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| 甘蔗尿苷二磷酸葡萄糖焦磷酸化酶基因(UGPase)转化拟南芥及转基因植株的生理特性分析 | |
| 引用本文： | 连玲,叶冰莹,陈如凯,谢华安,张建福,陈由强.甘蔗尿苷二磷酸葡萄糖焦磷酸化酶基因(UGPase)转化拟南芥及转基因植株的生理特性分析[J].农业生物技术学报,2012,20(5):481-488. |
| 作者姓名： | 连玲 叶冰莹 陈如凯 谢华安 张建福 陈由强 |
| 作者单位： | 1. 农业部华南杂交水稻种质创新与分子育种重点实验室;福州国家水稻改良分中心;福建省作物分子育种工程实验室;福建省水稻分子育种重点实验室;福建省农业科学院水稻研究所,福州350003;福建省作物种质创新与分子育种省部共建国家重点实验室培育基地,福州350003 2. 福建师范大学生命科学学院,福州350108;农业部甘蔗遗传改良重点实验室,福州350108 3. 农业部甘蔗遗传改良重点实验室,福州,350108 |
| 基金项目： | 国家自然科学基金项目，农业部现代农业产业技术体系建设专项资金 |
| 摘 要： | 尿苷二磷酸葡萄糖焦磷酸化酶(UDP-glucose pyrophosphorylase,UGPase)是植物糖代谢的主要参与酶之一,在植物的生长发育过程中起着重要作用。本研究将甘蔗(Saccharum officinarum)UGPase基因cDNA片段连接至载体pBI121,通过BamHⅠ和SacⅠ酶切鉴定及测序验证,结果表明,植物表达载体成功构建;通过农杆菌(Agrobacterium tumefaciens)的介导,采用浸花法转化拟南芥(Arabidopsis thaliana)。结合卡那霉素抗性筛选和PCR检测,获得了5株T0代转基因植株。对T1代转基因植株进行PCR及Southern blot分析,结果表明,目的基因已成功转入拟南芥中,并且不同的转化植株含有目的基因的拷贝数不同。对T2代转基因植株进行PCR和RT-PCR检测,结果表明,目的基因不仅能在自交系后代中稳定遗传,而且在RNA水平也有表达。同时,对T2代转基因植株的可溶性总糖、蔗糖及淀粉含量进行测定,结果表明,与野生型相比,转基因植株中可溶性总糖含量没有明显的变化,但蔗糖含量有所提高,并且差异明显,比野生型植株提高了50.85%~96.99%,而淀粉含量都较野生型植株的低,降低了9.69%~36.76%。说明UGPase在蔗糖与淀粉的转换过程中起着较为重要的作用,其催化的反应方向影响着组织中这两种产物(蔗糖和淀粉)的分配。 |
| 关 键 词： | 尿苷二磷酸葡萄糖焦磷酸化酶(UGPase) 载体构建 转化 糖含量测定 |
Transformation of UDP-glucose Pyrophosphorylase Gene (UGPase) from Saccharum officinarum into Arabidopsis thaliana and Analysis of Physiological Characteristics of Transgenic Plants |
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| LIAN Ling , YE Bing-Ying , CHEN Ru-Kai , XIE Hua-An , ZHANG Jian-Fu , CHEN You-Qiang.Transformation of UDP-glucose Pyrophosphorylase Gene (UGPase) from Saccharum officinarum into Arabidopsis thaliana and Analysis of Physiological Characteristics of Transgenic Plants[J].Journal of Agricultural Biotechnology,2012,20(5):481-488. | |
| Authors: | LIAN Ling YE Bing-Ying CHEN Ru-Kai XIE Hua-An ZHANG Jian-Fu CHEN You-Qiang |
| Institution: | 3,4* 1 Key Laboratory of Hybrid Rice Germplasm Innovation and Molecular Breeding of South China,Ministry of Agriculture/Fuzhou Branch,National Rice Improvement Center of China/Fujian Engineering Laboratory of Crop Molecular Breeding/Fujian Key Laboratory of Rice Molecular Breeding/Rice Research Institute,Fujian Academy of Agricultural Sciences,Fuzhou 350003,China;2 Incubator of National Key Laboratory of Fujian Germplasm Innovation and Molecular Breeding between Fujian and Ministry of Sciences & Technology,Fuzhou 350003,China;3 College of Life Sciences,Fujian Normal University,Fuzhou 350108,China;4 Key Laboratory of Sugarcane Genetics and Breeding,Ministry of Agriculture,Fuzhou 350108,China |
| Abstract: | UGPase(UDP-glucose pyrophosphorylase) is one of the enzymes which mainly involves in plant carbohydrate metabolism,and plays an important role in the process of plant growth and development.The plant expression vector was constructed by ligating the cDNA fragment of UGPase from Saccharum Officinarum into expression vector pBI121,then the recombinant plasmid pBI-UGP was confirmed by restriction enzyme digestion analysis with BamH Ⅰ /Sac Ⅰ,and further verified by DNA sequencing.The expression vector was introduced into Arabidopsis thaliana by floral-dip method,with Agrobacterium tumefaciens as media.Five T0 transgenic plants were selected by combining kanamycin screening with PCR detection.After selecting by kanamycin screening,T1 transgenic plants were identified by PCR and Southern blot,the results showed that the target gene was transformed into Arabidopsis thaliana successfully,and different transgenic plants contained various copy numbers of UGPase from Saccharum officinarum.T2 transgenic plants were identified by PCR and RT-PCR,the results indicated that the gene not only inherited stably in self-progeny,but also expressed at the RNA level.Meanwhile,content of soluble sugar,sucrose and starch were determinated in T2 transgenic plants and wild plants.Compared to wild plants,the soluble sugar content of transgenic plants had little change,however,sucrose content of transgenic plants improved obviously,increased by 50.85%～96.99%.Starch content of transgenic plants was lower than that of wild plants,decreased by 9.69%～36.76%.The research proved that UGPase plays a critical role in the process of converting sugar and starch,and the direction of its catalysis influneces distribution of these products in plant organization.It will lay foundation for further study of UGPase. |
| Keywords: | UGPase Vector construction Transformation Sugar content |
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