| Dangshan Pear is an important economic fruit in DangShan County,Anhui Province.In recent years,there are poor conditions such as rough taste of the flesh.Therefore,it is especially necessary to study the key enzyme genes 4CL and C3H of the pear lignin synthesis pathway.The WRKY transcription factor is a 4CL and C3H regulatory factor,so 7 kinds of economic fruits(pear,apple,cherry,peach,plum,strawberry,raspberry)are selected,using bioinformatics methods to analyze the evolutionary unity and difference of the WRKYI subfamily members of the seven species of Rosaceae,it is helpful to better study the molecular evolution of the WRKYI subfamily gene and its relationship with lignin metabolism pathway.The main results are as follows:1.Compared with wild-type Arabidopsis thaliana,overexpression of Pb4CL11 significantly increased the expression of Arabidopsis thaliana stems(AtPAL,AtC4H,At4CL,AtHCT,AtC3H,AtCCOMT,AtCCR,AtF5H,AtCOMT,AtCAD4 and AtCAD5),the expression level increased by 1.7 times or more,can significantly promote root elongation and plant height,but the determination of lignin content in the inflorescence stem and the cross-section staining observation were not significantly different from the wild type.2.Compared with wild-type Arabidopsis thaliana,overexpression of PbC3H1 had no significant effect on root length and plant height,and 4 true leaves and bolting could be grown 5-7 days earlier.The lignin content of the inflorescence stalk of Arabidopsis thaliana increased significantly,reaching 1.49 times.In addition to AtF5H,it could significantly increase the expression of(AtPAL,AtC4H,At4CL,AtHCT,AtC3H,AtCCOMT,AtCCR,AtCOMT,AtCAD4,and AtCAD5),the expression amount is more than 1.2 times.It can increase the number of cells in the interfiber fiber region and the thickening of the cell wall.3.By searching the local genome database,a total of 7,9,10,11,13,20 and 28 WRKY Ⅰ genes were identified in P.avium,P.persica,P.mume,R.occidentalis,F.vesca,P.bretschneideri and M.domestica,respectively.These genes were randomly and unevenly distributed on chromosomes.Constructing the ML phylogenetic tree,98 genes was divided into twelve subgroups(A-M),which belong to four clades(i.e.class Ⅰ-Ⅳ)with higher bootstrap values.The members in the same subgroup had the similar exon-intron structures,conserved motifs,indicating that it is conservative.In these seven Rosaceae genomes,the gene duplication events of WRKY Ⅰ gene family only identified in both P.bretschneideri and M.domestica:the P.bretschneideri genome had ten gene family members were found in the duplicated segments of chromosomes,the synteny of seven gene pairs was significant in the duplicated region of the genome and speculated to evolve from large-scale duplication events;the M.domestica genome had twenty-four gene family members were identified in the duplication region of the genome,four gene pairs were identified as tandem duplication events,twelve gene pairs were found to be located in the duplicated segments of chromosomes,and these pairs were identified to be evolved from segmental duplication events.4.Quantitative analysis of 18 Pb WRKY fluorescence in different developmental stages of pear fruit,found that the trend of 14 gene expression levels increased first and then decreased(PbWRKY01/04/06/08/09/10/11/13/14/15/16/17/18/19),in which(PbWRKY01/04/09/10/13/14/15/16/17)will rise again after the trend of the expression level at 63 d after flowering.The peaks of these 14 gene expressions(except PbWRKY14)appeared between 39d and 63d after flowering,both before the peak of stone cells peaked.WRKY transcription factor can bind to WRKY71OS,a key enzyme gene of lignin synthesis pathway 4CL and C3H promoter,indicating that WRKY transcription factor may be involved in the regulation of stone cell content during pear fruit development.However,the trend of PbWRKY02/03/05/12 expression is inconsistent with other gene expression trends,which may be involved in the regulation of other functions of pear fruit. |
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