Identification Of Mads-Box Gene Family In Soybean And Functional Analysis Of GmAP1 And GmSHPα

MADS-box genes are complex gene family in higher plants. MADS-box transcription factors are key regulators of several plant development processes. Besides being involved in floral organ specification, they have also been implicated in several aspects of plant growth and development. Soybean(Glycine max (L.) Merr.) is one of the most important economical crops, as it is the main source for vegetable oil and protein. The reproductive growth will directly affect seed yield and quality. Therefore, to unravel the genetic mechanisms underlying the regulation of flower and fruit is of great significance in improvement of soybean production.In this research, we identified 143 MADS-box genes in soybean genome by screening the available database using bioinformatics analysis. Based on the phylogenetic relationships of the conserved MADS domain, these transcription factors were divided into two types:type I and type II. The soybean type II MADS-box genes were further classified into MIKCc (64 genes) and MIKC* (7 genes) groups, and type I MADS-box genes were classified into Ma (36 genes), Mβ (11 genes) and My (25 genes) groups. Analysis of MADS-box genes in different groups were presented, including sequence characteristics, chromosome locations, gene structures, protein properties and phylogenetic relationships. EST search showed that most MADS-box genes were active and can be transcribed in soybean. Gene expression patterns were investigated by searching against microarray data. We found that the soybean MADS-box genes were primarily expressed in flower and fruit. The comparison of the MADS-box proteins of soybean and Arabidopsis helped to well understand the evolution of MADS-box gene family and also suggested that MADS-box genes might play important roles in soybean flower and fruit development.According to the analysis of MADS-box genes in soybean, one AP1-like gene was chosen for further study, named GmAPl. We isolated the genomic sequence and ORF of GmAP1 from soybean. GmAPl contained eight exons and seven introns, it was a single copy gene in soybean genome and located in Chr.16. The ORF of GmAP1 consisted of 711 bp which encoded a protein with 236 amino acids. Sequence comparison showed that GmAP1 was similar with other API-like proteins. It contained the conserved domains characterizing AP1-like proteins:MADS domain, K domain and euAPl-motif. The phylogenetic tree suggested that GmAP1 fell into AP1/SQUA family which belonged to A class in ABCDE model.RNA in situ hybridization analysis showed that GmAPl transcript was first detected at the early stage of meristem differentiation and sequentially accumulated in both the apical inflorescence meristem and lateral floral meristems of soybean, suggesting that GmAP1 might determine floral meristem and be involved in floral primordia identities. Real-time PCR and RT-PCR were used to detail the gene expression pattern. GmAP1 exclusively expressed in flower. During flower development, the expression was observed in the tiny flower bud and maintained until mature flower. In mature flower, expression of GmAP1 was restricted to the sepals and petals.GmAP1 was a nucleus localized transcriptional activator, suggesting it functioned as a transcription factor in gene regulatory network. The in planta function of GmAP1 was shown through expressing in tobacco and Arabidopsis. Compared with wild type, most 35S::GmAP1 transgenic plants started flowering earlier. Transgenic tobaccos showed dwarfism and apical dominance reduction. In some early flowering tobaccos, floral organ alterations were observed, such as abnormal feature of perianth, petal-like stamen and stamen-like petal. These results illustrated GmAP1 functioned on flowering control, floral meristem formation and floral organ identities.In this research, a SHP-like gene was also chosen for cloning and functional analysis, named GmSHPa. A cDNA full length of GmSHPa was obtained by RACE with a complete ORF of 729 bp. GmSHPa consisted of 242 amino acids and was located to the nucleus. Sequence alignment showed that GmSHPa was similar with other SHP proteins. It contained highly conserved domains:MADS domain and K domain. AG motif Ⅰ and Ⅱ were recognized in the C terminus. The phylogenetic tree showed that GmSHPa was close to SHP homologues in legume, such as Lotus and Pisum. It fell into PLE group of AG subfamily which belonged to C/D class in ABCDE model, suggesting a function on stamen and carpel identities and regulation of fruit development.Gene expression analysis showed that GmSHPa was highly expressed in reproductive organs of soybean, including flower and fruit. It can not be detected in vegetative organs. GmSHPa was expressed during the fruit development at a low level. Its transcript was also detected in seed coat. In mature flower organs, GmSHPa is mainly expressed in carpels. These findings suggested that the function of GmSHPa was involved in carpel and pod development. Over expression of GmSHPa in Arabidopsis altered the morphology of fruit and flower. Transgenic fruits were significantly smaller than wild type. A noticeable shrink of the valves were observed. The dehiscence zones were more fibrous. The valve margins showed more definition than those of wild type fruits. The pod shatter was promoted in 35S::GmSHPa transgenic fruits, but the germination of seeds was not affected. The transgenic line, in which GmSHPa was highly expressed, had no petal in all flowers. These results indicated that GmSHPa controlled fruit dehiscence and regulated flower organs development.