Cloning And Functional Analysis Of CpWRKY46 And CpWRKY71 Genes Of Chimonanthus Praecox

Wintersweet(Chimonanthus praecox)cut flower is popular for its unique flowering time,elegant color and pleasant aroma.In recent years,the wintersweet cut flower industry in Chongqing,Sichuan,Henan and Shanghai has developed rapidly.As a cut flower,the vase life directly affects its ornamental quality and market competitiveness.How to delay the senescence of wintersweet flowers has aroused widespread concern of researchers.At present,there are some studies on the physiology and preservation of wintersweet cut flowers,but there are few reports on the molecular regulation mechanism of wintersweet flower senescence.In order to understand the regulatory mechanism of wintersweet flower senescence,a flower transcriptome database was constructed earlier in our laboratory.In this study,by analyzing the transcriptome database,we found that some WRKY genes were significantly up-regulated during the flower senescence,and thus speculated that they may be related to the flower senescence of wintersweet.Here,in order to study the functions of WRKY genes in the senescence process of wintersweet flower,we isolated WRKY genes from wintersweet,and analyzed their basic characteristics,expression characteristics and biological functions.The main findings are as follows:(1)Based on the sequence information in the transcriptome database,c DNA sequence of two WRKY genes were isolated and named Cp WRKY46 and Cp WRKY71,respectively.The length of Cp WRKY46 c DNA is 1491 bp including a 1029 bp encoding frame,a 355 bp 5'-untranslated region(UTR),and a 107 bp 3'-UTR.The length of Cp WRKY71 c DNA is 1137 bp including a 951 bp encoding frame,a 93 bp 5'-untranslated region(UTR),and a 93 bp 3'-UTR.Subsequently,we isolated genomic g DNA fragment of Cp WRKY46 and Cp WRKY71 using genomic DNA as template.The length of Cp WRKY46 g DNA is 2143 bp,and it contains two introns of 87 bp and 565 bp.The length of Cp WRKY71 g DNA is 2231 bp,and it contains two introns of 417 bp and 677 bp.(2)Characterization of the deduced Cp WRKY46 and Cp WRKY71 protein.The molecular formula of Cp WRKY46 protein is C₁₆₄₆H₂₅₅₉N₄₆₃O₅₄₁S₁₂,the molecular weight of the protein was 37.87 k D,and the theoretical isoelectric point was 5.44.Amino acids Serine(Ser)had a proportion of 14.0%(48/342),the largest proportion in amino acid composition,followed by proline(Pro),which had a proportion of 7.9%(27/342).The protein with a Grand average of hydropathicity(GRAVY)of-0.698,is a hydrophilic protein.Sequence analysis showed that Cp WRKY46 contains a conserved WRKY domain and a C2HC-type zinc finger structure,is a member of the?subfamily.The molecular formula of Cp WRKY71 protein is C₁₅₁₀H₂₃₀₄N₄₃₄O₄₉₁S₁₇,the molecular weight of the protein was 34.93 k D,and the theoretical isoelectric point was 6.6.Amino acids Serine(Ser)had a proportion of 15.2%(48/316),the largest proportion in amino acid composition,followed by proline(Pro),which had a proportion of 9.2%(29/316).The protein with a Grand average of hydropathicity of-0.884,is a hydrophilic protein.Sequence analysis showed that Cp WRKY71 contains a conserved WRKY domain and a C2H2-type zinc finger structure,is a member of the?c subfamily.(3)To understand whether Cp WRKY46 and Cp WRKY71 have the basic characteristics of transcription factors,we analyzed the subcellular localization and transcriptional activity of Cp WRKY46 and Cp WRKY71.Transient expression analysis in tobacco epidermal cells showed that Cp WRKY46 and Cp WRKY71 were located in the nucleus.Analysis of the transcriptional activity in the yeast system showed that both Cp WRKY46 and Cp WRKY71 could transactivate the expression of HIS3 and Lac Z reporter genes in yeast cells,indicating that both Cp WRKY46 and Cp WRKY71 have transcriptional activation activity.(4)Gene expression patterns can reflect gene function to a certain extent.To understand the possible biological functions of Cp WRKY46 and Cp WRKY71 genes,q RT-PCR was performed to detect the expression patterns of Cp WRKY46 and Cp WRKY71genes in wintersweet.In different tissues,the expression of Cp WRKY46 in roots,stems,young leaves,and flowers was lower than that in senescent leaves;during flower development,the expression of Cp WRKY46 was highest in the early-withering stage,followed by that of sepal and pistil primordia differentiation stage.In different tissues,Cp WRKY71 was highest expressed in flowers,followed by senescent leaves;in different stages of flower development,Cp WRKY71 gene was highest expressed at early-withering stage.