Lei Jie, Miao Yingchun, Lan Yu, Han Xiuxiu, Liu Hongbo, Gan Yi, Niu Leilei, Wang Yanyan, Zheng Zhifu
School of Agriculture and Food Science, Zhejiang A & F University, Hangzhou, China.
School of Forestry and Biotechnology, Zhejiang A & F University, Hangzhou, China.
Front Plant Sci. 2018 Mar 19;9:353. doi: 10.3389/fpls.2018.00353. eCollection 2018.
The initial step in glycerolipid biosynthesis, especially in diverse allopolyploid crop species, is poorly understood, mainly due to the lack of an effective and convenient method for functional characterization of genes encoding glycerol-3-phosphate acyltransferases (GPATs) catalyzing this reaction. Here we present a novel complementation assay for quick and specific characterization of GPAT-encoding genes. Its key design involves rational construction of yeast conditional lethal ΔΔ double mutant bearing the heterologous gene whose leaky expression under repressed conditions does not support any non-specific growth, thereby circumventing the false positive problem encountered with the system based on the ΔΔ mutant harboring the native episomal gene whose leaky expression appears to be sufficient for generating enough GPAT activities for the non-specific restoration of the mutant growth. A complementation assay developed based on this novel mutant enables quick phenotypic screen of GPAT sequences. A high degree of specificity of our assay was exemplified by its ability to differentiate effectively GPAT-encoding genes from those of other fatty acyltransferases and lipid-related sequences. Using this assay, we show that AtGPAT1, AtGPAT5, and AtGPAT7 can complement the phosphatidate biosynthetic defect in the double mutants. Collectively, our assay provides a powerful tool for rapid screening, validation and optimization of GPAT sequences, aiding future engineering of the initial step of the triacylglycerol biosynthesis in oilseeds.
甘油脂生物合成的起始步骤，尤其是在多种异源多倍体作物物种中，目前仍知之甚少，这主要是由于缺乏一种有效且便捷的方法来对催化该反应的3-磷酸甘油酰基转移酶（GPAT）编码基因进行功能表征。在此，我们提出了一种用于快速、特异性地表征GPAT编码基因的新型互补分析方法。其关键设计包括合理构建携带异源基因的酵母条件致死ΔΔ双突变体，该异源基因在抑制条件下的渗漏表达不支持任何非特异性生长，从而规避了基于携带天然附加型基因的ΔΔ突变体系统所遇到的假阳性问题，该天然附加型基因的渗漏表达似乎足以产生足够的GPAT活性以实现突变体生长的非特异性恢复。基于这种新型突变体开发的互补分析方法能够对GPAT序列进行快速的表型筛选。我们的分析方法具有高度特异性，例如它能够有效地将GPAT编码基因与其他脂肪酰基转移酶及脂质相关序列的基因区分开来。利用该分析方法，我们发现AtGPAT1、AtGPAT5和AtGPAT7能够弥补双突变体中磷脂酸生物合成缺陷。总体而言，我们的分析方法为快速筛选、验证和优化GPAT序列提供了一个强大的工具，有助于未来对油料种子中三酰甘油生物合成起始步骤进行工程改造。
