| Allium cepa L. is two year diploid (2n=2x=16) vegetable crops, which is native to Asia, it is widely cultivated due to its characteristics of rich in nutrients and resistance to transport and storage, etc. To explain the molecular mechanism of floral organ development, we isolated and characterized six full length cDNA sequences of onion MADS-box genes from conventional onion varieties’RUPI’denoted as AcFUL, AcDEF, AcPI, AcAG, AcAGL6and AcSEP3by using RACE (rapid amplification of cDNA ends). In addition, investigation was also made to analyze sequence characteristics and phylogenetic relationships based on bio informatics methods. We analyzed the expression pattern of these genes in different organs of onion according to the semi-quantitative RT-PCR and real-time fluorescence quantitative PCR. The results of this study laid a solid foundation for further interpreting the action mechanism of onion MADS-box genes and molecular mechanism of floral development.(1) In this study we used conventional onion varieties’RUPI’as test materials, obtained six full length cDNA sequences contained open reading frames by the method of RACE and named AcFUL, AcDEF, AcPI、AcAG、AcAGL6'AcSEP3.(2) In order to interpretate the homology of the six genes to established MADS-box genes, We analyzed sequence characteristics and phylogenetic relationships of amino acid sequences corresponding to the genes using softwares of bio informatics. Sequence characteristics analysis show that AcFUL, AcDEF, AcPI, AcAG, AcAGL6and AcSEP3encode deduced proteins with242,205,224,229,241and240amino acids residues respectively, all of the amino acid sequences have a typical MIKC-type structural domain and C-terminal of these proteins have specific motifs of A, B, C. E-class MADS-box genes. Phylogenetic analysis indicated that AcFUL belongs to the FUL-like-lineage of the monocot A-function gene family; AcDEF and AcPI belong to the PI/GLO and AP3/DEF-lineage of the monocot B-function gene family respectively; AcAG belongs to the AG-like-lineage of the monocot C-function gene family; AcAGL6belongs to the AGL6-like-lineage of the monocot E-function gene family; AcSEP3gene belongs to the SEP3-like-lineage of the monocot E-function gene family.(3) Semi-quantitative RT-PCR and real-time PCR revealed that expression signal of the six onion MADS-box genes were detected in all floral organs, however in the vegetative organs either low level or no expression signal were detected. The expression analysis of AcFUL showed the highest level in the carpels and flowers, moderate level in membranous sheath, petals and stamens, very low level in the flower buds, leaves and scapes, no expression in roots nor cauloid, all of these revealed AcFUL was not only involved in deciding characteristics of floral organs, but also related with fruit and leaf development. The expression of AcPI and AcDEF is tissue-specific, with the highest level in the petals and stamens, moderate level in scapes, membranous sheath, flower buds and flowers, and very low level in the roots, cauloids and leaves, the expression pattern is corresponding to that B-class genes function in deciding characteristics of petals and stamens. AcAG specifically expressed in stamens and carpels with high level, expressed in membranous sheath, petals, flower buds and flowers with moderate level, confirmed that C-class genes mainly controled features of stamens and carpels. AcAGL6expressed in root, cauloids, leaves and scapes with low level, the expressing level increased from membranous sheath to flower. There are roughly identical expressing level of AcSEP3in four floral organs, flower buds and flowers are roughly identical, the expression pattern supported E-class genes are involved in the determination of all four whirl flower organs. |
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