Franks T K, Powell K S, Choimes S, Marsh E, Iocco P, Sinclair B J, Ford C M, van Heeswijck R
Cooperative Research Centre for Viticulture, SA, Australia.
Transgenic Res. 2006 Apr;15(2):181-95. doi: 10.1007/s11248-005-3737-7.
A multigenic trait (biosynthesis of the secondary metabolite, dhurrin cyanogenic glucoside) was engineered de novo in grapevine (Vitis vinifera L.). This follows a recent report of transfer of the same trait to Arabidopsis (Arabidopsis thaliana) using three genetic sequences from sorghum (Sorghum bicolor): two cytochrome P450-encoding cDNAs (CYP79A1 and CYP71E1) and a UDPG-glucosyltransferase-encoding cDNA (sbHMNGT). Here we describe the two-step process involving whole plant transformation followed by hairy root transformation, which was used to transfer the same three sorghum sequences to grapevine. Transgenic grapevine hairy root lines that accumulated transcript from none, one (sbHMNGT), two (CYP79A1 and CYP71E1) or all three transgenes were recovered and characterisation of these lines provided information about the requirements for dhurrin biosynthesis in grapevine. Only lines that accumulated transcripts from all three transgenes had significantly elevated cyanide potential (up to the equivalent of about 100 mg HCN kg(-1) fresh weight), and levels were highly variable. One dhurrin-positive line was tested and found to release cyanide upon maceration and can therefore be considered 'cyanogenic'. In in vitro dual co-culture of this cyanogenic hairy root line or an acyanogenic line with the specialist root-sucking, gall-forming, aphid-like insect, grapevine phylloxera (Daktulosphaira vitifoliae, Fitch), there was no evidence for protection of the cyanogenic plant tissue from infestation by the insect. Consistently high levels of dhurrin accumulation may be required for this to occur. The possibility that endogenous grapevine gene expression is modulated in response to engineered dhurrin biosynthesis was investigated using microarray analysis of 1225 grapevine ESTs, but differences in patterns of gene expression associated with dhurrin-positive and dhurrin-negative phenotypes were not identified.
多基因性状（次生代谢产物蜀黍氰苷的生物合成）在葡萄（欧亚种葡萄）中被从头设计。这是继最近一份关于利用高粱（双色高粱）的三个基因序列将同一性状转移到拟南芥（拟南芥）的报告之后：两个编码细胞色素P450的cDNA（CYP79A1和CYP71E1）以及一个编码UDPG - 葡萄糖基转移酶的cDNA（sbHMNGT）。在此，我们描述了一个两步过程，包括整株植物转化，随后是毛状根转化，该过程用于将相同的三个高粱序列转移到葡萄中。回收了积累来自无、一个（sbHMNGT）、两个（CYP79A1和CYP71E1）或所有三个转基因转录本的转基因葡萄毛状根系，对这些系的表征提供了关于葡萄中蜀黍氰苷生物合成需求的信息。只有积累了所有三个转基因转录本的系才有显著升高的氰化物潜力（高达约100毫克HCN千克(-1)鲜重），并且水平高度可变。对一个蜀黍氰苷阳性系进行了测试，发现其在浸渍时会释放氰化物，因此可被视为“产氰的”。在这种产氰毛状根系或非产氰系与专门吸食根、形成虫瘿的蚜虫样昆虫葡萄根瘤蚜（葡萄根瘤蚜，费奇）的体外双重共培养中，没有证据表明产氰植物组织受到昆虫侵害的保护。要发生这种情况可能需要持续高水平的蜀黍氰苷积累。利用对1225个葡萄ESTs的微阵列分析研究了内源性葡萄基因表达是否响应工程化的蜀黍氰苷生物合成而被调节，但未发现与蜀黍氰苷阳性和阴性表型相关的基因表达模式差异。
