WO2019007177A1 - 一种在真核细胞中表达固氮酶基因的方法 - Google Patents

一种在真核细胞中表达固氮酶基因的方法 Download PDF

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WO2019007177A1
WO2019007177A1 PCT/CN2018/089745 CN2018089745W WO2019007177A1 WO 2019007177 A1 WO2019007177 A1 WO 2019007177A1 CN 2018089745 W CN2018089745 W CN 2018089745W WO 2019007177 A1 WO2019007177 A1 WO 2019007177A1
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nitrogenase
gene
pvx
expressing
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程奇
孙文丽
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中国农业科学院生物技术研究所
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    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8203—Virus mediated transformation
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    • C12Y118/06—Oxidoreductases acting on iron-sulfur proteins as donors (1.18) with dinitrogen as acceptor (1.18.6)
    • C12Y118/06001—Nitrogenase (1.18.6.1)

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  • the invention belongs to the technical field of genetic engineering, and particularly relates to a method for expressing a nitrogenase gene in a eukaryotic cell.
  • Biological nitrogen fixation is the main source of nitrogen required by plants worldwide, accounting for about 75%.
  • the use of biological nitrogen fixation can reduce the use of nitrogen fertilizer.
  • One of the important measures to resolve this contradiction However, no eukaryotic organisms having nitrogen-fixing functions have been found so far, and known nitrogen-fixing organisms are limited to prokaryotic microorganisms such as bacteria and actinomycetes.
  • Biological nitrogen fixation refers to the biological process by which nitrogen-fixing microorganisms use their own nitrogen-fixing enzymes for nitrogen fixation. Nitrogenase is a catalytic reaction center for reducing nitrogen in the air.
  • the nitrogen fixation reaction of nitrogen-fixing organisms is achieved by the catalysis of nitrogenase.
  • the nitrogenase is very conserved and is encoded by the nif genome.
  • the nif genome in most nitrogen-fixing organisms includes 16-19 genes with a total length of 23 kb, including 8 essential genes: nifHDKENXBQ.
  • a typical nitrogenase consists of ferritin and molybdenum ferritin. It reduces ferritin by electron donors such as ferredoxin and flavodoxin, and transfers single electrons from ferritin to ferroferrin (depending on hydrolysis of MgATP). ), the last electron is transferred from the P cluster to the FeMo cluster substrate binding site inside the MoFe protein.
  • Each electron transfer requires a combination of ferritin and ferromolybdenum, and the two components are separated after the transfer.
  • all nitrogen-fixing organisms have a molybdenum-iron nitrogenase system. Under the condition of lack of molybdenum, some nitrogen-fixing organisms induce the synthesis of other cofactor nitrogen-fixases, such as vanadium-iron or iron-iron cofactor nitrogenase. Nitrogenase is sensitive to oxygen, and it is speculated that this is due to surface exposure of the 4FE-4S cluster between the two domains of the dimer.
  • the nitrogen fixation reaction of nitrogenase has the following basic characteristics: 1. Metal ions and ATP are required; 2. It is sensitive to oxygen and needs to function under anaerobic or hypoxic conditions. It is precisely because of the above characteristics of nitrogenase that all nitrogen-fixing organisms discovered so far are limited to prokaryotic microorganisms (bacteria and actinomycetes), and the common feature of these organisms is anaerobic, except for a special case, cyanobacteria. Cyanobacteria are a class of oxygen-emitting photosynthetic organisms. Under light, the oxygen concentration in the cells is increased by photosynthesis. However, it also has an anaerobic nitrogen-fixing system, which forms two kinds in the long-term evolution process. A unique antioxidant mechanism.
  • Some filamentous cyanobacteria can form a heteromorphic cell in the middle or at the end of the cell chain. They are bulky and have a thick outer membrane to prevent oxygen from entering the cell.
  • the heteromorphic cell has only photosynthetic system I, so photosynthesis does not release oxygen, so nitrogen fixation The enzyme can play a good role in nitrogen fixation in the heterogeneous cells;
  • the protection of nitrogen-fixing enzymes of non-heteromorphic cyanobacteria is diverse: some use time separation of nitrogen fixation and photosynthesis, nitrogen fixation in the dark, photosynthesis under illumination, such as cyanobacteria; The bundled population is formed, and the cells in the anaerobic environment at the center lose the photosynthetic system II, which is beneficial to the nitrogen fixation of the nitrogenase in the micro-oxygen environment, such as the genus Trachomatis; and some when the nitrogenase activity is high. The peroxidase and the activity of excess peroxidase in the cells for removing toxic peroxides are also increased, such as the genus Myxobacteria. Lucas J et al.
  • Cheng Qi proposed the idea of expressing nitrogen-fixing genes in chloroplasts.
  • Cheng Qi et al. transformed the ferrozyme gene nifH of Klebsiella pneumoniae into Chlamydomonas reinhardtii chloroplast and replaced the chlL of DPOR protein which plays a role in chlorophyll synthesis under dark conditions.
  • Chlamydomonas can still be synthesized under dark conditions.
  • Chlorophyll indicating that NIFH can function in the chloroplast of Chlamydomonas, is a successful early exploration of chloroplast expression of nitrogen-fixing enzymes.
  • the object of the present invention is to solve the deficiencies of the prior art, and to provide a method for expressing a nitrogenase gene in a eukaryotic cell, thereby realizing the expression of a nitrogenase gene in a plant, thereby laying a foundation for plant self-fixing nitrogen.
  • a method of expressing a nitrogenase gene in a eukaryotic cell comprising the steps of:
  • Step 1 amplifying the nitrogenase gene fragment
  • Step two constructing a recombinant vector: ligating the nitrogenase gene fragment to a viral transformation vector;
