| 拟南芥WUS基因的克隆及植物表达载体的构建与鉴定 |
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| 引用本文： | 毕政鸿,李哲.拟南芥WUS基因的克隆及植物表达载体的构建与鉴定[J].中国农学通报,2013,29(18):119-126. |
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| 作者姓名： | 毕政鸿 李哲 |
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| 作者单位： | 1. 海南大学农学院，海南儋州 571737; 中国热带农业科学院橡胶研究所/农业部橡胶树生物学重点开放实验室，海南儋州 571737
2. 中国热带农业科学院橡胶研究所/农业部橡胶树生物学重点开放实验室,海南儋州,571737 |
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| 基金项目： | 科技部国际科技合作项目“橡胶树易碎愈伤组织长期培养植株再生和超低温保存研究”(2008DFA32020)；农业部“948”项目“橡胶树易碎胚性愈伤组织长期继代培养、过表达Lec1基因促进胚状体发生和植株再生”(2010-S7)；国家自然科学基金项目“橡胶树胚性细胞悬浮培养及其植株再生的研究”(30760128)。 |
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| 摘 要： | 克隆得到正确的拟南芥WUS基因，成功构建WUS基因组成型植物表达载体和WUS-EGFP融合基因诱导型植物表达载体，分别转化了根癌农杆菌EHA105，并用WUS基因组成型植物表达载体转化橡胶树愈伤组织，得到组织化学检测阳性的愈伤组织，为深入研究WUS基因生理生化功能做准备。用双酶切切下植物表达载体PBI121的35S-GUS片段，将其连接到pCAMBIA2301上成为中间植物表达载体pCAMBIA2301-35S-GUS。提取拟南芥总RNA，通过RT-PCR方法获得WUS基因，经过TA克隆连接到pEASY-T1载体上，再用双酶切切下WUS基因，将其取代pCAMBIA2301-35S-GUS中的GUS片段，形成pCAMBIA2301-35S-WUS植物表达载体。同时以质粒pCAMBIA2301-EGFP为模板，PCR获得EGFP，用重叠延伸PCR技术（gene splicing by overlap extension PCR，简称SOE PCR）获得WUS- EGFP融合基因，经双酶切将融合基因连接到诱导型植物表达载体pER8上，构成pER8-WUS-EGFP诱导型植物表达载体。通过电击转化法将这两个载体成功转入根癌农杆菌EHA105中，经过双酶切检测、PCR检测与测序分析，检测结果皆完全正确，克隆的WUS基因与WUS-EGFP融合基因序列和NCBI上公布的序列也完全一致，说明这两个植物表达载体已经构建成功。另外用pCAMBIA2301-35S-WUS植物表达载体遗传转化橡胶树易碎胚性愈伤组织，经GUS染色为蓝色，说明愈伤组织转化成功。
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| 关 键 词： | 上海 上海 |
| 收稿时间： | 2012/11/7 0:00:00 |
| 修稿时间： | 2012/12/24 0:00:00 |
Cloning of Arabidopsis WUS Gene, Construction and Identification of Plant Expression Vectors |
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| Bi Zhenghong
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Li Zhe.Cloning of Arabidopsis WUS Gene, Construction and Identification of Plant Expression Vectors[J].Chinese Agricultural Science Bulletin,2013,29(18):119-126. |
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| Authors: | Bi Zhenghong Li Zhe |
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| Abstract: | Arabidopsis WUS gene was correctly cloned, and the WUS gene constitutive plant expression vector and WUS-EGFP fusion gene inducible plant expression vector were successfully constructed. They were transformed into Agrobactrium tumefaciens EHA105. The calli of rubber tree were transformed by pCAMBIA2301-35S-WUS. The positive results of histochemical staining had been obtained. The process were the following. 35S-GUS fragment was cleavaged from PBI121 by double enzymes digestion and was cloned into pCAMBIA2301 to form pCAMBIA2301-35S-GUS middle plant expression vector. After extraction of the total RNA of Arabidopsis thaliana, WUS gene was cloned by RT-PCR method and linked into pEASY-T1 vector, then the vector was doubly digested to create WUS fragment, which thus was inserted into pCAMBIA2301-35S-GUS instead of GUS fragment and formed pCAMBIA2301-35S-WUS plant expression vector. On the other hand, EGFP gene was amplified from pCAMBIA2301-EGFP vector by PCR, then the fusion DNA fragment of WUS and EGFP which were amplified by gene splicing by overlap extension PCR was cloned into pER8 inducible plant expression vector. These two plant expression vectors were successfully transformed into Agrobactrium tumefaciens EHA105 by electroporation method. Double enzymes digestion, PCR and sequencing detection verified that the results were correct. The sequences of the cloned WUS gene and WUS-EGFP fusion gene were the same as NCBI′s, it showed that these two vectors had been correctly constructed. Afterwards, friable embryogenic calli of rubber tree were transformed by the vector pCAMBIA2301-35S-WUS, and GUS staining showed that transformed calli had been obtained. These studies had provided a good base for further researching on the function of WUS gene in somatic embryogenesis and organogenesis of calli in Hevea brasiliensis. |
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| Keywords: | AtWUS gene EGFP gene fusion gene SOE PCR Hevea brasiliensis |
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