Study On Overexpression Of BCAT4,CYP79F1 And ESM1 To Increase Sulforaphane Content In Broccoli Cells

Sulforaphane(SFN),also known as rapidin,is a sulfur-containing organic compound that is one of the natural plant products with the best anticancer effect.It can induce the body to produce type Ⅱ detoxifying enzymes,making cancer cells programmed apoptosis and play a role in preventing cancer.Cruciferous Brassica plant broccoli is an important material for the production of SFN,but at present,the production of SFN in ordinary broccoli still cannot meet the huge market demand.The use of seedlings as production materials will greatly increase the production cost Production of SFN by broccoli sterile cell lines can shorten the production cycle and is a new production method.Previous research strategies to improve SFN synthesis via single genetic modification in plants were not ideal.In this study,three important SFN synthesis related genes,branched-chain amino acid transferase 4 encoding gene BCAT4,cytochrome P450 oxidase encoding gene CYP79F1 and epithiospecifier modifier 1 encoding gene ESM1,were cloned from broccoli.To get more SFN using cell suspension culture,Single gene introduction and triple gene serial introduction into broccoli calli were conducted mediated by Agrobacterium tumefaciens and the SFN content in the obtained transformed cell lines was compared in order to obtain a cell line with a higher SFN content,which lays the foundation for obtaining SFN through mass culture of cell lines.The results of this study are as follows:1、Target gene cloning:Trizol method was used to extract total RNA from wild-type broccoli seven-day-old seedlings and prepare cDNA by reverse transcription.Design specific primers for PCR amplification based on the CDS sequences of genes BCAT4(GenBank No.XMO 13728260.1),CYP79F1(GenBank No.MG012890.1),and ESM1(GenBank No.XM013728577.1).The target gene obtained by PCR amplification is purified and recovered.2、Construction of single gene overexpression vector:The single gene was ligated to the vector XF-350 through a recombination reaction to obtain a single gene recombination vector:XF350-ESM1,XF350-BCAT4,XF350-CYP79F1.The results of double enzyme digestion and sequencing showed that the vector was successfully constructed.3、Construction of triple gene tandem overexpression vector:Using the successfully constructed recombinant plasmids XF350-BCAT4,XF350-CYP79F1,XF352-ESM1 as templates,PCR amplification expression boxes:BCAT4N(BCAT4+His tag+Nos 3 ’UTR),SCYP79F1N(CaMV 35S+HA tag+CYP79F1+His tag+Nos 3 ’UTR),SESM1(CaMV 35S+HA tag+ESMZ).Three gene expression boxes were tandemly connected to the vector by homologous recombination to obtain a three-gene tandem overexpression vector:XF350-BCAT4N-SCYP79F1N-SESM1.The results of double enzyme digestion and sequencing showed that the vector was successfully constructed.4、Agrobacterium transformation and molecular biology detection of transgenic cells:Resistant broccoli cells were obtained through Agrobacterium transformation and antibiotic screening.Detection of DNA molecule level in resistant cells,preliminary demonstration of successful integration of the target gene with the genomic DNA of the host cell.RT-PCR results showed that the target genes BCAT4,CYP79F1,ESM1,and BCAT4N-SCYP79F1N-SESM1(B-C-E)could be overexpressed in the host cells.5、Detection of SFN content in transgenic cells:Detection of SFN content in each cell line by high performance liquid chromatography.The SFN content of BCAT4 and B-C-E gene overexpressed cell lines was 139.7 and 171.4 μg/g,which were 1.79 and 2.19 times of SFN in wild-type cell,respectively.And the difference was extremely significant.The SFN content of B-C-E gene overexpressed cell lines was 1.23 times of BCAT4 gene overexpressed cell lines.And the difference was significant.However,the overexpressed cell lines of CYP79F1 and ESM1 did not detect a higher SFN content than the control.In summary,by genetically modifying broccoli cells,the overexpression of single gene BCAT4 has a greater effect on SFN content than CYP79F1 and ESM1.The effect of multi-gene tandem co-expression on SFN content is better than that of single gene.