| Chrysanthemum is one of perennial herb which belongs to Compositae,has wide varieties and morphological diversity.It is one of the top-ten traditional famous flowers in China,and the top of the four most important cut flowers in the world[1],which possesses good ornamental,edible and medicinal value.In recent years,technology of molecular biology has been rapidly developed in flower breeding,and the resistance-related genes and their biological functions have been a hot-spot of research currently.In this study,we designed degenerate primers according to the conserved sequences of chitinase gene released by Gen Bank,and isolated a homologue of chitinase gene designated ChCHI from the leaves of Chrysanthemum,using RT-PCR and RACE technology,then analyzed the new gene by bioinformatics method.The expression of ChCHI gene in Chrysanthemum leaves under different concentration of SA treatment was determined by qRT-PCR method.The plant expression vector of Chrysanthemum gene was constructed and genetic transformation was studied preliminarily.1.Cloning and sequence analysis of the full cDNA sequence of ChCHI geneA full-length of 1 385 bp cDNA of ChCHI was obtained by RT-PCR and RACE amplification from the total RNA extracted from the leaves of chrysanthemum.The gene consisted of 960 bp ORF which encoded a protein of 319 amino acids,was designated as ChCHI?accession number: KX881373?.The ChCHI belongs to hydrophilic protein which molecular weight is about 35.5 kD,and the isoelectric point is about 6.38 that indicating it is a stable protein.The prediction of the secondary structure showed that the protein is mainly composed of ?-helix and random coil.Protein sequence alignment and phylogenetic tree analysis showed that the protein encoded by ChCHI belongs to the family of hydrolase family 19 chitinase,and it has a close relationship with Mikania micrantha?ACO25187.1?,that the sequence consistency was 87%.The 3D structure of the protein expressed by the ChCHI gene was predicted.2.Construction of the plant expression vector of pCAMBIA1300-ChCHIThe cloned ChCHI gene was connected with pGEM-T Easy Vector to construct the cloning vector pGEM-ChCHI.Double digest the plasmid pGEM-ChCHI andplasmid pCAMBIA1300 by restriction enzymes Xba?and Sma?,then connect the product of digestion using T4 DNA ligase.The plant expression vector p CAMBIA1300-ChCHI of chrysanthemum has been constructed successfully by colony screening,PCR detection and double enzyme digestion,which will be helpful for genetic transformation of the next experiments.3.Expression Analysis of ChCHI Gene from ChrysanthemumqRT-PCR was used to analyze the expression of ChCHI gene under different concentrations of SA.The results showed that after the induction with 0.5 mmol·L-1 and 0.1mmol· L-1 of SA,low concentration of SA(0.1mmol·L-1)can significantly promoted the expression of Chrysanthemum chitinase gene in a certain period of time?8h?.The overall trend is that the expression decreased on the 4 h and the highest expression level was achived at the 8 h,then with the treatment time prolonged,the expression decreased gradually to lower than that before induction.4.Genetic transformation to Chrysanthemum leavesBased on the results of previous studies,the conditions for the transformation of Chrysanthemum by Agrobacterium mediated method were explored,and a genetic transformation system was established. |
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