• ISSN 1008-505X
  • CN 11-3996/S
韩娇, 王莉, 何蕊, 李伟, 王冰, 黄升财, 孙永伟, 程宪国. CRISPR/Cas9系统构建的水稻OsPht基因突变体材料可用于养分转运评价[J]. 植物营养与肥料学报, 2017, 23(5): 1359-1369. DOI: 10.11674/zwyf.17177
引用本文: 韩娇, 王莉, 何蕊, 李伟, 王冰, 黄升财, 孙永伟, 程宪国. CRISPR/Cas9系统构建的水稻OsPht基因突变体材料可用于养分转运评价[J]. 植物营养与肥料学报, 2017, 23(5): 1359-1369. DOI: 10.11674/zwyf.17177
HAN Jiao, WANG Li, HE Rui, LI Wei, WANG Bing, HUANG Sheng-cai, SUN Yong-wei, CHENG Xian-guo. Rice mutant establishment of the mutated phosphate transporter OsPht gene by the CRISPR/Cas9 gene editing system for evaluating nutrient translocation[J]. Journal of Plant Nutrition and Fertilizers, 2017, 23(5): 1359-1369. DOI: 10.11674/zwyf.17177
Citation: HAN Jiao, WANG Li, HE Rui, LI Wei, WANG Bing, HUANG Sheng-cai, SUN Yong-wei, CHENG Xian-guo. Rice mutant establishment of the mutated phosphate transporter OsPht gene by the CRISPR/Cas9 gene editing system for evaluating nutrient translocation[J]. Journal of Plant Nutrition and Fertilizers, 2017, 23(5): 1359-1369. DOI: 10.11674/zwyf.17177

CRISPR/Cas9系统构建的水稻OsPht基因突变体材料可用于养分转运评价

Rice mutant establishment of the mutated phosphate transporter OsPht gene by the CRISPR/Cas9 gene editing system for evaluating nutrient translocation

  • 摘要:
    目的CRISPR/Cas9是细菌和古细菌为免受外来DNA片段侵袭形成的一种适应性免疫系统。CRISPR/Cas9已经被作为一种基因编辑的工具广泛应用到很多动物和植物的基因编辑。磷元素是植物生长过程中必需的营养元素,植物对磷的吸收主要由跨膜磷转运蛋白完成。本研究利用CRISPR/Cas9技术对Oryza Japonica Kitaake水稻中冰叶日中花磷转运蛋白McPht基因的同源体OsPht磷转运蛋白基因进行编辑,构建了McPht转运蛋白基因导入水稻OsPht缺失的突变体,初步验证了该突变体材料的功能。
    方法通过CRISPR/Cas9基因编辑手段,将水稻中与McPht蛋白同源性最高的磷转运蛋白基因进行编辑。首先将待编辑的基因片段连接到U3启动子驱动的pCXUN表达载体上,转化到农杆菌EHA105中,然后通过农杆菌转化到水稻幼胚,将获得的转化后再生T0代株系移栽到田间获得T1代种子,并将T1代株系进行盆栽培养,提取株系叶片基因组DNA用于PCR检测,所有株系PCR产物纯化后进行测序,依据序列变化判定目标基因的编辑位点,并对突变体进行生理指标测定。
    结果1) 基因编辑后的类型可分为两类,一类为目标编辑片段20个碱基中后6个碱基缺失,另一类为目标编辑片段20个碱基中后8个碱基缺失或突变。2) 编辑位点缺失6个碱基的株系占总突变株系的28.6%,编辑位点缺失8个碱基的株系占总突变株系的42.9%,GT碱基突变株系占总突变株系的28.6%。3) 水稻突变体的株高、鲜重、根系活力均小于野生型;根系扫描结果显示水稻突变体的根长、投影面积、表面积和侧根数均大于野生型;Cas-2的叶绿素和可溶性糖含量显著高于野生型,Cas-7的叶绿素和可溶性糖含量小于野生型,但差异不显著。
    结论CRISPR/Cas9基因编辑系统成功将水稻中McPht的同源体OsPht基因进行了编辑,所获得的水稻磷转运蛋白OsPht基因碱基缺失或突变不仅为所编辑基因的功能研究提供有效的科学依据,同时为冰叶日中花McPht磷转运蛋白基因导入OsPht基因功能缺失的水稻株系、验证其生理生化功能提供宝贵的评价材料支持。本研究表明基于CRISPR/Cas9介导的基因编辑系统在功能性评价植物养分转运蛋白基因方面是一条有效的途径。

     

    Abstract:
    ObjectivesThe CRISPR/Cas9 system, a generated adaptive immune system in bacteria and archaea bacteria by defending against the invasion from exogenous DNA fragments, is widely used nowadays as a gene editing tool in many animal and plant gene modification. Phosphorus is an essential nutrient element for plant growth, its absorption is mainly fulfilled through the transmembrane phosphate transporters in plants. In this study, the CRISPR/Cas9 gene editing technology was applied to modify rice phosphate transporter protein OsPht gene sharing the highest homology with the phosphate transporter protein McPht gene in Mesembryanthemum crystallinum, which would provide a reliable material for scientifically elucidating the function of McPht transporter protein.
    MethodsThe rice phosphate transporter protein OsPht sharing high homology with McPht protein was edited by CRISPR/Cas9 gene editing system. Briefly, based on the sgRNA mediation, the fragments generated by PCR using a pair of specific primers were inserted to the expression vector pCXUN with U3 promoter, and the resulting transformants were transformed into Agrobacterium EHA105 to infect the rice immature embryos of rice Oryza sativa L. japonica. cv. Kitaake by Agrobacterium transformation method. The T0 generating plant seedlings with anti-antibiotic were transplanted to the soils in field, and the T1 generation seeds were obtained and used for pot culture experiments, and the genomic DNA of rice seedling leaf was extracted for PCR detection, and all the PCR products were purified and sequenced, and the edited sites in the target gene fragments were determined according to the sequence changes, and the physiological measurements of mutant lines were performed.
    Results1) The mutants with the bases deletion or substitution in the OsPht gene were divided into two categories, which were represented by separately deleting 6 bases and 8 bases in the target editing fragment with 20 bases. 2) The mutants missing 6 bases accounted for 28.6% of the total mutant lines, and the mutants missing 8 bases accounted for 42.9% of the total mutant lines, and the mutants with mutation of GT bases accounted for 28.6% of the total mutant lines. 3) The plant height, fresh weight and root activity of mutant rice were lower than those of the wild type. The root length, shoot area, surface area and lateral root number were higher than those of the wild type. The contents of chlorophyll and soluble sugar of Cas-2 were higher than those of the wild type, and demonstrated remarkable differences, but the contents of chlorophyll and soluble sugar of Cas-7 were lower than those of the wild type, and no significant difference was observed.
    ConclusionsThe CRISPR/Cas9 gene editing system has successfully edited the rice homologous OsPht of the McPht, and these mutants with bases deletion or substitution in the OsPht gene not only establish a reliable materials basis for scientifically elucidating the function of the target gene, but also simultaneously provide valuable material supports, which the phosphate transporter McPht gene is introduced into the mutants with bases deletion or substitution in the OsPht gene, for systematically verifying physiological and biochemical functions of exogenous homologous from other plants. This study shows that the CRISPR/Cas9-mediated gene editing system is a scientific and effective pathway for the functional evaluation of nutrient transporter genes in plant.

     

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