In Vitro Conservation And Cloning And QPCR Expression Of Fc-MLP2Gene Of Plantlets In Fortunella Crassifolia Cv. Youxijingan And Other Citrus Germplasm Resources

In this experiment, in vitro plantlets of Fortunella crassifolia cv. Youxijingan and othercitrus germplasm resources were used as the materials for the studies of establishment ofregeneration system, conservation of germplasm resources, cloning and quantitative PCRexpression of Fc-MLP2and genetic transformation into tobacco and function identification ofFc-MLP2. The main results were as follows：1. Establishment of the in vitro regeneration system of Fortunella crassifolia cv.YouxijinganIn this experiment, the media for micropropagation, growth and rooting of tube plantlets wereoptimized in Fortunella crassifolia cv. Youxijingan. The results showed that KT had promotingeffect on proliferation, the best proliferation medium was G2treatment （MS+NAA0.05mg/L+KT1.5mg/L） under the action of KT and NAA, the order of the impact of the proliferation was BA>BA*NAA>NAA, the best proliferation medium was MS+6-BA1.2mg/L+NAA0.05mg/L, theproliferation coefficient reached97.8%; the best rooting medium was MS+NAA0.3mg/L+IBA0.2mg/L, the root induction rate reached83.33%and the mean number of rooting reached3.27.2. In vitro conservation of Fortunella crassifolia cv. Youxijingan plantlets andother citrus germplasm resourcesFortunella crassifolia cv. Youxijingan in vitro plantlets were used as the main materials. Theinfluences of different containers, different concentrations of PP₃₃₃, CaCl₂and mannitol on theconservation of Fortunella crassifolia cv. Youxijingan in vitro plantlets were compared. Theresults showed that: in the single-factor experiments, adding lower concentrations of CaCl₂on theMS medium not only increased the root-shoot ratio, but also was suitable for conservation ofFortunella crassifolia cv. Youxijingan in vitro plantlets; in multiple factor test, the bestproliferation medium was C4(MS+CaCl₂0.44g/L+mannitol0g/L+PP₃₃₃1mg/L), it was suitablefor in vitro conservation of Fortunella crassifolia cv. Youxijingan plantlets, and the seedlingsurvival rate reached100%without subculture for6months, the seedling survival rate reached97.8%without subculture for12months.In this experiment,25accessions of citrus germplasm resources were collected and thegrowth status of67accessions of citrus germplasm resources seedlings in vitro conserved in our lab were observed. The results showed that: the seedling growth of pomelo germplasm resourceswere robust, root developed, leaves was larger and thick green, but the excessive growth of theseedlings was serious. The wild citrus seedlings grew weaker generally, root and stem wereweaker, and we found that the rooting ability of this kind of seedlings was also very weak insubculture. The seedlings growth of kumquat and Citrus reticulata were between pomelo and thewild citrus seedlings.3. Cloning and Bioinformatics analysis of MLP2gene from the leaves in vitroplantlets in Fortunella crassifolia cv. YouxijinganThe Fc-MLP2cDNA sequence was cloned from the leaves using RT-PCR and RACEtechniques in Fortunella crassifolia cv. Youxijingan. The accession number was JX310276inGenBank. The cDNA sequence consisted of908bp with an intact open reading frame of669bp,encoding a polypeptide of223amino acids. Bioinformatics analysis showed that the protein was ahydrophobic secretory protein located in extracellular position, with two functional sites,13phosphorylation sites and a signal peptide. The secondary structure was made of the alpha helix,beta sheets, beta turns and coils. Phylogenetic analysis also indicated that the Fc-MLP2was closeto RlemMLP2of Citrus jambhiri in genetic relationship.4. Fc-MLP2gene expression analysis in the process of in vitro conservation ofFortunella crassifolia cv. Youxijingan by real time fluorescence quantitative PCRUsing18S rRNA gene as reference gene, the Fc-MLP2gene expression was analysed inmRNA transcription in the process of in vitro conservation of the plantlets of Fortunellacrassifolia cv. Youxijingan. The results showed that Fc-MLP2gene expressed at each stageduring in vitro conservation of Fortunella crassifolia cv. Youxijingan. On the4thmonth ofconservation, the Fc-MLP2gene expression was at the higher level; on the beginning and12thmonth of conservation， the expression levels were low； on the6thand8thmonth ofconservationhe，the level of Fc-MLP2gene expression rose sharply again; the expression level ofFc-MLP2gene in Fortunella crassifolia cv. Youxijingan was related to plantlet growth anddevelopment during in vitro conservation in Fortunella crassifolia cv. Youxijingan.5. Genetic transformation into tobacco and function identification of Fc-MLP2geneIn this experiment, the pCAMBIA1301-Fc-MLP2expression vector was constructed byXbaⅠand SalⅠ digestion technology, and transformed into Agrobacterium EHA105.Agrobacterium-mediated transformation of Nicotiana tabacum plants, and PCR and GUSdetection of resistant plants after hygromycin selection were performed. The results showed thatthe transformation frequency amounted to62.22%. The transgenic N. tabacum plants were treatedunder cold treatments at4℃, they were respectively sampled after0,3,6,12,24,48h for PCR detection. The results showed that after cold treatments, the expression levels of Fc-MLP2genedropped sharply; besides, after wound treatment for12h, the expression levels of Fc-MLP2geneincreased sharply. It was speculated that the Fc-MLP2gene played a role in withstandingenvironmental stress.