Bioinformatics Analysis And CtMADS24 Regulation Function Of MADS-box Gene Family In Carthamus Tinctorius L.

Carthamus tinctorius is a traditional Chinese herb with high economic and medicinal value.The differentiation rate and abortion of florets in the flower bulb of Carthamus tinctorius directly affect the seed yield during long-term cultivation,and promoting the normal development of florets has become one of the methods to improve its yield.Up to now,the molecular mechanism of the Carthamus tinctorius flowering process and flowering organ formation is not clear,and the MADS-box family genes play a key role in regulating the flowering process of plants.In this study,we predicted the evolutionary characteristics,gene structure,protein conserved structural domains,and other key information of the Carthamus tinctorius MADS-box family based on its genome sequencing data,and analyzed the expression patterns of different developmental stages and different floral organs of Carthamus tinctorius petals by q RT-PCR and RNA-seq techniques,and mined the AP1/FUL subfamily members of Carthamus tinctorius MADS-box family by the above methods.We also investigated the relationship between CtMADS24 and the regulation of Carthamus tinctorius flowering transformation and floral organ building.The main research results are as follows:1.77 MADS-box members in the Carthamus tinctorius genome were identified by used bioinformatics and classified into two major classes and 15 subfamilies.Amino acid structure and gene structure analysis indicated high structural conservation among the subfamily members,while GO annotation suggested that the Carthamus tinctorius MADS-box protein family members are functionally complex and mostly have typical transcription factors regulatory functions.2.Analysis of the cis-acting elements in the upstream 2000 bp promoter region of MADS-box family 77 genes revealed the possible regulatory relationships of the family genes.q RT-PCR and RNA-seq techniques were used to investigate the expression patterns of floral organs at different floral developmental stages and early floral development of Carthamus tinctorius.The results showed that the expression patterns of different subfamily genes in floral organs were specific,and combined with the results of bioinformatics and expression analysis,we found that CtMADS24,a subfamily of AP1/FUL of the Carthamus tinctorius MADS-box family,was expressed at a high level during the whole flower development and was specifically expressed in the early sepal development.3.The results showed that overexpression of CtMADS24 in Arabidopsis caused up-regulation of the expression level of some genes characteristic of flower organ development,resulting in shorter flowering time,reduced number of rosette leaves,and decreased endogenous growth hormone content;while the silenced strain Carthamus tinctorius showed a significant delay in flowering and an increase in endogenous growth hormone content,suggesting that CtMADS24 can positively regulate flower development but suppress plant nutritional development.4.The nuclear localization characteristics of CtMADS24 were clarified by the transient transformation of CtMADS24-GFP protein in tobacco,and the binding activity of CtMADS24 to CAr G elements was verified using yeast one hybrid system and dual-luciferase reporter systems.5.The promoter region of 1933 bp upstream of the coding region of CtMADS24 was successfully cloned,the possible transcription factor binding sites on pro CtMADS24 were predicted,and the regulatory roles between CtWRC,CtARF,and CtMADS24 were verified,providing theoretical support for the in-depth study of the molecular mechanisms of flowering transition and floral organ-building in Carthamus tinctorius.