无患子MADS-box基因家族全基因组鉴定及SmAP1基因功能研究

[Objective]To study the members and classification of the MADS-box gene family of Sapindus,clarify the expression rules and functions during its growth and development,and select the key gene SmAP1 for functional verification,in order to provide a regulatory molecule for Sapindus flowering.foundation for mechanism research.[Method]Based on the genome-wide data of Sapindus,this study identified the MADS-box gene family and conducted a comprehensive analysis,including gene structure,conserved motifs,phylogenetic evolution,prediction of cis-acting elements,protein properties and protein interaction networks of SmMADS genes.At the same time,the expression patterns of MADS-box in various vegetative organs and reproductive organs were analyzed,and RT-qPCR technology was used to verify the expression of key genes.The co-expression network of SmMADS genes during the flowering process was constructed by using Pearson correlation coefficient,the key gene SmAP1 was screened out,and the expression vector was cloned and constructed,and further genetically transformed in Arabidopsis thaliana to observe the phenotypic differences and preliminarily elucidate the SmAP1 gene functions in regulating flowering time and floral organ development.[Result]A total of 106 MADS-box gene members were identified in the whole genome data of Sapindus mukorossi.Based on Arabidopsis thaliana,grape(Vitis vinifera),soybean(Glycine max),peach(Prunus persica),apple(Malus pumila),poplar(Populus trichocarpa)and Sapindus mukorossi MADS protein data to construct a phylogenetic tree,and they were divided into two subfamilies,Type I and Type II.Type I can be divided into three subclass,Mα,Mβ,and Mγ,while Type II is divided into 18 subclass.Analysis of cis-acting elements can respond to photoperiod,hormones and environmental stress,and has certain functions in regulating abiotic stress.Different subclasses of SmMADS proteins have different regulatory functions.The expression of MADS-box gene members was significantly different in 26 tissue parts including vegetative organs(such as roots,stems,and leaves)and reproductive organs(pericarp,seeds,and male and female florets),especially male and female floral organs(such as petals,stamens,and ovaries).Type I MADS members were mainly expressed in the vegetative organs of roots,stems and leaves,and were significantly expressed in the cotyledon growth stage of fruit development.Type II MADS members are mainly involved in regulating the growth of male and female flowers and the development of floral organs in Sapindus.Fluorescence quantitative data indicated that the expression levels of SmSEP4,SmPI,SmAP3,and SmSOC2 were increased during the initiation of inflorescence differentiation.Additionally,SmPI and SmAP1 were highly expressed in the stamens,pistils,and petals development.Notably,SmAP3 played a vital role in the microspore formation process of stamens of different gender flowers.In this study,the full-length 726 bp coding region of SmAP1 gene was successfully cloned.Compared with the wild type,the bolting and flowering time of the 35S::SmAP1 Arabidopsis plants of the nine T2 lines were significantly earlier in different degrees,and the ovaries were thicker.The results of RT-qPCR experiments showed that in the floral tissue of 35S::SmAP1 Arabidopsis thaliana,flowering time-related integration factors(AtFT and AtSOC1),floral organ development-related factors(AtAP1,AtAP3 and AtPI)and flowering meristem regulators(AtLFY and AtCAL)were up-regulated,while flowering inhibitors(AtTFL1,AtAGL24 and AtSVP)were down-regulated.[Conclusion]The MADS-box genes exhibited different expression patterns during the eight growth stages of male and female flowers,as well as floral organ development.The overexpression of the SmAP1 gene in Arabidopsis thaliana results in the upregulation of flowering integrators and floral meristem regulators.Compared to the wild-type plants,the 35S::SmAP1 transgenic Arabidopsis showed varying degrees of early bolting and flowering.This result has laid the foundation for further research on the molecular mechanisms underlying flowering regulation in Sapindus.