衣藻叶绿体PsbA启动子的克隆及活性检测
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摘要
衣藻作为一种模式绿藻已应用于多种研究中。由于它具有多种优点:基因组序列已测序完成,遗传机制比较清楚;既能自养,又能异养;体内含有一个大的杯状叶绿体,容易被转化;生长周期短,光合效率高,因此素有“绿色酵母”之称,常用作宿主细胞来表达外源蛋白。衣藻是唯一一种在细胞核、叶绿体和线粒体中实现外源基因转化的生物。但外源基因在叶绿体中表达量低下一直是困扰衣藻叶绿体生物反应器商业化的瓶颈。影响外源基因表达量的最主要因素之一是启动子的强弱。强启动子能提高外源基因的转化频率,从而提高外源基因的表达量。衣藻叶绿体光系统II反应中心蛋白D1基因(PsbA)的启动子通常被认为是一种强启动子。本研究中,克隆了莱茵衣藻叶绿体PsbA启动子,以期PsbA启动子能提高外源基因在衣藻叶绿体中的转录效率。
本研究的主要内容如下:
首先,提取了莱茵衣藻DNA总基因组,设计PsbA启动子引物,并以总基因组为模板克隆了PsbA启动子。
其次,构建了质粒p64D-PsbA-aadA。将PsbA启动子连接到质粒p64D中壮观霉素抗性基因(aadA)的上游。转化大肠杆菌DH5α,经氨苄青霉素和壮观霉素的筛选,获得了阳性单克隆斑。初步证明了PsbA启动子在大肠杆菌中能调控外源基因的表达。
最后,构建了质粒psk-PsbA-gfp。将绿色荧光蛋白基因(gfp)连接到PsbA启动子的下游。转化大肠杆菌,经筛选后,获得了阳性单克隆斑。利用荧光显微镜,观察到了具有绿色荧光的大肠杆菌。证明了PsbA启动子具有启动活性。
本研究成果为衣藻叶绿体基因工程的进一步研究奠定了基础。
Chlamydomonas is used as a model green alga in many studies. Because it’s genome sequence has been sequenced, and the genetic mechanism is clear; it is a eukaryotic unicellular green algae; it is not only a autotrophic organism but also a heterotrophic organism; it contains a big cup chloroplast that can be easily transformed; it has short growth cycle and high photosynthetic efficiency, it is called“green yeaet”and usually is used as host cells to express foreign gene. The Chlamydomonas is the only organism that has been transformed successfully in nucleus, chloroplast and chondriosome. But the poor foreign gene expression level in chloroplast is a bottleneck that limits its using as bioreactor. The one of main factor affecting the foreign gene expression is promoter. The strong promoter can increase the transcription frequency and increase foreign genes expression level. The Chlamydomonas chloroplast endogenic photosystem II protein D1 promoter is usually considered as a strong promoter. In this research, the promoter above mentioned is cloned, and we hope that the promoter may be promote the foreign genes expesson in Chlamydomonas chloroplast.
The contents of this study is as following:
Firstly, the chlamydomonas total genome is extracted, and the PsbA primer is designed. With total DNA as templates, the PsbA promoter is cloned using the method of PCR.
Secondly, the plasmid p64D-PsbA-aadA is constructed. The PsbA promoter is ligated to the spectinomycin resistance gene (aadA) upstream. The construction is transformed into E.coli DH5α. After screening on ampicillin and spectinomycin plate, the positive clone is obtained. Which proves that the PsbA promoter has the promoter activity.
Finally, the plasmid psk-PsbA-gfp is constructed. The green fluorescent protein gene (gfp) is ligated to PsbA promoter downstream. After transforming E.coli DH5αand screening, the positive clone is obtained. Using the fluorescent microscope, the green fluorescence is observed in E.coli. This proves that PsbA promoter can regulate the green fluorescent protein gene expression in E.coli. The results of this research lay the foundation for further chlamydomonas chloroplast engineering study.
