莱茵衣藻BBS1基因RNAi载体的构建及应用

doi:10.13560/j.cnki.biotech.bull.1985.2015.07.016

生物技术通报 ›› 2015, Vol. 31 ›› Issue (7): 109-114.doi: 10.13560/j.cnki.biotech.bull.1985.2015.07.016

莱茵衣藻BBS1基因RNAi载体的构建及应用

陈利红李丽丽高利芬周俊飞李甜甜彭海

(江汉大学系统生物学研究院，武汉 430056)

收稿日期:2014-11-12 出版日期:2015-07-16 发布日期:2015-07-16
作者简介:陈利红，女，博士，助理研究员，研究方向：植物分子生物学；E-mail：clh101@126.com
基金资助:
武汉市人力资源和社会保障局“衣澡冷适应的系统生物学研究”项目

Construction and Application of the RNAi Vector for BBS1 Gene in Chlamydomonas reinhardtii

Chen Lihong, Li Lili, Gao Lifen, Zhou Junfei, Li Tiantian, Peng Hai

(Institute for System Biology，Jianghan University，Wuhan 430056)

Received:2014-11-12 Published:2015-07-16 Online:2015-07-16

摘要/Abstract

摘要：

巴德-毕氏综合症(Bardet-Biedl syndrome，BBS)是一种由多种基因突变造成、与原生纤毛功能缺失相关的疾病，包含12种致病基因，分别为BBS1-12。旨在通过对衣藻BBS1基因进行RNAi干扰来研究其在衣藻中的功能。首先搭建衣藻快捷简单的RNAi骨架载体；再用RT-PCR克隆莱茵衣藻BBS1基因的一段cDNA 序列于中间载体pEASY-T1上；测序后通过两次酶切连接到RNAi骨架载体上，经菌液PCR、酶切和测序验证，成功构建BBS1基因的RNAi干涉载体。经基因枪介导法转化莱茵衣藻CC503，转基因衣藻具有明显不同于对照的表现型，趋光性发生了改变。

关键词: 莱茵衣藻, RNAi载体, BBS1 基因, 趋光性

Abstract:

Bardet-Biedl syndrome is a disease caused by mutations of various genes and associated with afunction of primary cilia. There are 12 disease-causing genes of BBS1-12 respectively. This study aimed to understand the function of BBS1 gene in Chlamydomonas reinhardtii through RNAi interference technology. Firstly, the fast and simple RNAi backbone vector was constructed. Subsequently, a cDNA in BBS1 gene was cloned into media vector pEASY-T1 using RT-PCR. After sequencing, the target fragment was inserted into the RNAi backbone vector through the twice enzyme digestion. Verified by PCR testing, enzyme digestion and sequencing, the RNAi vector of BBS1 gene was successfully constructed. The phenotype of transgenic C. reinhardtii CC503 transformed into BBS1 RNAi plasmid by gene gun was significantly different from that of the control and their phototaxis changed compared to the control.

Key words: Chlamydomonas reinhardtii, RNAi vector, BBS1 gene, phototaxis

陈利红, 李丽丽, 高利芬, 周俊飞, 李甜甜, 彭海. 莱茵衣藻BBS1基因RNAi载体的构建及应用[J]. 生物技术通报, 2015, 31(7): 109-114.

Chen Lihong, Li Lili, Gao Lifen, Zhou Junfei, Li Tiantian, Peng Hai. Construction and Application of the RNAi Vector for BBS1 Gene in Chlamydomonas reinhardtii[J]. Biotechnology Bulletin, 2015, 31(7): 109-114.