(5)To understand the characteristics of expression regulation of Cp WRKY46,a 1671bp Cp WRKY46 promoter sequence Pro Cp WRKY46 was isolated.Sequence analysis showed that Pro Cp WRKY46 contained ethylene,salicylic acid,gibberellin,abscisic acid,and jasmonic acid response cis-elements.ACC,salicylic acid,gibberellin,abscisic acid,and jasmonic acid were used to treat wintersweet and Pro Cp WRKY46:GUS Arabidopsis seedlings,we found that the expression of Cp WRKY46 and GUS genes were affected by these hormone treatments.These results indicated that Cp WRKY46 might be involved in hormone-mediated biological processes.Meanwhile,GUS staining results of Pro Cp WRKY46:GUS transgenic plants showed that the GUS gene driven by Pro Cp WRKY46 is much less expressive in roots,stems,young leaves,flower buds,and the initiating bloom stage than older leaves,blooming flowers and the flower organ abscission position during flower senescence,indicating that the function of Cp WRKY46may be related to senescence and abscission.(6)In order to explore the function of Cp WRKY46 gene,Cp WRKY46 was transfected into Arabidopsis via floral dipping transformation method,and the phenotypic changes of transgenic plants caused by Cp WRKY46 were analyzed.Compared with the control plants,the transgenic plants exhibited earlier senescence phenotype,accompanied by a decrease in chlorophyll content and an increase in the expression of senescence related genes SAG12 and SAG13.Digital gene expression profiling technology was used to analyze the differencial-expressed genes in transgenic plants.We found that 6 senescence-related genes,12 ethylene pathway-related genes,and 6 salicylic acid pathway-related genes were up-regulated in the overexpression plants.To further confirm the mechanism of Cp WRKY46 in regulation of senescence,q RT-PCR was performed to detect the expression of the genes related to the ethylene,salicylic acid,abscisic acid and jasmonic acid pathways in over-expressed and control plants.The results showed that the expression levels of the ethylene and salicylic acid pathway genes were significantly up-regulated in the overexpression plants,while that of abscisic acid and jasmonic acid related genes did not change significantly,suggesting that Cp WRKY46 might cause the earlier senescence of transgenic plants through promoting the biosynthesis of ethylene and salicylic acid.(7)To analyze the expression regulation characteristics of Cp WRKY71,a 4170 bp Cp WRKY71 promoter sequence Pro Cp WRKY71 was cloned from wintersweet.Sequence analysis revealed that Pro Cp WRKY71 contains low temperature,high temperature,drought,salicylic,abscisic,and jasmonic acid response elements.After treating wintersweet and Pro Cp WRKY71:GUS with 4?,42?,PEG600,salicylic acid,abscisic acid,and jasmonic acid,we found that the expression of Cp WRKY71 and GUS genes were affected by these treatments,indicating that Cp WRKY71 may be involved in abiotic stress and hormonal response.At the same time,the GUS staining results of Pro Cp WRKY71:GUS plants showed that the expression level of GUS gene driven by Pro Cp WRKY71 in roots,stems and young leaves was much lower than that in senescent leaves,and the GUS activity in flower buds and initiating bloom stage was lower than that in bloom and senescence stage.This indicated that the function of the Cp WRKY71 gene may be related to senescence.(8)To study the function of the Cp WRKY71 gene,Cp WRKY71 was heterologously overexpressed in Arabidopsis.Compared with the control plants,the transgenic plants exhibited early flowering phenotype,accompanied by a decrease in rosette leaf number.Analysis of the expression levels of the key flowering genes in the transgenic plants revealed that the expression levels of FT,LFY,AP1,FUL,and CAL were significantly increased.In addition,Cp WRKY71 transgenic Arabidopsis also exhibited a premature senescence phenotype,with reduced chlorophyll content and increased expression of senescence-related genes SAG12 and SAG13.To fully verify the function of the Cp WRKY71 gene,Cp WRKY71 was transferred to petunia using the leaf disc method.Petunia over-expressing Cp WRKY71 showed a premature senescence of leaves and flowers.In summary,Cp WRKY46 and Cp WRKY71 are two WRKY transcription factor genes that were predominantly expressed in senescent leaves and flowers.Subcellular localization and transcriptional activity experiments prove that both WRKY proteins are located in the nucleus and have transcriptional activation activity.Heterologous expression of the Cp WRKY46 and Cp WRKY71 genes both caused the earlier senescence phenotype of transgenic plants,and accompanied by the up-regulation of senescence marker genes SAG12 and SAG13.Further analysis of the Cp WRKY46 transgenic plants revealed that the expression levels of salicylic acid and ethylene pathway-related genes were also up-regulated to varying degrees in the transgenic Arabidopsis.The expression patterns and phenotype of overexpression plants imply that both Cp WRKY46 and Cp WRKY71 might be involved in the regulation of the wintersweet flower senescence.