Cloning And Functional Analysis Of Flowering-related Genes From Gossypium Hirsutum L

In order to provide enough and good gene resources, we cloned flowering related genes in thisstudy and did function analysis for them.Genes belong to SQUA subfamily usually had function in promoting flowering, so we usedAtAP1and AtFUL as the query sequences to Blast cotton EST database. We assembled the obtainedESTs and got two genes with complete open reading frames (ORF). Primers were designed accordingto the sequences and crossing the whole ORF. The PCR products were named GhMADS22andGhMADS29separately. The two genes both had eight exons and seven introns from the start codon tothe stop codon according to the alignment between the obtained cDNA sequence and the Gossypiumraimondii L. genome sequence. The two genes expressed higher and higher with the apicesdeveloping. GhMADS22had high levels in leaves, buds and bracts, while almost did not expressed inovules and fibers. Similar to GhMADS22, GhMADS23also hardly expressed in ovules and fibers,while expressed very high in floral organs and apical buds. GhMADS22and GhMADS23expressioncould be promoted by GA treatment. The transgenic Arabidopsis which over expressed GhMADS22bolted earlier than wild type and converted the indefinite inflorescences to solitary flowers or terminalflowers. The heteromorphosis of floral organs also occurred in transgenic plants. Secondary flowerscould form at the bases of bracts or pistils. Some flowers had two pistils, seven petals and stamens.Some flowers had only three petals, eight stamens surrounding pistils randomly. There were alsoflowers without bracts. In addition, the floral organs were delayed to senesce and shed in transgenicplants over expressing GhMADS22. It was also found that GhMADS22could respond to abscisic acid.In summary, GhMADS22may have functions in promoting flowering, improving resistance anddelaying senescence for cotton. So GhMADS22could be a good candidate gene for promotingearly-maturation in cotton breeding.Mutation of SVP gene in Arabidopsis shortened the vegetative growth stage and promotingflowering. We cloned the SVP homologue gene GhMADS29from CCRI36upland cotton by the samemethod as above. Blast alignment showed that GhMADS29was most similar to SVP4from kiwifruit.Alignment between the obtained cDNA and genome sequence showed that GhMADS29had nineexons and eight introns. GhMADS29expressed highest in2SAM (shoot apical meristems at thesecond true leaf expanded stage) at which stage flower bud initiated differentiation. Expressionanalysis in different tissues showed that GhMADS29expressed very highly in leaves and apical buds.These results made us to speculate that GhMADS29may play a part in controlling flowering in thesame way as AtAP1. However, the transgenic experiment showed that the transgenic plants overexpressing GhMADS29did not show difference in bolting time under long day conditions. There maybe other homologues of SVP in cotton taking parts in regulating flowering.According to the GhMADS29genome sequence we designed primers to clone its promoter and1316bp was obtained from-19site of ATG upstream. Predomination of promoter elements indicatedthat this promoter sequence had the core and essential elements and some light, temperature, hormones, stress responded elements. Transient expression and transgenic experiments both provedthis sequence had promoter activity. GUS staining on different tissues of transgenic plantsdemonstrated that this promoter had activity in roots, leaves, trichomes, bracts, petals, and valves, butnot in stems, stamens and seeds.