  • Step 3 transfecting the eukaryotic cell with the recombinant vector constructed in the second step
  • Step 4 Screening for plant cells expressing the nitrogenase gene.
  • the nitrogenase gene fragment in step one is NifH, NifE, NifB and/or NifN.
  • primers for amplifying the nitrogenase gene fragment in the first step are SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, and SEQ. ID No. 6, SEQ ID No. 7 and SEQ ID No. 8.
  • PCR reaction system at the time of amplification described in the first step is:
  • the second step is: the NifH/NifE/NifB/NifN gene is ligated into the vector PVX expression vector PVX-HisG-MF1 by ClaI/SalI, EagI/SalI, EagI/SalI, ClaI/SalI restriction sites, respectively.
  • the PVX-HisG-MF1-NifH/NifE/NifB/NifN vector was obtained.
  • sequence of the PVX-HisG-MF1-NifH, PVX-HisG-MF1-NifE, P VX-HisG-MF1-NifN, PVX-HisG-MF1-NifB vectors described in the second step is SEQ ID No. 13
  • sequence of the NifH, NifE, Ni fB, NifN gene cloned as shown in SEQ ID No. 14, SEQ ID No. 15, and SEQ ID No. 16 is SEQ ID No. 9, SEQ ID No. 10, and SEQ ID No. .11, SEQ I D No.12.
  • the invention has the beneficial effects that the expression of the nitrogenase structure gene in the plant is realized: the invention realizes the expression of the nitrogenase structure gene in the plant, thereby laying a foundation for the plant self-fixing nitrogen.
  • the nitrogenase structural gene is only present in prokaryotes, and the expression of prokaryotic genes in eukaryotes is not particularly easy. So far, the nitrogenase structural gene that has been successfully expressed in plants has been reported to be only NifH.
  • Prokaryotic genes are divided into coding regions and non-coding regions. The genes on the non-coding region determine whether certain traits are expressed, how many times they are expressed, and when they begin to be expressed.
  • the coding region can be transcribed into the corresponding messenger RNA, which in turn directs the synthesis of the protein.
  • the non-coding region is the opposite, but the non-coding region is essential for the expression of genetic information because there are nucleotide sequences that regulate the expression of genetic information on the non-coding region.
  • the non-coding region is located upstream and downstream of the coding region. The most important of the nucleotide sequences that regulate expression of genetic information is the RNA polymerase binding site located upstream of the coding region. RNA polymerase catalyzes the transcription of DNA into RNA, which recognizes and binds to binding sites in regulatory sequences.
  • the T7 promoter a strong promoter from T7 phage that specifically reacts to T7 RNA polymerase, is a sequence that initiates transcription of the T7 phage gene.
  • T7 RNA polymerase selectively activates the transcription of the T7 phage promoter, which synthesizes mRNA about five times faster than the normal E. coli RNA polymerase.
  • the plant viral vector expresses a high level of expression of a foreign protein, a rapid expression rate, and a wide host range.
  • the present invention successfully expressed NifH/NifE/NifB/NifN in plant cells using a T7-initiated PVX vector.
  • Figure 1 is a PCR clone of the nifH, nifE, nifB, nifN genes in Heliobacterium chlorum.
  • Figure 2 shows PCR detection of PVX/HisG-nifH, PVX/HisG-nifE, PVX/HisG-nifB and PVX/HisG-nif N vectors.
  • G6 Total protein of tobacco leaves transfected with PVX-HisG-MF1-NifN vector
  • G7 Total protein of control tobacco leaves
  • HELIOBACTERIUM CHLORUM MED IUM 370 medium was cultured in HELIOBACTERIUM CHLORUM MED IUM 370 medium.
  • HELIOBACTERIUM CHLORUM MEDIUM 370 medium K2HPO4 1.0g
  • MgSO 4 x 7H 2 O 1.0 g, Yeast extract 10.0 g, Distilled water 1000.0 ml, pH 7.0 was adjusted. Nitrogen gas was charged, and sodium ascorbate (0.5 g) was added to continue to be filled with nitrogen. Adjust pH 6.8. Pour 45 ml of the medium into a 50 ml volumetric flask and tighten with a rubber stopper. Sterilize at 121 ° C for 15 minutes. Heliobacteriumchlorum is cultured in low light conditions.
  • Heliobacteriumchlorum genomic DNA was extracted according to the method of Ausubel et al. (1995).
  • the homologous sequence fragment of NifH/NifE/NifB/NifN in Heliobacteriumchlorum was searched at the NCBI website National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/), and primers were designed using Primer5 software.
  • the primer sequences are shown in Table 1.
  • the 857/1388/831/1306 bp NifH/NifE/NifB/NifN gene sequence was cloned, and the sequence showed 100% homology with the sequence published on the NCBI website.
  • the NifH/NifE/NifB/NifN gene was ligated into the vector Potato X pot virus (PVX) expression vector PVX-HisG-MF1 by ClaI/SalI, EagI/SalI, EagI/SalI, and ClaI/SalI restriction sites, respectively.
  • PVX-HisG-MF1-NifH/NifE/NifB/NifN vector was verified by sequencing and the gene was successfully cloned into an expression vector.
  • PVX-HisG-MF1-NifH/NifE/NifB/NifN was used in the transfection experiment of tobacco Nicotian abendhamiana after being subjected to an in vitro transcription kit. After transfection, tobacco was incubated for 2-3 weeks at 20-26 degrees, 12 hours light, and 12 hours dark condition. Tobacco leaves with virus infection spot symptoms were screened for protein SDS-PAGE and WESTERN hybridization.
  • the total protein of transgenic and non-transgenic tobacco leaves was extracted using a plant protein extraction kit.
  • the total protein of the obtained transgenic and non-transgenic tobacco leaves was detected by SDS-PAGE electrophoresis.
  • WESTERN hybridization assay was performed using an anti-his antibody (Invitrogen) antibody and a horseradish peroxidase-conjugated secondary antib ody (Invitrogen) secondary antibody. The results showed that both NifH/NifE/NifB/Nif N proteins were successfully expressed in tobacco.
  • NifH sequence (SEQ ID No. 9):
  • NifE sequence (SEQ ID No. 10):
  • NifB sequence (SEQ ID No. 11):
  • NifN sequence (SEQ ID No. 12):
  • PVX-HisG-MF1-NifH sequence SEQ ID No. 13:
  • PVX-HisG-MF1-NifE sequence SEQ ID No. 14:
  • PVX-HisG-MF1-NifN sequence SEQ ID No. 15:
  • PVX-HisG-MF1-NifB sequence SEQ ID No. 16:

