Identification Of U-box Gene Family In Citrus Genome And The CcPUB4 Gene Gditing Using CRISPR/Cas9

Salt stress is one of the major stress that harm plants,severely inhibiting plant growth and yield,and high salt may even cause plant die.Citrus is an important fruit species in the world and in southern of China.It is a sweet soil plant that is extremely sensitive to salt.Salt damage often occurs in the main citrus producing areas of China.Under salt stress,plants showed chlorosis and leaf tips.Litter,quality deterioration,and trees deed.With the increase of industrial pollution and the use of artificial fertilizers and other human factors,the area of saline soil is increasing,which also poses a serious threat to the citrus industry.Therefore,excavation of genes related to salt stress,understanding the response and adaptation mechanism of citrus to salt stress,and cultivating relevant stress-resistant varieties are of great significance for citrus production.U-box protein is widely distributed in the plant genome and its structure is highly conserved.It plays the role of E3 ubiquitin ligase in ubiquitin degradation pathway and has a important role in response to abiotic stress.Studies have shown that plant U-box protein can regulate the expression of salt-stress related genes,and then regulated Na~+and K~+homeostasis to maintain the balance of Na~+and K~+in cells to enhance the salt tolerance.Therefore,we analyze the citrus U-box gene family by bioinformatics,including gene structure,chromosomal localization and evolution analysis,expression analysis,the candidate gene CcPUB4,which may be related to salt stress,was screened.To study the relationship between citrus ubiquitin ligase gene CcPUB4 and stress resistance,the CRISPR/Cas9 vector was constructed for CcPUB4 gene for transgenic function verification.The main research are as follows:(1)56 CcPUBs gene family members were identified from the whole genome of Citrus clementina,which can be divided into 7 categories,namely U-box only,U-box+ARM-1,U-box+WD40-1,TPR+U-box,Kinase+U-box,U-box+ARM-2 and U-box+WD40-2,U-box only,and distributed unevenly on the 1st and 9th Scaffold;The physicochemical properties of the family protein such as theory isoelectric point and molecular weight are quite different;promoter analysis indicates that the CcPUBs gene family can respond to hormones not only in response to external environments such as light and drought..(2)The U-box gene family participates in abiotic stress response process.In this study,we analyzed the RNA-Seq data of Poncirus trifoliate Raf.under cold stress and found that the U-box gene family is involved in the response of plants to cold stress,and showed four different response patterns(first decline and then rise,first rise and then fall,always rising,falling all the time).one representative gene is selected from five different clusters of the citrus U-box gene family for qRT-PCR.One representative gene was selected from five different clusters of citrus U-box gene family for qRT-PCR.The results showed that U-box gene family expression was tissue-specific and could respond to hormonal and abiotic stresses;CcPUB4 was induced by Na+and its high expression played an important role in citrus response to salt stress.(3)CRISPR/Cas9 technology has been widely used in many species and achieved high editing efficiency.To further determine the relationship between CcPUB4 and stress resistance.Three suitable vectors pC1C.1C-gRNA-1,pP1C.1C-gRNA-6 and pP1C.1C-gRNA-1+gRNA-6 were constructed,designing appropriate targets site for CcPUB4 by online and in vitro assays.The CcPUB4 mutant plants(mainly base deletion)were obtained by transforming jincheng with Agrobacterium;Online design and in vitro digestion design gRNA can improve the editing efficiency of CRISPR/Cas9.