Cloning Research And Breeding Prospects Of MbCFAT1 Gene In Melaleuca Bracteata

Melaleuca bracteata,s evergreen tree of Myrtaceae,Melaleuca L.Melaleuca bracteata has golden or yellowish green leaves,graceful tree shape and strong resistance.It is one of the popular new tree species at present.Melaleuca bracteata leaves have high extraction rate of essential oil,and its essential oil has antibacterial and anti-inflammatory properties,which is mainly composed of Methyl eugenol.Coniferyl alcohol acyltransferase（CFAT）,as a key enzyme in the synthesis of Methyl eugenol,can catalyze Coniferyl alcohol to form Coniferyl acetate,and then undergo a series of reactions such as reduction and methylation catalyzed by EGS and EOMT to form Methyl eugenol.At present,the function of coniferyl alcohol acyltransferase in the synthetic pathway of Methyl eugenol has not been reported.In this paper,on the basis of analyzing the content of Methyl eugenol in wild-type strawberries,using Melaleuca bracteata seedlings as experimental materials,a CFAT gene was screened from the transcriptome database of differential expression in the synthetic pathway of essential oil established in the laboratory and cloned.The function of MbCFAT1 was studied by bioinformatics analysis,correlation analysis,subcellular localization,prokaryotic expression and eukaryotic expression analysis.It laid a foundation for strawberry disease resistance breeding in the future,and provided a theoretical basis for further study on the molecular metabolic mechanism of natural products and the quality improvement of essential oil.Results are as follows:1.A CFAT gene,named MbCFAT1,was cloned from the leaves of Artemisia angustifolia by RT-PCR,which is 1428bp,encodes 475 amino acids,molecular weight is 51.30KDa,isoelectric site is 8.72.the results of amino acid sequence alignment and phylogenetic tree analysis show that the amino acid sequence encoded by MbCFAT1 has the highest similarity with Ph CFAT of Petunia.There are conserved domains HXXXD and DFGWG in its amino acid sequence,which belongs to BAHD acyltransferase family and is closely related to strawberry Fa AAT2.2.Spraying exogenous Me JA and using q RT-PCR technique,the results showed that MbCFAT1 gene expression increased after different concentrations of Me JA treatment,and the increase of MbCFAT1 gene expression was the most significant after Me JA treatment of0.01mmol·L^-1.GC-MS analysis showed that the content of Methyl eugenol increased under different concentrations of Me JA treatment,and the correlation analysis between MbCFAT1 gene expression and methyl eugenol content showed that there was a positive correlation between MbCFAT1 gene expression and Methyl eugenol content under different concentrations of Me JA treatment.It is speculated that MbCFAT1 can catalyze the formation of cypress acetate from pine and cypress alcohol,which is the key gene in the synthesis of Methyl eugenol.3.The pNC-Cam1304-sub N vector was used to construct a fusion protein pNC-Cam1304-MbCFAT1-GFP carrying GFP,which was transformed into Agrobacterium-mediated transient expression in tobacco.The subcellular localization results were observed by laser scanning confocal microscopy,which showed that MbCFAT1 was localized in the nucleus.with the cytoplasmic membrane.4.The pET-32a（+）vector was used to construct the prokaryotic expression vector of MbCFAT1,and the recombinant protein pET-MbCFAT1 was obtained.The target protein with a molecular weight of about 69KDa was successfully expressed in Escherichia coli BL21（DE3）by IPTG induction;PAGE detected the samples and found that MbCFAT1 protein was mainly expressed in the form of inclusion bodies.5.The overexpression vector pNC-Cam1304--35s was constructed by using NC clone vector pNC-Cam1304-MbCFAT1-35s and transformed into yellow fruit forest strawberry mediated by Agrobacterium tumefaciens.The relative expression of MbCFAT1 gene was detected by q RT-PCR,and the content of Methyl eugenol in strawberry fruit was detected by GC-MS.The results showed that the expression of MbCFAT1 in transgenic strawberry fruit was 39.03 times higher than that of the control group,and the content of methyl eugenol was 138%higher than that of the control group.