本研究的主要内容如下:
首先,提取了莱茵衣藻DNA总基因组,设计PsbA启动子引物,并以总基因组为模板克隆了PsbA启动子。
其次,构建了质粒p64D-PsbA-aadA。将PsbA启动子连接到质粒p64D中壮观霉素抗性基因(aadA)的上游。转化大肠杆菌DH5α,经氨苄青霉素和壮观霉素的筛选,获得了阳性单克隆斑。初步证明了PsbA启动子在大肠杆菌中能调控外源基因的表达。
最后,构建了质粒psk-PsbA-gfp。将绿色荧光蛋白基因(gfp)连接到PsbA启动子的下游。转化大肠杆菌,经筛选后,获得了阳性单克隆斑。利用荧光显微镜,观察到了具有绿色荧光的大肠杆菌。证明了PsbA启动子具有启动活性。
本研究成果为衣藻叶绿体基因工程的进一步研究奠定了基础。
Chlamydomonas is used as a model green alga in many studies. Because it’s genome sequence has been sequenced, and the genetic mechanism is clear; it is a eukaryotic unicellular green algae; it is not only a autotrophic organism but also a heterotrophic organism; it contains a big cup chloroplast that can be easily transformed; it has short growth cycle and high photosynthetic efficiency, it is called“green yeaet”and usually is used as host cells to express foreign gene. The Chlamydomonas is the only organism that has been transformed successfully in nucleus, chloroplast and chondriosome. But the poor foreign gene expression level in chloroplast is a bottleneck that limits its using as bioreactor. The one of main factor affecting the foreign gene expression is promoter. The strong promoter can increase the transcription frequency and increase foreign genes expression level. The Chlamydomonas chloroplast endogenic photosystem II protein D1 promoter is usually considered as a strong promoter. In this research, the promoter above mentioned is cloned, and we hope that the promoter may be promote the foreign genes expesson in Chlamydomonas chloroplast.
The contents of this study is as following:
Firstly, the chlamydomonas total genome is extracted, and the PsbA primer is designed. With total DNA as templates, the PsbA promoter is cloned using the method of PCR.
Secondly, the plasmid p64D-PsbA-aadA is constructed. The PsbA promoter is ligated to the spectinomycin resistance gene (aadA) upstream. The construction is transformed into E.coli DH5α. After screening on ampicillin and spectinomycin plate, the positive clone is obtained. Which proves that the PsbA promoter has the promoter activity.
Finally, the plasmid psk-PsbA-gfp is constructed. The green fluorescent protein gene (gfp) is ligated to PsbA promoter downstream. After transforming E.coli DH5αand screening, the positive clone is obtained. Using the fluorescent microscope, the green fluorescence is observed in E.coli. This proves that PsbA promoter can regulate the green fluorescent protein gene expression in E.coli. The results of this research lay the foundation for further chlamydomonas chloroplast engineering study.
引文
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[3]胡鸿钧,殷春涛.拟衣藻属一新种-中华拟衣藻及其生活史的研究[J].武汉植物学研究,2001,19(6):453-457.
[4] Muqing Cao,Yu Fu,Yan Guo,et.al. Chlamydomonas reinhardtii(Chlorophyceae)colony PCR[J]. Protoplasma,2009,235:107-110.
[5]李修岭,李夜光.衣藻有性生殖的分子机制[J].西北植物学报,2009,29(7):1490-1495.
[6]张洪涛,艾山江·阿不都拉,徐田枚等.莱茵衣藻叶绿体生物反应器研究进展[J].生物技术通报,2007(2):27-32.
[7]徐田枚,张洪涛.莱茵衣藻Nfr-4突变株中类胡萝卜素含量的变化及其对藻生长的影响[J].植物生理学通讯,2007,43(3):417-420.
[8] Eric A,Johnson. Altered expression of the chloroplast ATP synthase through site-directed mutagenesis in Chlamydomonas reinhardtii[J].Regular Paper,2008,96:153-162.
[9] Ling Xiong,Richard T,Sayre.Engineering the chloroplast encoded proteins of Chlamydomonas[J]. Photosynthesis Research,2004(80):411-419.
[10] Jocab P,Monod J.Genotic regulatory mechanisais in the synthesis of protein [J ].Journal of Molecular Bilogy,1961,3:318-356.
[11]潘卫东,吕玉民,张贵星,等.莱茵衣藻叶绿体atpA启动子在杜氏盐藻中转录活性的检测[J].郑州大学学报(医学版),2004,39(1):35-38.
