Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice
Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice
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农杆菌介导的遗传转化、转基因生产及其在水稻雄性生殖发育研究中的应用
DOI：
10.3791/61665
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Dabing Zhang
中科院分区：
文献类型：
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作者：
Dawei Xu;Palash Ch;ra Mondol;Muhammad Uzair;Matthew R. Tucker;Dabing Zhang
雄性不育是杂交种子生产的重要农艺性状，通常以雄性生殖器官/配子的功能缺陷为特征。 CRISPR-Cas9基因组编辑技术的最新进展可以实现高编辑效率并节省时间地敲除特定位点的内源候选基因的突变。此外，农杆菌介导的水稻遗传转化也是基因修饰的关键方法，已被许多公共和私人实验室广泛采用。在本研究中，我们应用CRISPR-Cas9基因组编辑工具，通过对粳稻品种OsABCG15进行靶向基因组编辑，成功生成了三个雄性不育突变株系。我们使用了改良的农杆菌介导的水稻转化方法，该方法可以为水稻杂交种子生产提供优良的遗传去雄手段。转基因植物可以在2-3个月内获得，并通过PCR扩增和桑格测序进行基因分型来筛选纯合转化体。雄性不育纯合系的基本表型特征通过对水稻雄性生殖器官的显微观察、通过碘化钾(I2-KI)染色的发育花药的半薄横截面进行花粉活力分析来进行。
Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive organs/gametes. Recent advances in CRISPR-Cas9 genome editing technology allow for high editing efficacy and timesaving knockout mutations of endogenous candidate genes at specific sites. Additionally, Agrobacterium-mediated genetic transformation of rice is also a key method for gene modification, which has been widely adopted by many public and private laboratories. In this study, we applied CRISPR-Cas9 genome editing tools and successfully generated three male sterile mutant lines by targeted genome editing of OsABCG15 in a japonica cultivar. We used a modified Agrobacterium-mediated rice transformation method that could provide excellent means of genetic emasculation for hybrid seed production in rice. Transgenic plants can be obtained within 2–3 months and homozygous transformants were screened by genotyping using PCR amplification and Sanger sequencing. Basic phenotypic characterization of the male sterile homozygous line was performed by microscopic observation of the rice male.reproductive organs, pollen viability analysis by iodine potassium iodide (I2-KI) staining semi-thin cross-sectioning of developing anthers.