a vital role in abiotic stresses.However,there are few studies on USP in gymnosperms,and this assay was using USP of Picea willsonii as the experimental material.RACE technology was used to obtain the cDNA length of the PwUSP1 gene from the cDNA Library of Picea wilsonii before,besides PwUSP1 gene was analysed by bioinformatics technology.However,the molecular mechanism of the PwUSP1 remains unclear,in order to further explore the function of Pw USP1 gene in abiotic stresses and pollen growth,We transfered PwUSP1 into Picea wilsonii pollens and Arabidopsis thaliana.The main results are as follows1.To examine the tissue specific expression of PwUSP1 in Picea wilsonii.We use the RT-qPCR experiement to test the expression level,the results showed that the expression of PwUSP1 in pollen is much higher than that in other tissuess,which implys that PwUSP1 plays an important role in the pollen2.To investigate the role PwUSP1 plays in abiotic stress.We researched the relative expression of PwUSP1 under different stress treatments by RT-qPCR.These results showed that PwUSP1 responded to low temperature 4℃,high temperature 42℃,H2O2,drought,NaCl,MeJA and ABA treatments.Among them,Pw USP1 was obviously upregulated by salt and drought treatments3.To investigate subcellular localization of PwUSP1.We use the biolistic technology to transfer PwUSP1 into epidermal cells of onion.Results showed that the PwUSP1 was located in the nucleus and cytoplasm Besides,the yeast two hybrid experiment shows that PwUSP1 itself can form homologous two polymer.4.To futher determine the function of PwUSP1 in abiotic stresses.We transfered PwUSP1 into Arabidopsis by foral inflorescence impregnation.And detected the expression of PwUSP1 on DNA level,cell level and protein level.by PCR technology,detecting GFP signal of root cell and Western blotting technology separately.After the stable expression of transgenic Arabidopsis was obtained,it showed that GFP signal located in nuclear which was consistent with the results of the subcellular localization in onions.5.The mutant plant has 19 bp absence from 1913 to 1932 base site,and expression level has decresased.6.Seed germination experiments showed that overexpression PwUSPl could improve germination rate of seeds under NaCl and mannitol treaments.Root length test showed that oxPwUSPl plants had longer root length than wild type plants.Besides,it was also found that The content of chlorophyll,CAT and POD in overexpressed plants were higher than those of wild plants.The results of DAB staining showed that oxPwUSPl plants has lower ROS contents than those in wild tipe plants under NaCl and mannitol treatments.These results showed that PwUSPl could enhance resistance of plants under NaCl and mannitol treatments.7.To determine the potential pathway of PwUSP1 to improve the resistance of Arabidopsis we selected several related genes and detected the relative expression by RT-qPCR.Results showed that PwUSP1 was more likely to be involved in abiotic stress process through the ABA pathway.8.To determine the function of PwUSP1 in pollen.We investigated the relative expression of PwUSP1 by RT-qPCR transfered Pw USP1 in pollens.Germination experiment showed that PwUSP1 was induced by calcium ion during pollen germination process,and it was found that PwUSP1 is likely to changed the growth direction and germination rate of pollen tube,however,the specific mechanism still needs further exploration and validation.