[12]孙晓红,陈明杰,潘迎捷.启动子克隆概述[J].食用菌学报,2002,9(3):57-62.
[13]张学成,郭楠,宋晓金.藻类基因启动子结构与功能研究进展[J].中国海洋大学学报,2008,38(3):404-412.
[14] V.G.Ladygin. The Transformation of the Unicellular Alga Chlamydomonas reinhardtii by Electroporation[J].Microbiology,2003,72(5):658-665.
[15]苏忠亮,程江峰,梁成伟,等.外源基因在衣藻叶绿体中高效表达的研究[J].海洋通报,2008,27(4):101-105.
[16] Ishikura K,Takaoka Y,Kato K,et al. Expression of a Forgeign Gene in Chlamydomonas reinhardtii Chloroplast [J].Journal of Bioscience and Bioengineering,1999,87(3):307-314.
[17] Rochaix J-D,Dillewijn van J.Transformation of the green algae chlamydomonas reinhardtii with yeast DNA[J].Nature(Lond),1982,290:70-72.
[18] Kindle KL.High-frequence nuclear transformation of Chlamydomonas reinhardtii[J].Methods in Enzymology,1998,197:27-38.
[19]吴志华,黎双飞,胡章立.莱茵衣藻线粒体外源基因表达系统的初步研究[M].深圳:学术研讨会论文集,2010,62-63.
[20] kindle K L,Richards K L,Stern D B.Engineering the chloroplast genome:Techniques and capabilities for ckloroplanst transformation in Chlamydomonas reinhardtii[J].Proc Natl Acad Dci USA,1991,88:1721-1725.
[21] WANG Chaogang,HU Zhangli,HU Wei. Expression and molecular analysis of phbB gene in Chlamydomonas reinhardtii[J].Chinese Science Bulletin,2004,49(16):1713-1717.
[22]陈颖,李文,孙勇如.藻类基因工程的研究技术及方法[J].植物学通报,1999,16(4):321-331.
[23] BOYNTON J E,GILLHAN N W,HARRIS E H,et.al. Chloroplast transformation in Chlamydomonas with high velocity microprojectiles[J ].Science,1988,240:1534-1538.
[24]张中林,山松,陈曦.丙肝病毒融合抗原基因NS3-C定点整合入衣藻叶绿体基因组的研究[M].遗传,1999,21(6):1-6.
[25] Elizabeth Specht,Shigeki Miyake-Stoner,Stenhen Mayfield.Micro-algae come of age as a platform for recombinant protein production[J].Biotechnol Lett,2010,(32):1373-1383.
[26] Arthur R.Grossman[J].Plant Physiology,2005,137:410-427.
[27]赵雅坤,史贤明,张忠明.人白细胞介素4在衣藻叶绿体中的高效转化及表达[J].华中农业大学学报,2006,25(2):110-116.
[28]杨宗岐,李轶女,陈凤,等.人可溶性TRIL蛋白在衣藻叶绿体中的表达[J].科学通报,2006,51(12):1400-1405.
[29]徐琴.莱茵衣藻CAO基因与CBN1基因功能对比分析的研究[M].新疆,新疆大学学报,2004:10-11.
[30]龚丽丽,郭晶晶,许晓明. Cr6+胁迫对莱茵衣藻光合作用的影响[J].西北植物学报,2010,30(6):1166-1172.
[31]李洪涛,朱珂,刘清岱,等.不同培养条件下莱茵衣藻细胞中SGAT酶活性的变化[J].植物生理学通讯,2010,46(4):359-364.
[32] Anja Hemschemeier,Swanny Fouchard,Laurent Cournac.Hydrogen production by Chlamydomonas reinhardtii: an elaborate interplay of electron sources and sinks[J].Planta,2008,227:397-407.
[33] Anastasios Melis,Thomas Happe. Trails of green alga hydrogen research-from Hans Gaffron to new frontiers[J]. Photosynthesis Research,2004(80):401-409.
[34]潘丽霞,杨登峰,梁智群.绿藻高效制氢影响因素的研究[J].中国生物工程杂志,2007,27(4),146-152.
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