Genome-Wide Identification And Analysis Of MADS And AP2 Gene Families In Phalaenopsis Aphrodite

The floral structure of orchids exhibits unique evolutionary features such as the specialization of labellum and the formation of gynostema.These specialized flower organs provide orchids with great appreciation,and the mechanism of flower organ development has been a hot research topic in botany and horticulture.Phalaenopsis is the orchid species with the largest planting scale,the widest popularity and the most solid molecular biology research foundation.In this study,we identified two genomewide gene families related to flower organ development in Phalaenopsis,MADS-box and AP2,and analyzed their phylogeny,gene structure and collinearity.The basic attributes of these two gene families and the corresponding direct homologous genes of flower organ attribute genes in Arabidopsis ABCE model in Phalaenopsis were identified.Then,the expression levels of these genes in sepals,petals,labellum and pistil column of Phalaenopsis were analyzed to predict the flower organ development model of Phalaenopsis.In addition,the codon bias of organ development genes was analyzed and the optimal codon of these genes was screened.The specific results are as follows:(1)Identification and analysis of PaMADS gene families: A total of 57 PaMADS members were identified by two ways BLAST and conserved domain verification,which were divided into type I(29 members)and type II(28 members).Most members of type I contain 1 or 0 introns,while members of type II contain 0～2 introns.The MADS family members of type II are mostly involved in the development of flowers,fruits and seeds of plants,which is related to the reproduction and evolution of plants,requiring more precise and flexible regulation.This may be the reason why the introns of MADS family members of type II are significantly more than those of type I in Phalaenopsis.All members of PaMADS family contained MADS-box domain(motif1),and most of type II proteins contained K-box domain(motif2,motif4,motif6).The promoter region of this family of genes contains many cis-acting elements related to abiotic stress,light,plant hormones and growth and development.Only 3 pairs of PaMADS family members were colinear,indicating that the gene family did not have a large expansion,which may also be one of the reasons for the small number of members of the gene family.In addition,the collinearity with the At MADS family is weak,and the amplification of MADS gene family in angiosperms may occur before the species differentiation of Phalaenopsis and Arabidopsis.After the speciation of Arabidopsis and Phalaenopsis,the MADS-box gene of the former underwent several rounds of important chromosome rearrangement and fusion,while the gene family did not undergo a mass replication event in Phalaenopsis.(2)Identification and analysis of PaAP2 gene family: A total of 121 AP2 family members were obtained from Phalaenopsis.Phylogenetic analysis showed that the AP2 family could be divided into 5 subfamilies,namely RAV subfamily(29),AP2 subfamily(42),DREB subfamily(19),ERF subfamily(26)and Soloist subfamily(5),There are3-7 motifs in PaAP2 family members,and motif1 is shared by all members.The special motifs of the RAV subfamily are motif7 and motif8.The special motifs of the AP2 subfamily are motif2 and motif3.The characteristic domains of ERF subfamily are motif5,motif6,motif14,and motif20.The number of introns in PaAP2 family is small,most of them are 0～2,and 7 members have more than 2 introns.The length of g DNA of only 15 members was more than 5000 bp.The collinearity between the two PaAP2 genes and At AP2 in Arabidopsis thaliana suggests that the amplification of MADS gene family members may occur before its differentiation with Arabidopsis thaliana.A total of 11,498 cis-acting elements were found in the PaAP2 family,which can be roughly divided into four categories: light responsive elements,hormone responsive elements,stress responsive elements and growth and development related elements.Among them,there are 1318 optical response elements,more than 10% of the total number;Hormonal response elements accounted for 7.3% of the total.It is noteworthy that in the AP2 subfamily,not only TATA elements and related enhancers related to protein regulation,but also some members of the promoter region have elements related to hormone regulation and regulatory elements related to light response.The traditional view is that the members of the AP2 family only play a regulatory role in growth and development,but it can be inferred that the members of the AP2 family are also involved in the response of plants to stress.(3)Expression model and codon bias analysis of organ attribute genes of Phalaenopis: phylogenetic analysis identified 30 organ attribute genes of Phalaenopis.Among them,The corresponding genes in Arabidopsis flower organ development model ABCE model were PAXXG045840(corresponding to At AP1),PAXXG201440(corresponding to At AP2),PAXXG070630(corresponding to At AP3),PAXXG193450(corresponding to At PI),PAXXG220840(for At AGAMOUS)and PAXXG314420(for At SEP1).The expression levels of PaAP1 and PaAP2 remained high in sepals and petals,but decreased in labellum and pistils.The expression level of PaAP3 was high in petal and labellum,but low in sepal and antherium column.PaPI is somewhat similar,except that it maintains the highest level of expression only in the labellum.The expression level of PaAG gene was the highest in synthium column,while PaSEP was highly expressed in sepals and petals,but very low in labellum synthium column,similar to PaAP1 and PaAP2.The CAI average value of 30 organ attribute genes was0.21,far less than 1,indicating that the codon bias of genes was weak.Nine optimal codons were determined by △RSCU method,among which 7 codons △RSCU≥0.5,5 codons ending in G/C and 2 codons ending in A/T(U).Combined with the content of GC of the third codon,it is inferred that the organ attribute genes of Phalaenopis prefer codons ending in G/C,especially those ending in C.Similar to the preferences of most monocotyledons.The analysis of odd-even bias,neutral plots and ENC plots further indicated that organ attribute genes of Phalaenopis were affected by both selection and base mutation,and the former was more important.This study through PaMADS and PaAP2 genome-wide identification and analysis of the two gene families,made clear the two family system development,gene structure,contains all the family of the promoter regulatory elements,for further mining from the two family plant or adversity stress response genes and development,have important reference value.In addition,30 floral organ development genes were identified from PaMADS and PaAP2 gene families.The expression analysis of direct homolog of ABCE gene in floral organs revealed that PaPI,a direct homolog of Class B genes in Phalaenopsis,was directly related to the development of labellum.The expression pattern of class E genes is different from that of traditional genes,and more similar to that of Class A genes.In addition,the codon bias characteristics of Phalaenopis organ development gene were identified,which also laid a solid foundation for the innovation of Phalaenopis flower type through transgenic.