Cloning Of CtCP1 Gene In Safflower And Its Role In Leaf Senescence

Safflower is a traditional cash crop in China,which can be used for edible,medicinal and oil purposes,and has great development potential.Leaf senescence of safflower is a normal phenomenon in the development process,but in the safflower cultivation,leaf premature senescence will lead to quality degradation and economic loss.So far,the molecular mechanism of safflower premature aging process is not clear.Papaya cysteine protease(CP)is widely involved in plant growth and development,and it is often associated with plant senescence.CP complete genome sequence has been identified in more and more plants,but has not been reported in safflower.In this study,bioinformatics analysis of CP family of C.carthamus was conducted based on genome sequencing data,and the expression patterns of different tissues of C.carthamus were analyzed by qRTPCR,and CtCP1 gene which was positively correlated with leaf aging was screened out.The prokaryotic expression vector of Escherichia coli was constructed to analyze the differences in enzyme activity of common substrates.Plant expression vectors were constructed to obtain T3 generation overexpressed Arabidopsis Thaliana and transient transformed safflower strains,which were subjected to low temperature and salicylic acid stress to identify the function of CtCP1 gene in leaf aging process.Specific research results are as follows:1.Twenty-five CtCP candidate genes were identified by bioinformatics and named CtCP1-CtCP25.According to phylogenetic analysis,they were divided into 9 subclasses(RD21,CEP,XCP,XBCP3,THI,SAG12,RD19,ALP and CTB).The analysis of gene structure showed that the structure conserved among different subfamily members was lower,but the same subfamily members had higher conserved structure.The results of promoter analysis showed that there were ABRE abscisic acid response,TCA-element salicylic acid response,GT1-motif,G-box light response element,LTR low temperature stress,ARE antioxidant stress,and TC-rich in the promoter of CtCP gene family repeats defensive response,seed-specific regulation and endosperm expression.2.Fluorescence quantitative PCR was used to analyze the expression of candidate genes.The results showed that the expression of different subfamily genes has discrepancy in different tissues,with the highest expression of CtCP1 in leaf tissues,CtCP3 in root and flower tissues,and CtCP6 in stem tissues.Among them,the expression level of CtCP1 gene showed a trend from low to high during the early stage to the late stage of leaf aging,which was consistent with the expression level from the bottom to the top of the leaf.3.CtCP1 gene coding region 1096 bp was successfully cloned.The recombinant pET32a-CtCP1 protein with the size of 62.03 kDa was expressed by E.coli prokaryotes.The activity of CtCP1 protein on traditional protease substrates was different.4.Transgenic Arabidopsis thaliana was obtained by tidbits infection method,and two high-expression strains were identified in T3 transgenic materials.Two abiotic stresses,salicylic acid and low temperature,were carried out on wild-type strains,high-expression strains and mutant strains.The results showed that: After treatment with 100mmol/L salicylic acid or low temperature,the expression of CtCP1 gene was significantly up-regulated in high expression Arabidopsis thaliana,and the indicators closely related to aging(expressions of superoxide dismutase,catalase and malondialdehyde content)were positively correlated.The closely related indexes of antioxidant(peroxidase expression,chlorophyll,soluble sugar,proline content)decreased significantly,and the number of dead cells increased.The mutant is the opposite,and the wild-type strains are in between.Based on the changes of three Arabidopsis thaliana strains,it is speculated that CtCP1 gene plays a role in promoting leaf senescence,and presents higher sensitivity to salicylic acid and low temperature stress.5.The transient transformation system of safflower leaves was constructed,and the phenotypic changes were observed under salicylic acid and low temperature stress using overexpression and gene silencing techniques(VIGS).The results showed that the transient overexpression of CtCP1 gene was significantly up-regulated,which was positively correlated with the aging related genes CtSAG12 and CtSAG29 and the chlorophyll-related genes CtNYC1,and the chlorophyll content decreased,superoxide anion and dead cells increased.Silenced plants are the opposite of that.The overall results were similar to those of Arabidopsis thaliana,again confirming that CtCP1 gene plays a role in promoting plant aging.