利用基因编辑手段研究植酸酶合成基因
植酸酶能够破坏植酸对矿物元素强烈的亲和力，从而水解得到单胃动物可用的无机磷。因此，提高饲用作物籽粒中植酸酶的含量可以提高动物对饲料磷的利用率，减少碳酸氢钙添加，减轻动物高磷粪便造成的环境污染。大麦内源的HvPAPhy_a是植酸酶合成途径的关键基因，丹麦奥胡斯大学的研究人员利用基因编辑手段证实了其对植酸酶合成的关键作用并分析了调控该基因表达的关键元件。
利用CRISPR/Cas9和TALENs技术，研究人员敲除了大麦中的HvPAPhy_a基因，结果显示大麦成熟籽粒中植酸酶活性显著降低，这表明了HvPAPhy_a基因对植酸酶合成及活性的关键作用。HvPAPhy_a基因启动子区域的三个调控元件GCN4、Skn1、RY对基因的表达至关重要。此外，如果突变位点发生在GCN4/Skn1/RY模体下游，HvPAPhy_a基因的表达将进一步降低，这也表明还有另外的元件控制着HvPAPhy_a基因的表达。
该研究显示出基因编辑突变手段在研究基因功能和调控模式领域的强大作用。
Plant Mol Biol. 2017 Jul 28.
Evaluation of the mature grain phytase candidate HvPAPhy_a gene in barley (Hordeum vulgare L.) using CRISPR/Cas9 and TALENs
Author
Holme IB*, Wendt T, Gil-Humanes J, Deleuran LC, Starker CG, Voytas DF, rinch-Pedersen H.
*: Department of Molecular Biology and Genetics, Research Centre Flakkebjerg, Aarhus University, Denmark.
Abstract
In the present study, we utilized TALEN- and CRISPR/Cas9-induced mutations to analyze the promoter of the barley phytase gene HvPAPhy_a. The purpose of the study was dual, validation of the PAPhy_a enzyme as the main contributor of the mature grain phytase activity (MGPA), as well as validating the importance of a specific promoter region of the PAPhy_a gene which contains three overlapping cis-acting regulatory elements (GCN4, Skn1 and the RY-element) known to be involved in gene expression during grain filling. The results confirm that the barley PAPhy_a enzyme is the main contributor to the MGPA as grains of knock-out lines show very low MGPA. Additionally, the analysis of the HvPAPhy_a promoter region containing the GCN4/Skn1/RY motif highlights its importance for HvPAPhy_a expression as the MGPA in grains of plant lines with mutations within this motif is significantly reduced. Interestingly, lines with deletions located downstream of the motif show even lower MGPA levels, indicating that the GCN4/SKn1/RY motif is not the only element responsible for the level of PAPhy_a expression during grain maturation. Mutant grains with very low MPGA showed delayed germination as compared to grains of wild type barley. As grains with high levels of preformed phytases would provide more readily available phosphorous needed for a fast germination, this indicates that faster germination may be implicated in the positive selection of the ancient PAPhy gene duplication that lead to the creation of the PAPhy_a gene.
