| 摘要: |
| 【目的】获得转人源透明质酸酶基因PH20(hPH20)红花，为hPH20药用蛋白的生产奠定基础。【方法】人工合成hPH20基因，并在两端引入NcoⅠ/Hind Ⅲ酶切位点。将合成的hPH20基因与pUC57载体连接，构建克隆载体pUC57-hPH20，对其进行双酶切鉴定。用NcoⅠ/HindⅢ酶切pUC57-hPH20获得hPH20基因，将其插入到植物油体高效表达载体pOBT上，构建重组质粒pOBT-hPH20，进行PCR和双酶切鉴定。采用冻融法将重组质粒pOBT-hPH20转入根瘤农杆菌EHA105中，通过农杆菌介导法转化红花，对转hPH20基因红花植株进行PCR检测。【结果】成功构建了hPH20基因的植物油体表达载体pOBT-hPH20，获得了3株PCR检测呈阳性的转hPH20基因红花植株。【结论】建立了完善的红花再生体系，获得了转hPH20基因的红花植株。 |
| 关键词: 红花 透明质酸酶hPH20 油体 |
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| 基金项目:“十二五”国家高新技术研究与发展计划项目(2011AA100606)；中小企业创新基金项目(13C26212201223)；长春市重大科技攻关项目(2014077)；吉林省教育厅项目(201458/2015190) |
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| Expression of human hyaluronidase gene in oil body of safflower |
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YANG Jing,LI Hong-rui,YI Shan-yong,et al
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| Abstract: |
| 【Objective】The transgenic safflower was obtained to express human hyaluronidase gene (hPH20) and lay foundation for development of hPH20 products.【Method】The hPH20 gene was synthesized and NcoⅠ/Hind Ⅲ enzyme sites were introduced at both ends.Then the hPH20 gene was inserted into plant expression vector pOBT by NcoⅠ/Hind Ⅲ.The recombinant plasmid was named pOBT-hPH20 and was transferred into Agrobacterium tumefaciens EHA105.The hPH20 gene was introduced into safflower via Agrobacterium-mediated method and positive transgenic plants were determined by PCR analysis.【Result】The recombinant plasmid pOBT-hPH20 was successfully constructed.PCR confirmed that hPH20 gene was integrated into the genome of safflower plant and three transgenic plants were obtained.【Conclusion】The safflower regeneration system was constructed successfully and hPH20 gene was successfully transformed into safflower plants. |
| Key words: safflower hyaluronidase hPH20 oil body |
