| Wintersweet(Chimonanthus praecox)is an endemic shrub in China,which belongs to the genus Chimonanthus praecox in the class of Magnolia Ranunculus.Because of its elegant anthocyanin and sweet fragrance,it is widely used in bonsai,garden greening,cut flower production,road greening,etc.The domestic industry of wintersweet has also developed rapidly.However,the rapid development of Chimonanthus praecox industry is faced with problems such as long childhood and uneven flowers.Studies have shown that flower induction is the key to affecting the number and quality of flower buds,determining the flowering period of flowering plants,and the amount and yield of flowers in the coming year.The research on the molecular mechanism of the induction of wintersweet is beneficial to solve the problems existing in the development of the industry of wintersweet.At present,the research and understanding of the molecular mechanism of flowering induction are mainly concentrated in a few one-year-old model plants and two-year-old model plants,such as Arabidopsis thaliana and rice,and the understanding of the molecular mechanism of flowering induction in perennial plants,especially woody plants,is still very limited.The wintersweet is a deciduous shrub of the class Magnolia,and the class Magnolia is considered to be the most primitive angiosperm.The research on the molecular mechanism of the flower induction of wintersweet can provide a reference for the revealing of the molecular mechanism of the flower induction of woody plants and even angiosperms,and for the production of to shorten the childhood period,cultivate early-flowering lines,and speed up the breeding process to provide a theoretical basis.We obtained a CCCH-type zinc finger protein CpC3H3 in the wintersweet developmental transcriptome database.Expression analysis found that the CpC3H3 gene was lowly expressed in the vegetative organs and highly expressed in the floral organs.It is speculated that this gene may play an important role in the flowering induction of wintersweet,but its function remains to be studied.In order to explore the role and mechanism of CpC3H3 gene in the induction of wintersweet flowers,this experiment was the first time to clone and obtain the CpC3H3 CCCH-type zinc finger protein gene of wintersweet interacting proteins were studied.The experimental content and main results of this paper are as follows:(1)Cloning and molecular structure analysis of CpC3H3 gene.The cDNA sequence of CpC3H3 gene was screened from the transcriptome database.Editseq software was used to analyze the full length of CpC3H3 gene.It was found that the CpC3H3 gene contained 447 amino acids and the complete maximum open reading frame was 1341bp.(2)Structural Characterization of Proteins Encoded by CpC3H3 Gene.ProtParam software predicts CpC3H3 protein molecular formula online is C2106H3255N591O669S27,protein molecular weight is 48.42kD,theoretical isoelectric point is 8.37;protein instability coefficient is 53.82,the overall average hydrophilicity is-0.579,it is an unstable hydrophilic protein.Subcellular localization results showed that CpC3H3 protein was localized in the cytoplasm.In the protein secondary structure,a helix accounted for 5.61%,extended chain accounted for 25.34%and random curl accounted for 69.06%.Analysis of the conserved domain of CpC3H3 gene showed that CpC3H3 protein was a member of CCCH type zinc finger protein family and contained five CCCH type zinc finger proteins,all of which were C-X8-C-X5-C-X3-H.(3)Analysis of cis-acting elements in the promoter of CpC3H3 gene.PlantCare online software was used to analyze and predict the cis-acting element of 2142bp upstream of ATG of CpC3H3 gene.The results showed that the cis-acting elements of the CpC3H3 gene promoter were divided into four categories:light-responsive elements,hormone-responsive elements,stress-responsive elements and other elements.There are 16 light-responsive elements,including I-box,G-box,Box 4,etc.There are 12 hormone response elements,including ABRE and MYC in response to abscisic acid,and ERE in response to ethylene.There are 10 stress response elements,including MBS in response to drought and TC-rich repeats in response to defense and stress.Other elements include core promoter elements TATA-box,CAAT-box,etc.(4)Analysis of the expression characteristics of CpC3H3 gene in wintersweet.The expression characteristics of CpC3H3 gene in different tissues and different developmental stages were studied by fluorescence quantitative PCR.The results showed that CpC3H3 gene was expressed in roots,stems,leaves,flowers and other tissues,and the expression in flower organs was higher than that in vegetative organs such as roots,stems and leaves.At the same time,the expression level of CpC3H3 gene in the flower meristem formation stage was higher than other stages.(5)Preliminary functional analysis of CpC3H3 gene.The Arabidopsis thaliana pC AMBIA13 00-CpC3H3 overexpression vector was constructed,transformed into Colombian wild-type Arabidopsis thaliana,and screened for Hyg resistance.DNA detection of CpC3H3 gene transgenic plants in T0 generation and fluorescence quantitative analysis of homozygous transgenic plants in T3 generation were performed.A total of 14 homozygous Arabidopsis thaliana lines that had been transformed into CpC3H3 gene were obtained in T3 generation.Phenotypic observations and follow-up studies were carried out for each line(OE-10,OE-14,OE-18).Fluorescence quantitative analysis of the key flowering genes in Arabidopsis transgenic CpC3H3 gene showed that the expression of CpC3H3 gene in Arabidopsis thaliana enhanced the expression of endogenous FT,SOC1,LFY,AP1,and FLC genes in Arabidopsis thaliana.Through the observation of the representative type of T3,it was found that the transgenic Arabidopsis thaliana thaliana stalked 1 cm earlier than wild-type Arabidopsis thaliana,and the number of rosette leaves at the same time was more than that of the wild-type.Analysis of RNA-sequencing data from wild-type and transgenic Arabidopsis revealed 123 differentially expressed genes in the transcriptome data,15 of which were associated with flowering function.ARR4,HBI1,PAR1,GATA21,BEE1,EPR1,AtNF-YC-4,CGA1 genes were involved in the flowering of photoperiod pathway.ARR7,ARR5,ARR15,GASA6,ARR6,ARR9,and IPT3 are also associated with other hormone-regulated flowering.(6)Preliminary Analysis of CpC3H3 Interacting Proteins in Prunus chinensis.Construct a bait plasmid Y-BD-CpC3H3-1 that can be used for CpC3H3 protein interaction verification.CpC3H3-1 is 549 bp in length and can translate 183 amino acids,including the first three structures of the CpC3H3 gene from the 5’ end to the 3’ end area.Using the bait plasmid Y-BD-CpC3H3-1 and 6 genes that may interact with CpC3H3 protein screened by transcriptome data,including Cs04g01506(homologous to ARR5),Cs03g01023(homologous to HBI1),Cs04g00438(homologous to MYB50),Cs09g01407(homologous to EBP1),Cs03g02822(homologous to SRG3),Cs05g02579(homologous to ARR4)were used for protein interaction verification.The results showed that there was no direct interaction between the CpC3H3 gene and these six genes,and the target proteins related to the CpC3H3 gene needed to be further screened and studied. |
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