摘要
提高种子含油量是油料作物育种的重要目标。为了对含油量相关基因的功能进行深入分析,本研究克隆或合成了催化整个三酰甘油合成途径的含油量相关的5个基因。于同一遗传转化载体平台,采用农杆菌介导法,在模式植物烟草中完成了5个目标基因的功能比较研究。
利用同源序列法从油菜中克隆含油量相关基因BnGPDH、BnGPAT、BnDGAT的全长cDNA序列,对它们的序列特征进行了生物信息学分析;人工合成来自酵母的2个含油量相关基因ScGPDH、ScLPAT,并对它们的序列特征进行了生物信息学分析。构建由种子特异启动子Napin调控的5个目标基因转化油菜和烟草的载体,并将5个基因转化野生型烟草植株,均成功获得大量阳性植株。气相色谱分析结果表明,野生型烟草种子含油量为12.68%,种子脂肪酸主要由棕榈酸(C16:0)、硬脂酸(C18:0)、油酸(C18:1)、亚油酸(C18:2)、亚麻酸(C18:3)组成,不含超长链脂肪酸芥酸等。作为油菜含油量转基因理论研究的材料,具有本底低、背景干净的优势。可考虑以烟草为模式植物进行油料作物超长链脂肪酸的超量表达和异位表达,以获取工业用优良脂肪酸的研究工作。
转基因烟草T0代植株种子含油量的分析结果表明:1)单独转化BnDGAT和ScLPAT基因可使野生型烟草种子含油量显著提高;2)单独转化BnGPAT、ScGPDH以及BnGPDH基因可使野生型烟草种子含油量极显著提高。
转基因烟草T1代植株种子含油量的分析结果表明: 1)BnGPAT基因转化烟草T1代种子含油量分布在14.26%-18.03%间,平均值为15.91%; 2)BnDGAT T1代植株种子含油量分布于13.93%-18.88% ,平均值为16.05%; 3)ScGPDH T1代植株种子含油量分布于14.18%-19.58%,平均值为16.50%; 4)ScLPAT T1代植株种子含油量分布于13.80%-20.64%,平均值为16.93%; 5)除一株种子含油量为11.73%,相对降低含油量7.51%外,BnGPDH基因转化烟草植株T1代种子的含油量为13.50%-19.85%,平均值为16.13%; 6)5个基因转化提高种子含油量的平均值依次为25.47%、26.58%、30.13%、33.52%和27.21%。合成的2个基因ScLPAT和ScGPDH提高含油量的程度稍稍领先,之后依次是BnGPDH、BnDGAT和BnGPAT。
以上结果表明,来自油菜的3个基因和来自酵母的2个基因,都能够在烟草种子中有效表达且稳定遗传。5个基因绝大多数后代均在一定程度上提高了种子含油量,但各基因的表现存在差异,不同转化事件的表现也存在显著差异。本研究为后期鉴定纯合体完成杂交试验,研究相关基因杂交组合以期进一步提高烟草种子含油量的研究提供了依据,也为油菜高含油量的基因调控提供了理论与实践支持。
Oil content is the key object of oilseed breeding. It is important to investigate the genes related to oil content. In this study, 5 genes involved in oil content during triacylglycerol biosynthesis were cloned or synthesized in order to elucidate through transgenic over-expression how different the 5 genes are on the oil content.
To investigate the function of the genes involed in oil content, the full cDNA sequences of BnGPDH、BnGPAT and BnDGAT genes were cloned from rapeseed, as well as ScGPDH、ScLPAT were synthesized from yeast. These 5 sequences were aligned and analyzed by using bioinformatic tools and then incorporated into rapeseed and tobacco transgenic vectors regulated by Napin promoter. A lot of positive tobacco transformants of these genes were obtained through agrobacterium-mediated transformation. The following results were drawn through the analysis of oil content in seed by gas chromatography.
The results of wild type tobacco showed that the oil content of WT is 12.68% with the fatty acids C16:0, C18:0, C18:1, C18:2, C18:3 and free of long chain fatty acid. The T0 transformants of tabacco showed significantly higher oil content in seed than WT tobacco when transformed separately with BnDGAT and ScLPAT gene and showed highly significant higher oil content in seed when transformed separately with BnGPAT, ScGPDH and BnGPDH gene.
In T1 generation, all the transformants from the four genes BnGPAT, BnDGAT, ScGPDH and ScLPAT showed significantly higher oil content with the rang 14.26%-18.03%, 13.93%-18.88%, 14.18%-19.58% and 13.80%-20.64%, and the mean 15.91%, 16.05%, 16.50% and 16.93%, respectively, corresponding to an oil content increase by 25.47%,26.58%,30.13% and 33.52%. Transformants from BnGPDH showed larger variation in oil content ranging from 11.73% to 19.85%, among which only one transformant showed a 7.51% drop in oil content.
In conclusion, all the 5 genes derived from rapeseed and yeast can increase seed oil content significantly and they ranked from ScLPAT, ScGPDH, BnGPDH, BnDGAT to BnGPAT according to the magnitude of the increase in oil content. This study provides a basis for the further experiment by stacking these genes into a single rapeseed cultivar.
引文
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