BCAT4、CYP79F1和ESM1过表达提高西兰花细胞萝卜硫素含量

萝卜硫素(sulforaphane,SFN)是目前防癌抗癌效果最好的植物天然产物之一。十字花科芸薹属植物西兰花是生产SFN的重要材料。然而普通西兰花中SFN产量仍无法满足巨大的市场需求。之前单一基因改造提高植物SFN含量的策略效果并不理想。该研究从西兰花中克隆了SFN合成相关的支链氨基酸转移酶4编码基因BCAT4、细胞色素P450氧化酶编码基因CYP79F1和环硫修饰酶编码基因ESM1。分别以单基因导入和三基因串联导入的方式,通过农杆菌法转化西兰花愈伤组织,对所获得的转化细胞系中SFN的含量进行比较,以期获得SFN含量更高的细胞系,为通过细胞系大量培养获取SFN奠定基础。实验结果表明,目的基因BCAT4、CYP79F1、ESM1和B-C-E(BCAT4NSCYP79F1N-SESM1)都成功整合于宿主细胞基因组。BCAT4和B-C-E基因过表达细胞系中SFN含量为139.7和171.4μg/g,分别是野生型细胞系SFN含量的1.79倍和2.19倍,差异极显著(**P<0.01);B-C-E基因过表达细胞系SFN含量是BCAT4基因过表达细胞系的1.23倍,差异显著(*P<0.05)。而CYP79F1和ESM1过表达细胞系未检测到高于对照的SFN含量。综上所述,单基因BCAT4的过表达对SFN含量的影响大于CYP79F1与ESM1。多基因串联共表达对SFN含量的影响效果好于单个基因的过表达。

Overexpression of BCAT4,CYP79F1 and ESM1 Promote Sulforaphane Yield in Broccoli Cells

SFN(sulforaphane)is one of the plant natural product with best anti-cancer effect at present.Broccoli,a Brassicaceae plant,is an important material for the production of SFN.However,the yield of SFN in broccoli is very low,and far from meeting market demand.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 yield of SFN in the transformed cell line was compared with wild type cells.The results showed that the target genes BCAT4,CYP79F1,ESM1 and BCAT4N-SCYP79F1N-SESM1(B-C-E)were successfully integrated with the genomic DNA of the host cell.The SFN content of BCAT4 and BC-E gene overexpressed cell lines was 139.7 and 171.4μg/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,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.