参考文献

[1] Goodenough UW. Green yeast[J]. Cell, 1992, 70(4)：533-538.
[2] Merchant SS, Prochnik SE, Vallon O, et al. The Chlamydomonas genome reveals the evolution of key animal and plant functions[J]. Science, 2007, 318(5848)：245-250.
[3] 柳林, 纪伟. 纤毛疾病和与之相关的基因[J]. 现代生物医学进展, 2012, 12(2)：373-375.
[4] Lechtreck KF, Johnson EC, Sakai T, et al. The Chlamydomonas reinhardtii BBSome is an IFT cargo required for export of specific signaling proteins from flagella[J]. The Journal of Cell Biology, 2009, 187(7)：1117-1132.
[5] 潘俊敏. 衣藻、纤毛与“纤毛相关疾病”[J]. 中国科学C辑：生命科学, 2008, 38(5)：399-409.
[6] Oeffner F, Moch C, Neundorf A, et al. Novel interaction partners of Bardet-Biedl syndrome proteins[J]. Cell Motility and the Cytoskeleton, 2008, 65(2)：143-155.
[7] Napoli C, Lemieux C, Jorgensen R. Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans[J]. The Plant Cell, 1990, 2(4)：279-289.
[8] Bass BL. Double-stranded RNA as a template for gene silencing[J]. Cell, 2000, 101(3)：235-238.
[9] Kusaba M. RNA interference in crop plants[J]. Current Opinion in Biotechnology, 2004, 15(2)：139-143.
[10] Wesley SV, Helliwell CA, Smith NA, et al. Construct design for efficient, effective and high-throughput gene silencing in plants[J]. The Plant Journal：For Cell and Molecular Biology, 2001, 27(6)：581-590.
[11] Simon DF, Descombes P, Zerges W, et al. Global expression profiling of Chlamydomonas reinhardtii exposed to trace levels of free cadmium[J]. Environmental Toxicology and Chemistry / SETAC, 2008, 27(8)：1668-1675.
[12] 李兴涵, 费小雯, 邓晓东. 莱茵衣藻磷酸果糖激酶RNAi载体构建及其对莱茵衣藻油脂积累的影响[J]. 广东农业科学, 2014, 10：155-159.
[13] 邓晓东, 蔡佳佳, 费小雯. 莱茵衣藻磷脂二脂酰甘油酰基转移酶3 在三酰甘油合成中的功能研究[J]. 水生生物学报, 2014, 38(4)：745-750.
[14] Kulaga HM, Leitch CC, Eichers ER, et al. Loss of BBS proteins causes anosmia in humans and defects in olfactory cilia structure and function in the mouse[J]. Nature Genetics, 2004, 36(9)：994-998.
[15] Ou G, Blacque OE, Snow JJ, et al. Functional coordination of intraflagellar transport motors[J]. Nature, 2005, 436(7050)：583-587.
[16] Kim YH, Epting D, Slanchev K, et al. A complex of BBS1 and NPHP7 is required for cilia motility in zebrafish[J]. PLoS One, 2013, 8(9)：e725.

莱茵衣藻BBS1基因RNAi载体的构建及应用

Construction and Application of the RNAi Vector for BBS1 Gene in Chlamydomonas reinhardtii

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 10

编辑推荐

Metrics

本文评价

[1]	李旺宁, 张豪杰, 李亚男, 梁梦静, 季春丽, 张春辉, 李润植, 崔玉琳, 秦松, 崔红利. 莱茵衣藻蓝光受体植物类型隐花色素CRY突变体的表型鉴定[J]. 生物技术通报, 2023, 39(2): 243-253.
[2]	葛欣, 崔天琦, 李兴旺, 谢录翰, 辛琪. 接合菌蓝光受体蛋白研究进展[J]. 生物技术通报, 2018, 34(4): 43-50.
[3]	宋燕子, 贾彬, 林柏成, 胡章立, 黄瑛. 莱茵衣藻酰基辅酶A合成酶cDNA克隆及其酵母表达[J]. 生物技术通报, 2015, 31(9): 119-124.
[4]	王潮岗, 陈爱娜, 胡章立, 于珊珊. 莱茵衣藻基因组文库的构建 [J]. 生物技术通报, 2013, 0(4): 85-89.
[5]	牛丽红;耿丽丽;张蕊;孙长坡;张杰;. 莱茵衣藻ω-3脂肪酸脱氢酶基因在毕赤酵母中的表达与活性分析[J]. , 2011, 0(12): 96-101.
[6]	冯瑜;马丽娟;闫楠;何芳莹;柴友荣;. 芸薹属原花青素跨膜转运蛋白TT12、AHA10基因家族RNA干扰载体的构建[J]. , 2010, 0(09): 116-122.
[7]	王五姐;魏渊源;刘兆普;杨志敏;. 莱茵衣藻血红素加氧酶1过表达载体的构建和遗传转化[J]. , 2010, 0(02): 157-161.
[8]	刘国宪;沈桂芳;胡英考;. 衣藻叶绿体表达系统的研究[J]. , 2009, 0(09): 28-33.
[9]	吕慧贞;陈新;穆清泉;张士刚;张元湖;. 苹果α-法尼烯合成酶基因双链RNAi载体的构建[J]. , 2007, 0(03): 122-125.
[10]	韩四海;胡章立;王潮岗;. 外源基因在莱茵衣藻叶绿体中的表达[J]. , 2007, 0(01): 89-94.