Floral Related Genes GhSPL3And GhLFY Research In Upland Cotton

Cotton is an important economic crop. Upland cotton, one of the most extensivelycultivated cotton species, has come to dominant world cotton commerce. The contradictionbetween large amount of people and small area of farmland as well as the conflict betweengrain and cotton for land is becoming more and more serious in China. So, early maturitybecame a basic breeding objective in cotton crop.Studies suggested that flowering is closelyrelated with early maturity. LFY, located in the center of flower regulation net, intergrates thesignal of flower induction and also activates the expression of floral organ identity gene. Asone of intergration factors in floral development, LFY gene is called as "flowering trigger"and played major role in floral regulation. SPL3is one member of SPL transcription factorfamily especially in plant. In Arabidopsis, SPL3acts in the upstream of LFY and regulate itsexpression positively to then influence flowering. From upland cotton CCRI, GhSPL3,GhLFY are cloned and analyzed with the amplication of qRT-PCR and subcellular location.To further understand the function of GhLFY, we transformed it to the Arabidopsis plantsincluding wild type and mutants background through agrobactetium-mediated gene transfermethod. Chromatin imnuprecipitation technology was used to detect the regulationmechanism of GhLFY by GhSOC1. The main results obtained from this research are asfollow:1.By bioinformation and RT-PCR Technology, GhSPL3was cloned from upland cottonand the ORF length of that is426bp which encodes141amino acid residues. Sequenceanalysis suggested that GhSPL3contains a typical SBP domain and a conservative nuclearlocalization signal. Construction the phylogeny tree using members of SPL family inArabidopsis and GhSPL3revealed that GhSPL3and AtSPL3are in a clade.Subcellularlocalization study revealed that GhSPL3localized in the nuclei and it suggested that GhSPL3protein have nulei lacolization function. Quantitative RT-PCR technology was performed toanalysis the expression pattern in of GhSPL3in different tissue and the development stage ofshoot apical. The results revealed that the expression level of GhSPL3is different in varioustissues. The expression of GhSPL3in the flowers and shoot apexes are the highest, the lowerwas in stem, and the lowest was in the root and leaves. Among different development stages of shoot apexes, the expression of GhSPL3was the highest at the third true leaf expansionstage in shoot apexes. These results indicated that GhSPL3might play an important role inbud differentiation, the transition of the growth phase and flowers formation.2. LFY/FLO homologs in other species, such as vitis, Arabidopsis and so on, are retrivedfome GenBank as queries to perform BLAST program in the genomic database of Gossypiumraimondii. By splice on line and bioinformation analysis, several usefull fragments were usedas templates for amplication in CCRI36. Combiding3'RACE technology, the complete ORFof LFY/FLO homologs gene in cotton named GhLFY was cloned and GhLFY contains1221base pair encoding426amino acids.To evaluated the evolutionary between GhLFY and otherFLO/LFY-like proteins, a phylogenetic tree was constructed using the protein sequences. TheGhLFY protein is more closely related to other FLO/LFY dicot proteins than to their monocotproteins. Among the dicots, GhLFY, HbLFY from Hevea brasiliensis and PtLFY fromPopulus tomentos formed a clade. It suggested that Gossypium hirsutum are nearly relationwith Hevea brasiliensis and Populus tomentos in the evolutionary among the speciesinvolved.To examine the subcellular localization of GhLFY, we introduced an expressionconstruct,GhLFY-GFP fusion encoding full-length GhLFY protein fused to GFP, into theonion epidermal cells. The35S::GhLFY-GFP fusion protein was observed exclusively in thenuclei of onion epidermal cells and in contrast, the35S::GFP protein was detected throughoutthe cells as expected. The results suggest that GhLFY is localized to the nuleus and it isconsistent with the function of LFY as a transcription factor.The transcription level of theendogenous GhLFY in Upland Cotton was examined using quantitative Real timeRT-PCR(qRT-PCR). In different tissues, low level of the GhLFY expression was detected inroots, stem and leaves, which is in contrast to the high level expression in shoot apexes at asignificantly elevated level. With the development of shoot apexes, the expression of GhLFYincreased sharply and reached a peak when the third true leaf fully expanded and thendecreased. These results suggested that GhLFY play an important role in transiton frominflorescence meristem to flower meristem. We transformed the gene of GhLFY to wild-typeand mutant Arabidopsis plants. The results showed that overexpression of GhLFY in wild-typebackground can cause early flowering. Complementation experiment revealed that GhLFYcan restore the flower organ phenotype.3. The transcriptional regulation study of GhLFY. Using genome walking technology,2538bp upstream sequence of GhLFY was obtained. Sequence analysis revealed that itcontains five typical CArG box motives. Comparasion the expression pattern between GhLFYand GhSOC1in shoot apex of upland cotton by qRT-PCR suggested that the expression trendof GhLFY is consistent with GhSOC1and so we inferred that GhSOC1and GhLFY could exist regulation relationship. Monoantibody of GhSOC1protein was customed made inAbMart and western blot was performed to testify that the antibody is available. ChIP resultsrevealed that GhSOC1protein was enrich in the CArG box region of GhLFY promoter and itsuggested that GhSOC1likely regulated the expression of GhLFY by binding its promoter asin Arabidopsis.