Kajla Sachin, Mukhopadhyay Arundhati, Pradhan Akshay K
Department of Genetics, University of Delhi South Campus, New Delhi, India.
Centre for Genetic Manipulation of Crop Plants, University of Delhi South Campus, New Delhi, India.
PLoS One. 2017 Aug 7;12(8):e0182747. doi: 10.1371/journal.pone.0182747. eCollection 2017.
Sinapine is a major anti-nutritive compound that accumulates in the seeds of Brassica species. When ingested, sinapine imparts gritty flavuor in meat and milk of animals and fishy odor to eggs of brown egg layers, thereby compromising the potential use of the valuable protein rich seed meal. Sinapine content in Brassica juncea germplasm ranges from 6.7 to 15.1 mg/g of dry seed weight (DSW) which is significantly higher than the prescribed permissible level of 3.0 mg/g of DSW. Due to limited natural genetic variability, conventional plant breeding approach for reducing the sinapine content has largely been unsuccessful. Hence, transgenic approach for gene silencing was adopted by targeting two genes-SGT and SCT, encoding enzymes UDP- glucose: sinapate glucosyltransferase and sinapoylglucose: choline sinapoyltransferase, respectively, involved in the final two steps of sinapine biosynthetic pathway. These two genes were isolated from B. juncea and eight silencing constructs were developed using three different RNA silencing approaches viz. antisense RNA, RNAi and artificial microRNA. Transgenics in B. juncea were developed following Agrobacterium-mediated transformation. From a total of 1232 independent T0 transgenic events obtained using eight silencing constructs, 25 homozygous lines showing single gene inheritance were identified in the T2 generation. Reduction of seed sinapine content in these lines ranged from 15.8% to 67.2%; the line with maximum reduction had sinapine content of 3.79 mg/g of DSW. The study also revealed that RNAi method was more efficient than the other two methods used in this study.
芥子碱是一种主要的抗营养化合物，它在十字花科植物的种子中积累。摄入后，芥子碱会使动物的肉和奶产生粗糙的味道，使褐壳蛋鸡的蛋产生鱼腥味，从而影响了富含蛋白质的种子粕的潜在利用价值。芥菜种质中的芥子碱含量在6.7至15.1毫克/克干种子重量（DSW）之间，显著高于规定的允许水平3.0毫克/克DSW。由于自然遗传变异有限，传统的降低芥子碱含量的植物育种方法大多未成功。因此，采用转基因方法进行基因沉默，靶向两个基因——SGT和SCT，它们分别编码参与芥子碱生物合成途径最后两步的酶UDP - 葡萄糖：芥子酸葡萄糖基转移酶和芥子酰葡萄糖：胆碱芥子酰转移酶。从芥菜中分离出这两个基因，并使用三种不同的RNA沉默方法，即反义RNA、RNA干扰和人工微小RNA，构建了八个沉默载体。通过农杆菌介导的转化培育出了转基因芥菜。在使用八个沉默载体获得的总共1232个独立的T0转基因事件中，在T2代中鉴定出25个显示单基因遗传的纯合系。这些品系种子中芥子碱含量的降低幅度在15.8%至67.2%之间；降低幅度最大的品系芥子碱含量为3.79毫克/克DSW。该研究还表明，RNA干扰方法比本研究中使用的其他两种方法更有效。
