| 绿竹BoGPIAP基因克隆与异位表达研究 |
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| 引用本文： | 董丽莉,孙化雨,赵韩生,娄永峰,王丽丽,高志民.绿竹BoGPIAP基因克隆与异位表达研究[J].林业科学研究,2015,28(5):627-633. |
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| 作者姓名： | 董丽莉 孙化雨 赵韩生 娄永峰 王丽丽 高志民 |
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| 作者单位： | 国际竹藤中心, 竹藤科学与技术重点开放实验室, 北京 100102;国际竹藤中心, 竹藤科学与技术重点开放实验室, 北京 100102;国际竹藤中心, 竹藤科学与技术重点开放实验室, 北京 100102;国际竹藤中心, 竹藤科学与技术重点开放实验室, 北京 100102;国际竹藤中心, 竹藤科学与技术重点开放实验室, 北京 100102;国际竹藤中心, 竹藤科学与技术重点开放实验室, 北京 100102 |
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| 基金项目： | 国家科技支撑计划专题"分子辅助育种技术集成与示范"(2012BAD23B0504)、"竹藤优异种质创制创新与种苗培育标准化示范"(2015BAD04B01)。 |
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| 摘 要： | 糖基化磷脂酰肌醇锚定蛋白(GPIAP)因其结构和功能的多样性,决定了它在各种生物学过程中都发挥着重要作用。采用同源克隆的方法从绿竹(Bambusa oldhamii)中获得一个GPIAP同源基因,命名为BoGPIAP,cDNA全长1 772 bp,其中包括1 356 bp的开放阅读框,编码一个451 aa的的蛋白。蛋白结构分析表明,该蛋白包含1个典型的GPIAP家族保守区域(47-211)和1个CCVS结构域,在N-端和C-端分别具有1个跨膜信号肽和1个GPI锚定信号肽,属于GPIAP家族。构建BoGPIAP与GFP融合的表达载体,在洋葱表皮细胞中瞬时表达,结果显示BoCOBL::GFP融合蛋白定位于细胞膜上,证明BoGPIAP基因编码的蛋白为膜蛋白。分别构建BoGPIAP的正义、反义表达载体并转化烟草(Nicotiana tabacum)。PCR检测结果表明,BoGPIAP已转入烟草。与野生型相比,转反义基因植株细弱,纤维细胞壁明显变薄;而转正义基因植株粗壮,纤维细胞壁明显变厚。表明BoGPIAP可能对竹子纤维细胞壁的发育具有调控作用。
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| 关 键 词： | 绿竹 糖基化磷脂酰肌醇锚定蛋白基因 亚细胞定位 异位表达 纤维细胞 |
| 收稿时间： | 2014/7/31 0:00:00 |
Isolation and Ectopic Expression of BoGPIAP from Bambusa oldhamii |
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| DONG Li-li,SUN Hua-yu,ZHAO Han-sheng,LOU Yong-feng,WANG Li-li and GAO Zhi-min.Isolation and Ectopic Expression of BoGPIAP from Bambusa oldhamii[J].Forest Research,2015,28(5):627-633. |
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| Authors: | DONG Li-li SUN Hua-yu ZHAO Han-sheng LOU Yong-feng WANG Li-li and GAO Zhi-min |
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| Institution: | International Center for Bamboo and Rattan, Key Laboratory on the Science and Technology of Bamboo and Rattan, Beijing 100102;International Center for Bamboo and Rattan, Key Laboratory on the Science and Technology of Bamboo and Rattan, Beijing 100102;International Center for Bamboo and Rattan, Key Laboratory on the Science and Technology of Bamboo and Rattan, Beijing 100102;International Center for Bamboo and Rattan, Key Laboratory on the Science and Technology of Bamboo and Rattan, Beijing 100102;International Center for Bamboo and Rattan, Key Laboratory on the Science and Technology of Bamboo and Rattan, Beijing 100102;International Center for Bamboo and Rattan, Key Laboratory on the Science and Technology of Bamboo and Rattan, Beijing 100102 |
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| Abstract: | Glycosylphosphatidylinositol-anchored protein (GPIAP) plays an important role in many biological processes because of its diversity of structure and function. A GPIAP gene was isolated from Bambusa oldhamii using homologous cloning method, and designed as BoGPIAP. The full-length cDNA of BoGPIAP was 1772 bp including an open reading frame of 1 356 bp. The predicted protein encoded by BoGPIAP was 451 amino acids with a transmembrane structure at the N-end and a signal of GPI-anchor at the C-end, and also containing a typical conserved domain (47-211) and a CCVS motif, which indicated that it was a membrane protein belonging to GPIAP family. The plant expression vector with BoGPIAP::GFP was constructed and transformed into onion epidermal cells. The results of fluorescent microscope showed that the fused proteins were mainly located on the cytoplasmic membrane, which revealed that the protein encoded by BoGPIAP was a membrane protein. The expression vectors of sense and antisense BoGPIAP were constructed into the multiple cloning sites of pBI121 respectively, and transformed into tobacco mediated with agrobacterium. The transgenic plants of BoGPIAP were confirmed by PCR method. The phenotypes showed the antisense transgenic plants were thin, while the sense transgenic ones were stout comparing with the wild type. The average thickness of fibrous cell wall in the antisense transgenic plants was thinner, while that of the sense transgenic ones was significantly thicker than that of wild type, indicating that the BoGPIAP may play a regulatory role in the wall development of fibrous cell in bamboo. |
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| Keywords: | Bambusa oldhamii glycosylphosphatidylinositol-anchored protein gene subcellular localization ectopic expression fibrous cell |
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