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文章采用代谢工程方法培育东莨菪碱高产转基因颠茄。根癌农杆菌遗传转化颠茄,过量表达东莨菪碱合成途径中限速酶基因h6h(Hnh6h),卡那霉素筛选获得转基因植株,快繁炼苗后,田间栽培至果期。基因组PCR鉴定转Hnh6h基因颠茄。SPSS软件分析转基因与非转基因颠茄株高、顶宽、主茎粗、叶长、叶宽、分支数和鲜重等性状的差异;HPLC测定其根、茎、叶、果中莨菪碱及东莨菪碱含量;采用qPCR技术测定各组织Hnh6h基因表达量。结果所获得的5个转基因颠茄株系(A8,A11,A12,C8,C19)都能同时检测到Kanr和Hnh6h基因,在非转基因颠茄中没有检测到这2个基因;与非转基因颠茄比较,转基因颠茄株系的株高、顶宽、主茎粗、叶长、叶宽、分支数和鲜重等都没有降低,且部分性状优于对照。转基因颠茄叶片中东莨菪碱含量最高,且高于莨菪碱;不同转基因颠茄株系叶片中东莨菪碱含量依次为C8>A12>C19>A11>A8,C8的东莨菪碱质量分数(2.17 mg.g-1DW)比非转基因颠茄(0.42 mg.g-1DW)提高4.2倍;在转基因颠茄根、茎、叶和果中都检测到Hnh6h基因表达,且在叶片中表达量最高,非转基因颠茄没有检测到Hnh6h基因表达。因此,过表达Hnh6h基因能够打破颠茄的东莨菪碱生物合成的限速反应,从而推动代谢流向东莨菪碱合成方向流动,提高了东莨菪碱合成能力,最终获得了东莨菪碱高含量的转基因颠茄。
The article uses metabolic engineering methods to cultivate scopolamine high yield transgenic belladonna. Agrobacterium tumefaciens was transformed into Belladonna, the hshhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh [ Genome PCR Identification of Hnh6h Belladonna. SPSS software was used to analyze the differences of plant height, top width, main stem diameter, leaf length, leaf width, branch number and fresh weight of transgenic and non-transgenic belladonna. The contents of scopolamine and scopolamine in root, stem, Content; using qPCR technology to determine the Hnh6h gene expression in each tissue. Results The five transgenic strains belonged to Helicoverpa armigera (A8, A11, A12, C8, C19) were able to detect both Kanr and Hnh6h genes and none of the two genes were detected in non-transgenic belladonna; The results showed that the plant height, top width, main stem diameter, leaf length, leaf width, branch number and fresh weight of transgenic belladonna lines were not decreased, and some traits were better than the control. The content of scopolamine in the leaves of transgenic belladonna leaves was the highest and higher than that of scopolamine. The contents of scopolamine in the leaves of different strains of belladonna belonged to C8> A12> C19> A11> A8, scopolamine of C8 (2.17 mg.g-1DW) Which was 4.2 times higher than that of non-transgenic belladonna (0.42 mg.g-1DW). Hnh6h gene expression was detected in the roots, stems, leaves and fruits of the transgenic plants, and the expression level was the highest in leaves. Non-transgenic belladonna was not detected To Hnh6h gene expression. Therefore, overexpression of Hnh6h gene can break the rate-limiting reaction of scopolamine biosynthesis in belladonna so as to promote the flow of metabolism toward scopolamine synthesis and improve the scopolamine synthesis ability. Finally, scopolamine high content of transgenic belladonna was obtained.