| 摘要: |
| 高粱（Sorghum bicolor）是世界上仅次于小麦、水稻、玉米和大麦的重要粮食作物之一，虽然高粱基因组已经完成了测序，但是针对高粱测序品种BTx623，遗传转化方法的缺乏限制了高粱遗传育种和功能基因组研究的发展。而原生质体瞬时表达技术，则因为其高效、快速的特性，在功能基因组研究中具有重要的作用。为了在高粱品种BTx623中建立原生质体瞬时表达体系，本研究以BTx623幼苗为材料，对原生质体分离过程中的渗透压、酶液成分、酶解时间进行研究。结果表明：BTx623幼苗的原生质体分离过程中，最佳酶解液组成为1%纤维素酶、0.25%离析酶、0.6 mol/L甘露醇、10 mmol/L 吗啉乙烷磺酸、1 mmol/L CaCl2、0.1%小牛血清蛋白和5 mmol/L β-巯基乙醇，并获得了每毫升1×107个的高质量原生质体，所获原生质体活性在90%以上。之后利用PEG介导的转化方法，将含有35S：：egfp的质粒导入到原生质体中，并通过荧光显微观察统计，遗传转化率达到（61.31±3.91）%。本研究通过优化高粱品种BTx623原生质体制备及瞬时转化的条件，成功建立了其原生质体瞬时表达体系，为进一步开展高粱品种BTx623功能基因组的研究奠定了基础。 |
| 关键词: 高粱 BTx623 原生质体分离 瞬时表达 |
| DOI：10.14188/j.ajsh.2021.01.006 |
| 分类号:Q943.2 |
| 基金项目:三峡区域珍稀植物遗传发育与种质创新重点实验室开放基金（2018KZW01） |
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| The establishment of protoplasts isolation and transient expression system of sorghum cultivar BTx623 |
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ZENG Gongjian, CHENG Yunwei, HAN Shaopeng, LÜ Yang, LU Yelei, ZHOU Chao, ZHANG Dechun, SHEN Xiangling
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Key Laboratory of Three Gorges Regional Plant Genetics & Germplasm Enhancement/Biotechnology Research Center， Three Gorges University， Yichang 443002， Hubei， China
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| Abstract: |
| Sorghum is one of the important crops next to wheat， rice， maize and barley in the world， although the sorghum genome has been sequenced， the lack of genetic transformation methods for the sorghum sequenced cultivar BTx623 has limited the development of sorghum genetic breeding and functional genome research. Because of the efficiency and rapidity， the protoplast transient expression technology plays an important role in the research of functional genome. In order to establish the protoplast transient expression system in BTx623， in this study BTx623 seedlings were used as materials to research the osmotic pressure， composition of enzyme solution and the time of digestion in the process of protoplast isolation. The results showed that in the process of protoplast isolation in BTx623 seedlings， the optimal composition of the enzyme solution is 1% cellulase， 0.25% mecerozyme， 0.6 mol/L mannitol， 10 mmol/L MES， 1 mmol/L CaCl2， 0.1% BSA and 5 mmol/L β-mercaptoethanol， then 1×107/mL protoplasts with high vitality were obtained and the protoplast activity was higher than 90%. After that， the plasmids containing 35S：：egfp was introduced into the protoplasts by PEG mediated transformation， from the observation under fluorescence microscope， the genetic transformation rate was （61.31±3.91）%. In this study， by optimizing the conditions of protoplast preparation and transient transformation in BTx623， the protoplast transient expression system was successfully established， which laid a foundation for further study on functional genomics in sorghum cultivar BTx623. |
| Key words: sorghum BTx623 protoplast isolation transient expression |
