Expression and vector construction of ZjPG gene in Ziziphus jujuba Mill.
[Objective]This study aimed to clone the polygalacturonase(PG)gene from Chinese jujube(Ziziphus ju-juba Mill.),analyze the expression pattern of ZjPG gene and construct an overexpression vector,providing a theoretical reference for exploring the molecular mechanism of ZjPG gene in regulating fruit cracking and breeding crack-resistant jujube varieties.[Method]Using Z.jujuba Mill.cv.Huping as experimental material,the ZjPG gene(ID:LOC107426373)was cloned.The ZjPG protein was analyzed bioinformatically by online tools such as ExPASy,SOPMA and SWISS-MODEL.A phylogenetic tree based on PG amino acid sequence was constructed by using MEGA 7.0 software,and the cis-acting elements of the ZjPG promoter were analyzed.The relative expression levels of the ZjPG gene were detected by quantitative real-time PCR(qRT-PCR)in different tissues of Z.jujuba Mill.cv.Huping(young leaves,mature leaves,flowers,fruits at expansion period,white mature period,crisp ripening period and full-ripening periods)as well as nor-mal and cracked fruits at the crisp ripening period,which had been all treated with different concentrations of methyl jas-monate(MeJA,0.2 and 0.4 mol/L)and abscisic acid(ABA,0.2 mol/L).The ZjPG gene overexpression vector was con-structed.[Result]The total length of coding region(CDS)sequence of ZjPG gene in Z.jujuba Mill.cv.Huping was 1353 bp,with 99.70％CDS sequence similarity and 99.33％amino acid sequence similarity to the ZjPG gene in NCBI database,in-dicating the successful cloning of the ZjPG gene.The molecular weight of ZjPG protein was 49145.28 Da,encoding 450 amino acids residues,with a theoretical isoelectric point of 6.27.It was a stable hydrophilic protein without transmem-brane structural domains,containing a conserved sequence of Pgul.Its secondary structure consisted of 12.67％α-helix,32.22％extended strand,8.00％β-sheet and 47.11％random coil.Phylogenetic evolutionary tree showed that ZjPG was closely related to AtPG fromArabidopsisthaliana and VvPG from Vitis vinifera.ZjPG and PG protein motifs of other nine species were highly conserved.The ZjPG gene promoter contained multiple hormone-responsive cis-acting elements.The qRT-PCR analysis showed that ABA and MeJA could induce the expression of ZjPG gene.The relative expression level of the ZjPG gene was significantly higher in cracked fruits than in normal fruits(P＜0.05).The ZjPG gene was expressed in leaves,flowers and fruits of Z.jujuba Mill.cv.Huping,with the highest relative expression level in full-ripening period fruits.The pCAMBIA-1300-ZjPG expression vector was successfully constructed.[Conclution]The CDS sequence of the ZjPG gene from Z.jujuba Mill.cv.Huping is successfully cloned and its overexpression vector is successfully constructed.The PG protein sequences are highly conserved among different species.The expression of the ZjPG gene is regulated by ABA and MeJA signals,and highly expressed in cracked fruits.The ZjPG gene expression shows tissue and spatio-temporal specificity.
Ziziphus jujuba Mill.cv.HupingZjPG genefruit crackingexpression featurevector construction
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