| The crape myrtle(Lagerstroemia indica L.)is a woody plant of the Lythraceae family and the genus Lagerstroemia,with beautiful tree posture,gorgeous flowers,long flowering period,and anti-pollution characteristics.It is not only suitable for planting in gardens,parks,and roads,but also for indoor bonsai and cut flowers viewing.It is an important summer ornamental tree species in our country and is popular among people.Improving its ornamental value is the primary concern in crape myrtle breeding,especially the research on flowering crape myrtle,which can provide theoretical basis for crape myrtle breeding to a certain extent,and promote the innovation and application of crape myrtle breeding research.Therefore,this thesis starts with the cloning,expression pattern analysis,prokaryotic expression,subcellular localization and transgenic Arabidopsis of its floral organ development genes,to isolate and preliminary functional verification of its flora organ identity genes.To reveal that the molecular mechanism of crape myrtle flowering provides a certain scientific basis.The main research contents of this paper are as follows:(1)On the basis of the Crape myrtle transcriptome data,RT-PCR was employed to cloned four MADS-box genes from the flower buds of Crape myrtle,named LiFUL1,LiAP3,LiAG,LiAGL11.Multiple sequence alignment and phylogenetic analysis showed that the obtained sequences all had a highly conserved MIKC domain.LiFULl belonged to the euFUL evolutionary branch of AP1/FUL subfamily,and its C-terminal had a euFUL domain.LiAP3 is a class B gene with a conserved euAP3 motif at the C terminal.LiAG belongs to the class C gene,the ends of C with conservative AG motif I and AG motif II;LiAGL 11 belong to class D,respectively,and the homologous amino acid sequences of other species are relatively conserved.Cluster analysis revealed that the four genes isolated from Crape myrtle were all clustered with Punica granatum into a reliable branch,indicating that these 4 MADS-box gene which regulate the development of Crape myrtle flower organs are closely related to Punica granatum.(2)The expression patterns of the four genes were analyzed by RT-qPCR,and the results explained that LiFULl was expressed not only in flower organs,but also in stems and leaves.The other four genes were all tissue-specific and only expressed in flower bud and flower organs.The expression levels of the four genes in different varieties of crape myrtle 'Hongye' and 'Xiangyun' were different.(3)LiFUL1,LiAP3 and LiAG gene and pCAMBIA130'0 vector that carrying green fluorescent protein(GFP)were selected to construct recombinant plasmid,and the tobacco was transformed by Agrobacterium-mediated method.The results demonstrated that the fluorescence of target genes were located in the nucleus.(4)The LiFUL1-pCold-TF recombinant protein was successfully expressed in E.coli BL21(DE3).There was a large amount of protein enrichment in the liquid after ultrasonic treatment,indicating that the induced expression of the target protein was soluble protein.It exists in the form,and the use of different concentrations of IPTG inducers has a certain effect on the expression of the protein,and the obtained protein bands are consistent with the predicted protein molecular weight.According to the principle that imidazole competitively binds to His tag protein,the target protein was purified.This lays the foundation for studying the function of genes at the protein level.(5)The recombinant plasmid of LiFUL1 and LiAP3 genes and plant expression vector PBI121 was constructed and transformed into Aturobacterium,and Arabidopsis thaliana was transformed by inflorescence dispersal method.The T0 generation seeds were collected,and the green color normal growth seedlings were screened on the resistance screening medium,while the non-resistant seedlings were greenened and turned white.This experiment provides basic data for in-depth study of molecular mechanisms related to the development of crape myrtle flower organs. |
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