Studies On In Vitro Lfowering And Related Molecular Mechanism In Tagetes Erecta L.

In this experiment, in vitro plantlets of Tagetes erecta L. were used as the materials for thestudies of establishment of regeneration system, in vitro flowering, gene cloning and quantitativeexpression of LFY and cab genes which were probably related to floral differentiation in Tageteserecta L., The main results were as follows:1Establishment of regeneration system of Tagetes erecta L.The stems of in vitro plantlets were used as materials to study the proliferation and rooting ofTagetes erecta L.. The results showed that the order of the impact of the proliferation was6-BA>sugar> NAA, the best proliferation medium was the MS medium supplemented with0.1mg/L6-BA,0.1mg/L NAA and30g/L sugar.Serious vitrification phenomenon was found in the above experiments. MS with0.1mg/L6-BA and0.1mg/L NAA was used as the basal medium to study the effects of sugar, NH4NO3multiples in MS medium, activated carbon, penicillin G sodium and the illumination time on theproliferation and vitrification of Tagetes erecta L.. The results showed that:①highconcentration of sugar could inhibit the occurrence of vitrification, but also led to the decline ofthe proliferation coefficient, the optimum concentration was50g/L.②The decline of NH4NO3multiples in MS medium would promote the occurrence of vitrification, so it was not theimportant factor affecting vitrification of Tagetes erecta L..③Activated carbon could inhibitvitrification phenomenon effectively, but the vitrification would occur easier when addingconcentration was too high, the optimum concentration was approximately1-2g/L.④PenicillinG sodium had a certain role on promoting the proliferation, but improved the vitrification ratesignificantly, so it should be removed.⑤The suitable illumination time could promote thegrowth of plantlets and reduce the vitrification rate, the best treated time was15h/d.2Study on in vitro flowering in Tagetes erecta L.The plantlets of Tagetes erecta L. were used as the materials to investigate the effects of theKH2PO4multiples in MS medium, KT, GA3, and the combined effects of PP333and sugar on thefloral bud induction. The results showed that: in the single-factor experiments, higher KH2PO4multiples in MS medium not only suppressed the floral bud differentiation, but also causedabnormalities of plantlets growth and development; among the range of0.1-0.8mg/L, the more amout of KT was added, the higher inducing rate was found, the medium adding0.8mg/L KT hadthe highest inducing rate; lower concentration of GA3could promote floral bud formation, higherconcentration just had opposite effect, while the MS medium supplemented with1.0mg/L was thebest for the floral bud induction. The randomized experiment showed that PP333influenced thefrequency of floral bud induction significantly as well as sugar, and the impact of PP333wasgreater than that of sugar on floral bud induction. The medium supplemented with0.3mg/L PP333and50g/L sugar was the best, the inducing rate was100%, the floral bud and the fully developednormal flowers could be observed as little as15-20days or about90days, respectively.3Cloning of LFY and cab genes in Tagetes erecta L.In this experiment, the full-lengths of cDNA of LFY and cab genes in Tagetes erecta L. werecloned successfully as in vitro plantlets were used as materials, and the bioinformatics predictionof the corresponding amino acid sequences was studied. The results showed that：①Thefull-length cDNA sequence of LFY gene was1486bp, containing20bp5’UTR,155bp3’UTR, and3’-end involved25poly (A) tails; the open reading frame had1311bp, encoding436amino acids.The nucleotide and the encoded amino acid sequences of LFY gene was highly homologous withother plants reported in GenBank. The gene was named LEAFY gene, and the accession numberwas JF825877in GenBank. The bioinformatics analysis results showed that: LFY protein was ahydrophilic protein, with a transmembrane helix and coil structure, without signal peptide. Thesecondary structure of this protein was mainly constituted by random coil and α-helix containing aleucine zipper motif at the89-107amino acid interval; the protein was located in the cytoplasmand the mitochondrial matrix space, containing20phosphorylation sites. These resultsdemonstrated that the LFY protein had multiple functions, and it might play an important role inthe process of signal transduction and gene transctiption.②The full-length cDNA sequence ofcab gene was916bp, containing43bp5’UTR,75bp3’UTR, and3’-end involved19poly (A)tails； the open reading frame had798bp encoding265amino acids. The nucleotide and encodedamino acid sequences of cab gene was highly homologous with other plants reported in GenBank.The gene was named CAB protein gene, and the accession number was JQ083583in GenBank.The bioinformatics analysis results showed that: CAB protein was also a hydrophilic protein, witha transmembrane helice model constituted by three sections and with N-terminal extracellual,without signal peptide. The main secondary structure was random coil and α-helix. The proteinwas located in the chloroplast stroma, chloroplast thylakoid membrane and chloroplast thylakoidlumen or peroxisome, containing7phosphorylation sites. These predictions demonstrated that theCAB protein gene had a number of functional sites, playing a relevant role in the photosythesisand energy metabolism pathway of Tagetes erecta L.. 4The expression analysis of LFY and cab genes at the transcription level inTagetes erecta L. by fluorescence quantitative PCRUsing18S rRNA gene as reference gene, the expressions of the LFY and cab genes inTagetes erecta L. during floral bud differentiation were investigated by using fluorescencequantitative PCR technology, the results showed that:①LFY gene expressed in each periodduring the floral bud differential process. The expression level was low when floral bud did not beinduced out, while it increased significantly after the floral bud was differentiated successfully;with the further development of the floral bud, the expression level increased, but it decreased atthe stage of flower finally, and expression level of each period was very low, suggesting that LFYshould express in the apical meristem mostly, with tissue-specific, the gene have roles indetermination and maintenance of the floral meristem, but its expression be inhibited by othergenes in the floral bud induction phase, its role in promoting floral organ formation be acumulative effect.②Cab gene had different expression levels during the floral bud differentialprocess. When the seedings were transferred to the induction medium, the expression levelappeared a downward trend, and it was lowest at the floral bud1stage, with no expression nearly.But when the floral bud was further developed, the level was higer, and it was the highest inflower. These results suggested that low level of cab should promote the floral bud induction,whereas high level of cab is conducive to the development of flower. The different expressiontrends of LFY and cab genes suggested that they should play different roles in the floral buddifferentiation process, and flower mechanism is a complex problem from the side.In summary, this paper made studies on the establishment of regeneration system and in vitroflowering of Tagetes erecta L., and the roles of LFY and cab in the flowering. In vitro plantlets ofTagetes erecta L. were firstly used as the materials for the cloning of LFY and cab genes, and thequantitative expression analysises of LFY and cab genes in the process of floral bud differentiationby the real time fluorescence quantitative PCR technique. These studies made importantfoundations for the follow-up researches of in vitro flowering and related molecular mechanism inTagetes erecta L. and other Asteraceae plants.