Cloning And Functional Analysis Of Fm4CL Gene In Fraxinus Mandshurica

In this study,Fraxinus mandshurica was used as materials.15 Fraxinus mandshurica 4CL genes were cloned and named Fm4CL1-Fm4CL15,which has been uploaded to the NCBI.Bioinformatics and expression pattern of 15 genes of Fraxinus mandshurica were analyzed The plant expression vector pBI121-Fm4CL2 and pB1121-Fm4CL4 were constructed and successfully transformed to tobacco plants.Lignin,cellulose and hemicellulose content were determined,respectively.And the content of POD and PAL were also measured in transgenic tobacco.The difference size and shape of cell morphology on the xylem in transgenic tobacco was compared with non-transgenic tobacco by microscopic observation.The study initially revealed of the expression pattern and functional analysis of Fm4CL in xylem development.The main results were as follows:1?Bioinformatics analyse of the Fm4CL in Fraxinus mandshurica15 Fm4CL full-length genes were performed bioinformatics analysis.Online software was uesd for analyzes the physical and chemical properties,the same as the protein structure.The results showed that Fm4CL2,Fm4CL3,Fm4CL6,Fm4CL 10,Fm4CL 14 were unstable protein,others were stable protein.Fm4CLl,Fm4CL3,Fm4CL4,Fm4CL7,Fm4CL8,Fm4CL10 were hydrophilic protein,the others were hydrophobic protein.All the Fm4CL proteins had the transmembrane domain,which showed that Fm4CL proteins had the ability of transmembrane.All the Fm4CL proteins were made of a helix,extended-chain,random coil,and distributed throughout the protein.And phylogenetic trees were also constructed.2?Expression pattern analysis of Fm4CL in Fraxinus mandshuricaFm4CL gene expression levels in different tissues(leaf,xylem,skin,flowers,buds,seeds)were analyzed.Fm4CL gene expression levels at different times(May,June,July,August,September)were also researched.The results showed that Fm4CL gene expression spatiotemporal specificity.Tension wood of Ash was sampled by the first day,the third days,and the seventh day treatment.The materials were cooected from tension wood and the opposite wood at the same time.The results showed that the expression level of Fm4CL gene responsed to the tension treatment.The study preliminarily revealed Fm4CL may be involved in synthesis of cell wall and lignin.3?The use of In-Fusion technology for the construction of expression vectors and the factors that affect the efficiency of the transformation in tobaccoThe In-Fusion cloning techniques were used in pBI121-Fm4CL2 and pBI121-Fm4CL4,The optimum differentiation medium in tobacco transformation was MS+0.05mg/L NAA+0.5mg/L 6-BA,infection bacterial concentration of OD600=0.3.The optimum infection time was 4 min,while the optimum time for the co-culture was3 days.After co-culture,the initial de-bacterial concentration and screening concentrations were:cefotaxime 500mg/L,kanamycin 25mg/L.After one week of culture bacteria off the kanamycin concentration increased to 50mg/L.When the shoot was 3cm,it was transferred into screening rooting medium to obtain transgenic tobacco.4?Functional analysis of Fm4CL2 and Fm4CL4Transgenic tobacco were detected by GUS and molecular identification.It proved to be successful to obtain transgenic tobacco plants.lignin content was higher in transgenic tobacco than non-transgenic control,while cellulose and hemicellulose content reduced in control.It illustrated the synthesis of cellulose and lignin in plants can compensate for each other.Meanwhile,the content of PAL decreased,which is located at the entrance way of benzenepropanoic acid.The content of POD(synthesis of a key enzyme in lignin)increased.Microscopic observation tobacco paraffin sections can clearly show transgenic tobacco xylem cells was smaller and closer than non-transgenic tobacco.Moreover,the single cell area in Fm4CL2 and Fm4CL4 were 26.5%and 23.5%lower than control.