Molecular Cloning And Functional Analysis Of Two MADS-box Genes From Bitter Gourd (Momordica Charantia L.)

Bitter gourd is not only a vegetable crop but also an important medicinal herb. Flowerdevelopment of bitter gourd is not common. Bitter gourd prosperous stage lasts about threeor four months with a number of flowers and male flowers preponderate in the flower of thebitter gourd. Whereas the processes of flower development about bitter gourd also present ahermaphroditic flower stage. These characteristics of flower development are veryprominent in the monoecious plants. In order to understand and exploit the molecularmechanisms that control flower development in bitter gourd, we have begun to clone andanalyze MADS-box genes from buds in bitter gourd. In this study we describe cloning andexpression analyses of two MADS-box genes (McAG2 and McAG6). Furthermore McAG6 isfirst AGL6-1ike gene in Cucurbitaceae.1 Cloning and characterization of bitter gourd AG-like MADS-box gene(McAG2)A combined 3'RACE and 5'RACE strategy was used to isolate MADS-box genes frombitter gourd. Full-length MADS-box gene designated McAG2 (GenBank accession No.DQ299943) was isolated. McAG2 cDNA is 1 003 bp in length and contains an ORF of 693bp, which encodes a 231 amino acids protein with an N-terminal extension preceding the MADS domain, Mw 26.5 kDa, deduced isoelectric point 9.59. The deduced amino-acidsequence of McAG2 was compared with other protein sequences found on the GenBankdatabase using the BLAST search program. McAG2 had high similarities with the deducedAG-like proteins. Multiple sequence alignments demonstrated that the McAG2 deducedprotein had the typical MIKC-type domain structure. The C-terminus of McAG2 containstwo highly conserved regions, which are AG motifâ… and AG motifâ…¡. Phylogenyreconstruction indicated that the McAG2 fell into a group of AG-like genes which belong tothe eudicot C lineage. RT-PCR and Quantitative real-time RT-PCR analysis of McAG2showed that the expression of McAG2 was very low or none in petal, calyx, and shoot apex,high in carpel and moderate in stamen and young fruit. The recombinant plasmidpET28a-McAG2 was expressed in E. coli. BL21. The molecular mass of expressionproducts, checked by SDS-PAGE, was 26 kDa. The over expression of 35:McAG2 blockthe second whorl development.2 Cloning and expression analysis of bitter gourd AGL6-like MADS-boxgene (McAG6)McAG6 (GenBank accession No. DQ431247) was isolated from bitter gourd using acombined 3'RACE and 5'RACE strategy. McAG6 cDNA is 1 061 bp in length and containsan ORF of 741 bp, which encodes a 247 amino acids protein, Mw 28.4 kDa, deducedisoelectric point 9.3. The deduced amino-acid sequences of McAG6 was compared withother protein sequences found on the GenBank database using the BLAST search program.McAG6 had high similarities with the deduced AGL6-1ike proteins. Multiple sequencealignments demonstrated that the McAG6 deduced proteins had the typical MIKC-typedomain structure. Similarity is also found at the C-terminus of the McAG6. All C-terminusof AGL6-1ike proteins presented two short, highly conserved regions, which were namedAGL6 motifâ… and AGL6 motifâ…¡. Both of the motifs were composed of 10 amino acidresidues. Phylogeny reconstruction indicated that the AGL6-1ike genes formed three clades.Clade 1 was distinctly divided into two groups: eudicots and gymnosperms, named clade 1Aand clade 1B; the monocots formed the clade 2; and the" basal angiosperms, such asMagnolia, formed the clade 3. In this dendrogram, McAG6 fell into calde 1A and was closer to VvMADS3 than other AGL6 homology. RT-PCR and Quantitative real-time RT-PCRanalysis of McAG6 showed that the expression of McAG6 was found very low or none inyoung fruit, carpel, and stamen, high in shoot apex, and moderate in calyx and petal.3 Cloning of the McAG2 gene promoter using DNA walking technologyTwo pairs of specific nested PCR primers were designed according to thepublished sequence of MADS-box gene (McAG2) in bitter gourd. The promoter(McAG2P) of McAG2 gene with a length of 1417bp was cloned by DNA walkingtechnology. McAG2P had the typical TATA box, CAAT box, regulator elements, etal. In order to establish a foundation for further study of McAG2P function inpromoting McAG2 gene specific expressions in higher plant reproductive organs,eight McAG2P absence sequences McAG2P1-McAG2P8 were obtained by PCR andinserted into vector pBI221 to take place of CaMV35S promoter to get eightexpression vector.