Molecular Cloning And Expression Pattern Analysis Of MADS-box Genes And Selection Of Reference Genes In Cycas Elongata

MADS-box genes are important transcription factors involved in flowers development in plants. Related researches have made a great progress in the past 25 years, such as the famous flower development ABCE model, but the origin of flowers is still unknown. Gymnosperms which are the most near relatives of angiosperms are regarded as an important point to study the molecular mechanism of the origin of flowers. Researches for MADS-box gene in gymnosperms were mainly focused on the Ginkgo, conifers and gnetophytes, which was not available in cycads. In this study 12 MADS-box genes(Ce MADS1-12) were chosen from the transcriptome database of Cycas elongate and were set for gene expression analysis in 8 different organs by q RT-PCR. Two genes belonged to M?+M?clade of Type I MADS-box gene, and the remaining ten genes belonged to MIKCC type which were divided into 9 clades:AG, GGM13(Bsister), AGL6, TM8, GGM4, Gp MADS4, TM3, SVP, GGM19 and GGM7. The two Type I genes, Ce MADS11 and Ce MADS12, expressed in vegetative organs and reproductive organs. As to the MIKCC type genes, Ce MADS1, 3, 4 were involved in reproductive organs development; Ce MADS2 and Ce MADS5-10 were detected in vegetative organs and reproductive organs. Gp MADS4 clade was only found in gymnosperm too, and functions of other clades of MADS-box genes in this study were relatively conserved. Especially what we got from the gene expression of B, C and E class genes depicted that these three clades of genes might emerge at least 300 million years ago and that they would have regulated the reproductive organ development in the common ancestor of gymnosperms and angiosperms.Quantitative reverse-transcription PCR(q RT-PCR) has been a key enabling technology of gene expression analysis in numerous molecular biology applications. The q RT-PCR is considered with high sensitivity, accuracy, reproducibility and high throughput capability for quantification of transcript levels. However, some variables such as the integrity, amount and purity of the RNA, as well as enzyme efficiency during c DNA synthesis and PCR amplification make it necessary to normalize the data for an accurate and reliable result. As we planed to do MADS-box gene expression analysis, and to date, reliable reference genes in C. elongata have not been well characterized, we chosen 13 reference genes(ACT7, TUB, UBQ, EIF4, EF1, CLATHRIN1, PP2 A, RPB2, GAPC2, TIP41, MAPK, SAMDC and CYP) from the transcriptome database of C. elongata, and evaluated them in 8 different organs samples. Results obtained from three programs, Norm Finder, Ge Norm and Best Keeper, suggested that GAPC2 and RPB2 are the most stable reference genes, while the ACT7 is the least stable one among the tested genes.To our knowledge, this work presents the first time to obtain MADS-box genes in C. Elongate, which fills the gaps of MADS-box genes of cycads in gymnosperms, and makes it way to getting better understanding of the flower origin in molecular level. Besides this study represents the first attempt to clone, sequence and evaluate commonly used reference genes and will facilitate further gene expression analyses of target genes in different tissues of C. elongate.