| Mei is a traditional Chinese famous flower.It has been cultivated for more than3,000 years with important ornamental value and broad market prospects.Mei possess rich flower colors and changeable flower types during long-term natural selection and artificial cultivation.There are currently 486 Mei cultivars,which contain lots of single-flower and double-flower cultivars.However,the molecular mechanism of the formation of double-flower Mei has not been understood.Therefore study the formation of double-flower Mei can not only help us to comprehend the underlying mechanism of Rosaceae woody plants,but improve Mei ornamental traits directionally.MADS-box genes are the genes closely related to flower development,which have very conserved MADS-box and K domain.We cloned PmAG and PmTOE3 genes,compared the nucleotide sequence of the PmAG and PmTOE3 between cultivars.PmAG gene expression between cultivars was analyzed by qRT-PCR.The flower bud differentiation period of the hybrid of ‘Xue Mei’ and‘Fenpi Gongfen’ was determined by paraffin sections,at the same time PmAG expression in different flower bud differentiation stages was analyzed by qRT-PCR.We constructed the PmAG overexpression vector,which was transferred into Arabidopsis for functional verification.The second intron of the PmAG gene was cloned and the cis-element analysis was analyzed.Taking the cultivars in ‘Green Calyx’ Form as the research object,we analyzed the A,B,and E class gene expression between cultivars.The main research results are as followed:1.The PmAG and PmTOE3 genes were cloned from ‘Xue Mei’.PmAG encodes243 amino acids and PmTOE3 encodes 474 amino acids.The nucleotide sequence alignment of PmAG and PmTOE3 genes between different cultivars showed that the PmAG is conserved.In ‘Da Gongfen’ and ‘Bian Lv’e’,there is a SNP in the mi R172 binding site of the PmTOE3 gene sequence,which might be a candidate site for the formation of double-flower Mei.2.The qRT-PCR results showed that the expression of PmAG gene of single-flower Mei cultivars was significantly higher than that of double-flower Mei.Taking the flower buds of the hybrid progeny of ‘Xue Mei’ and ‘Fenpi Gongfen’ as the research object,the flower bud differentiation period was divided into 9 stages through paraffin sections,and qRT-PCR of the PmAG gene was performed at the same time.It was found that the PmAG gene was not expressed at early stages,continued to increase from the beginning of the third round of floral organ formation to the completion of pistil development,and gradually decreased after entering the dormant state.3.The PmAG overexpression vector and CRISPR vector were constructed,and the PmAG overexpression vector was transformed into wild-type Arabidopsis.The petals of the transgenic plants became smaller with degenerated petals and stamens,and aborted flowers and pods.qRT-PCR showed that the expression of PmAG gene increased,and the single-copy line with PmAG gene was obtained.4.The second intron of PmAG gene was cloned and cis-element were analyzed.It was found that the second intron of PmAG gene contained LFY,WUS,TOE1,TOE2,AG and other gene binding sites related to flower development.5.Taking the ’Green Calyx’ Form as the object,we ananlyze the gene expression between cultivars.The results showed that PmAP1,PmFUL2,PmPI,PmSEP2,PmSEP4 had significant differences between different flower type cultivars,indicating that the expression of these genes may be related to double flower traits,but their underlying mechanisms still need to be further verified. |
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