Cloning And Functional Analysis Of Cpcor413pm1 Promoter From Chimonanthus Praecox (L.)

Wintersweet [Chimonanthus praecox(L.)Link] is an important winter ornamental flower in China.In winter with relatively few flower resources,wintersweet is an important fresh cut flower and bonsai material,which has high ornamental value and economic significance.COR gene(Cold-Regulated Gene,COR)is a kind of low temperature stress response gene unique to plants.In our laboratory,a gene related to cold resistance has been cloned from wintersweet,named Cpcor413pm1.This study cloned the upstream promoter fragment of the Cpcor413pm1 gene,hoping to explore the mechanism of the gene in the formation of cold resistance(freezing)by analyzing the function of the Cpcor413pm1 gene promoter.The main findings are as follows:1.q RT-PCR analysis of Cpcor413pm1 genes in wintersweet.Wintersweet for different tissues,flowers at different stages of development and the seedlings with different induction treatments of Cpcor413pm1 gene were subjected to q RT-PCR analysis.The results showed that the expression of the gene was relatively high in roots and flowers,and it was possible to participate in the regulation of root and flower development.the Cpcor413pm1 gene was expressed in all stages of wintersweet flower development,and the amount of expression in the decay stage was significantly higher than that in other periods.it was speculated that the gene might be involved in the aging regulation mechanism of wintersweet flower;Cpcor413pm1 gene could be induced to express under 4 ℃,42 ℃,abscisic and ethylene.it may participate in the regulation process of wintersweet.2.Amplification and cloning of the promoter sequence of Cpcor413pm1 gene.1815bp upstream fragment of Cpcor413pm1 gene was obtained by chromosome walking and named Cpcor413pm1 pro.Bioinformatics analysis showed that there were some abiotic stress and hormone response elements such as MYB,ABRE and ERE in the promoter sequence addition to the basic promoter elements TATA-box and CAAT-box.3.Activity validation and stable expression of the promoter of Cpcor413pm1 gene.According to the prediction results of the cis-acting elements,3 segments(Cpcor413pm1pro-P1/P2/P3)of the promoter fragments were analyzed,and the plant expression vectors of each promoter fragment were constructed,and then the recombinant vectors were transformed into tobacco for instantaneous activity verification,and the full-length fragment Cpcor413pm1 pro was transformed into Arabidopsis thaliana for stable expression.The results showed that all the promoter segments had promoter activity,and the Cpcor413pm1 pro T2 generation lines of Arabidopsis thaliana were obtained by resistance screening.4.Tissue specificity analysis of the promoter of Cpcor413pm1 gene.GUS histochemical staining annalysis of Cpcor413pm1 pro showed that genetically modified Arabidopsis thaliana and the growth period of seeds after germination can be colored,and it is inferred that the Cpcor413pm1 pro has no tissue expression specificity.The results of GUS gene q RT-PCR and enzyme activity analysis were consistent with the results of histochemical staining: the expression of GUS was found in all tissues of transgenic plants,and the GUS activity from high to low was root,stem,leaf,fruit and flower,indicating that Cpcor413pm1 pro was active in all tissues of plants and was a constitutive promoter.5.Stress response analysis of the promoter of Cpcor413pm1 gene.The Cpcor413pm1 gene promoter transgenic Arabidopsis seedlings were subjected to stress treatmnet of 4 ℃,42 ℃,ABA and ACC.The results of GUS gene q RT-PCR and GUS enzyme activity analysis showed that GUS gene were responsive to4 ℃,42 ℃,ABA and ACC driven by the Cpcor413pm1 pro promoter.It is suggested that the promoter may regulate the expression of downstream genes when the wintersweet encounters abiotic stresses.