Allen Robert S, Miller James A C, Chitty Julie A, Fist Anthony J, Gerlach Wayne L, Larkin Philip J
CSIRO Plant Industry, PO Box 1600, Canberra, ACT 2601, Australia.
Plant Biotechnol J. 2008 Jan;6(1):22-30. doi: 10.1111/j.1467-7652.2007.00293.x. Epub 2007 Sep 13.
We demonstrate that both over-expression and suppression of the gene encoding the morphinan pathway enzyme salutaridinol 7-O-acetyltransferase (SalAT) in opium poppy affects the alkaloid products that accumulate. Over-expression of the gene in most of the transgenic events resulted in an increase in capsule morphine, codeine and thebaine on a dry-weight basis. The transgenic line with the highest alkaloid content had 41%, 37% and 42% greater total alkaloids than the control in three independent trials over 3 years. DNA-encoded hairpin RNA-mediated suppression of SalAT resulted in the novel accumulation of the alkaloid salutaridine at up to 23% of total alkaloid; this alkaloid is not detectable in the parental genotype. Salutaridine is not the substrate of SalAT but the substrate of the previous enzyme in the pathway, salutaridine reductase. RNA transcript analysis of 16 primary T0 transformants and their segregating T1 progeny revealed an average reduction in SalAT transcript to about 12% of the control. Reduction in SalAT transcript was evident in both leaves and latex. Reverse transcriptase PCR and high-performance liquid chromatography analyses confirmed cosegregation of the expressed transgene with the salutaridine accumulating phenotype.
我们证明，在罂粟中，编码吗啡喃途径酶salsutaridinol 7 - O - 乙酰基转移酶（SalAT）的基因的过表达和抑制都会影响积累的生物碱产物。在大多数转基因事件中该基因的过表达导致胶囊中吗啡、可待因和蒂巴因的干重增加。在3年的三项独立试验中，生物碱含量最高的转基因系的总生物碱含量比对照分别高出41%、37%和42%。DNA编码的发夹RNA介导的SalAT抑制导致生物碱salsutaridine的新积累，其含量高达总生物碱的23%；在亲本基因型中无法检测到这种生物碱。Salsutaridine不是SalAT的底物，而是该途径中前一种酶salsutaridine还原酶的底物。对16个初级T0转化体及其分离的T1后代进行RNA转录本分析，结果显示SalAT转录本平均减少至对照的约12%。SalAT转录本的减少在叶片和乳汁中均很明显。逆转录酶PCR和高效液相色谱分析证实了表达的转基因与salsutaridine积累表型的共分离。
