Cloning,tissue-specific Expression Analysis And Functional Identification Of PmWRKYs Gene Potentially Involving In Resin Synthesis Of Masson Pine

Pine resin is an important secondary metabolite of conifers,plays a role in plant self-defense,and is widely used in industrial chemicals and biofuels.Masson pine is an important coniferous tree species and the main tallow tree species in Chin.Because of its vast land and abundant resources,it has great social,economic and ecological value.In addition to the regulation of key enzyme genes for terpene synthesis,the synthesis of Pinus massoniana pine resin is also regulated by transcription factors.WRKY transcription factor is one of the most important transcription factor families in higher plants.It plays a role in plant growth and development,biotic and abiotic stress responses,regulating the expression of key genes in the synthesis of rosin and other secondary metabolites,and affecting the yield of plant metabolites.Plays an important role.There are few reports on the regulation of terpenoid synthesis by transcription factors in Masson’s pine..Therefore,the discovery of WRKY transcription factor genes related to masson pine pine resin synthesis and analysis of the molecular mechanism of masson pine resin production can provide molecular theoretical basis for the breeding of high-yielding masson pine varieties in my country.In this study,based on the transcriptome data of the high and low lipid production of Masson pine in the laboratory,four differentially expressed WRKY transcript sequences were selected,and the full-length sequences of four WRKY transcription factor genes were cloned and analyzed by bioinformatics.The fluorescence quantitative PCR analysis was used.Tissue-specific expression of WRKY transcription factor,and explore the effect of exogenous methyl jasmonate treatment on WRKY transcription factor gene expression;construct plant hyperexpression vector and perform functional verification.The main results are as follows:1.The qRT-PCR technique was used to analyze the expression of 4 WRKY transcription factor genes in high-and low-fat masson pine.The results showed that the expression levels of PmWRKY1610,PmWRKY2870 and PmWRKY3069 in the needles and xylem of high-fat masson pine were significantly higher than those of low-fat yield.It is consistent with the results of transcriptome analysis.The expression level of PmWRKY2336 in the xylem of high-lipid-yielding germplasm was higher than that of low-lipid-yielding germplasm,but there was no significant difference among needles.These 4 PmWRKY may be involved in the regulation of terpenoid metabolites of masson pine.2.The full-length sequences of PmWRKY1610,PmWRKY2336,PmWRKY2870 and PmWRKY3069 were cloned.Based on their homology with Arabidopsis WRKY family genes and PmWRKY1-31 protein in Masson pine WRKY family,they were named PmWRKY2(WRKY2336)and PmWRKY32(WRKY3069)respectively.,PmWRKY33(WRKY2870)and PmWRKY34(WRKY1610).The total length of PmWRKY2 is 2,193 bp,the ORF is 1,428 bp,and the total code is 475 aa;the total length of PmWRKY32 is 2,368 bp,the ORF is 2,058 bp,and the total code is 685 aa;the total length of PmWRKY33 is 2,879 bp,the ORF is 2,785 bp,and the total code is908 aa;PmWRKY34 A total of 1,142 bp,ORF is 816 bp,a total code of 271 aa.3.PmWRKY2,PmWRKY32,PmWRKY33 and PmWRKY34 are unstable hydrophilic proteins without transmembrane structure and signal peptide,indicating that these 4 PmWRKY proteins are not secreted proteins;conservative structure and homology analysis show that 4 PmWRKY have all The complete and highly conserved WRKY domain has a high similarity to Masson pine,Arabidopsis and other plants in the conserved regions of the WRKY family.Among them,PmWRKY32 and PmWRKY33 belong to the group I members of the WRKY family,and PmWRKY2 belongs to the group IIc.Member,PmWRKY34 is a member of group IIb.Phylogenetic tree analysis showed that PmWRKY2 and PmWRKY10,PmWRKY32 and PmWRKY20,PmWRKY33 and At WRKY20,PmWRKY34 and PmWRKY24 are closely related.4.Expression analysis showed that the expression of four PmWRKY genes was tissue specific.The expression levels of PmWRKY32 and PmWRKY33 are as follows:stem>root>leaf;PmWRKY2 and PmWRKY34 are the highest in roots,and in stems and leaves,PmWRKY2 is expressed in leaves.The expression of PmWRKY34 in stems and leaves is similar.Under the treatment of methyl jasmonate,the four PmWRKY transcription factor genes are regulated and up-regulated in response to its induction.Among them,PmWRKY32 and PmWRKY33 are expressed at 0-24 h.An upward trend,reaching a peak at 24 h,and a decrease in expression at 24-48 h.The expression level of PmWRKY34 showed an upward trend at 0-6 h after treatment,and reached a maximum at about 6 h.After 6 h,the response regulation showed a downward trend.The expression level of PmWRKY2 showed an overall upward trend.At 48 h,PmWRKY2 still responded to the induced regulation of methyl jasmonate.5.The p BWA(V)HS-PmWRKY33-GLosgfp plant overexpression vector was successfully constructed.The subcellular location showed that PmWRKY33 was located in the nucleus.The genetic transformation of Nicotiana benthamiana successfully obtained 2 strains of transgenic tobacco seedlings.After qRT-PCR analysis,strain 1(The expression level of T1)is higher than that of strain 2(T2),which is 6.95 times that of T2.Expression analysis showed that the expression of four PmWRKY genes was tissue specific.The metabolites of transgenic tobacco and wildtype tobacco were detected by liquid chromatography-mass spectrometry(LC-MS),and the genes related to terpenoid synthesis of tobacco were studied.The results showed that transgenic tobacco with PmWRKY33 gene could improve the synthesis of isoppentadiene lipids and affect the up-regulated expression of Nb DXS1 and Nb DXS2.These results indicated that PmWRKY33 could regulate the synthesis of terpenoids.