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Abstract

提供了一种在真核细胞中表达固氮酶基因的方法，包括以下步骤：1）扩增所述固氮酶基因片段；2）将所述固氮酶基因片段连接至病毒转化载体构建重组载体；3）用2）中构建的重组载体转染所述真核细胞；4）筛选表达固氮酶基因的植物细胞。本发明实现了固氮酶基因在植物中的表达，从而为植物自主固氮奠定基础。

Description

一种在真核细胞中表达固氮酶基因的方法 技术领域
本发明属于基因工程技术领域，具体涉及一种在真核细胞中表达固氮酶基因的方法。
背景技术
氮肥的使用与我国粮食、能源、环境等可持续发展重大问题的矛盾日益尖锐，生物固氮是全球植物所需氮素的主要来源，约占75％，利用生物固氮，可减少氮肥的使用，是解决该矛盾的重要措施之一。然而，迄今为止没有发现任何具有固氮功能的真核生物，已知的能够固氮的生物只限于原核微生物，如细菌和放线菌。生物固氮是指固氮微生物利用其自身的固氮酶进行固氮反应的生物过程。固氮酶是将空气中的氮气还原的催化反应中心，是一种复杂的多蛋白复合体，至今仅在原核生物中发现，没有在植物中发现固氮酶基因的报道，也没有发现任何具有自主固氮能力的植物。因此开展固氮基因在植物中的表达研究，达到提高生物固氮效率、扩大宿主范围，发挥生物固氮在农业生产中的作用，将确保我国的主要农作物生产在继续提高产量、改良品质同时，大幅度地减少化肥、农药的施用量，实现我国农业可持续发展。
固氮生物的固氮反应是在固氮酶的催化作用下实现的。固氮酶非常保守，由nif基因组编码。多数固氮生物中的nif基因组包括16-19个基因，总长度为23kb，其中包括8个必需基因：nifHDKENXBQ。典型的固氮酶由铁蛋白和钼铁蛋白两部分组成，通过铁氧化还原蛋白和黄素氧还蛋白等电子供体还原铁蛋白，将单电子从铁蛋白转移到钼铁蛋白(要依赖MgATP水解)，最后电子在MoFe蛋白内部从P簇转移到FeMo簇底物结合位点。每一次电子转移都要求铁蛋白与钼铁蛋白组成一个复合体，转移结束后两个组分再分开。目前所有的固氮生物均有一个钼-铁固氮酶系统，在钼缺少的条件下，一些固氮生物会诱导其它辅助因子固氮酶的合成，如钒-铁或铁-铁辅因子固氮酶。固氮酶对氧敏感，有人推测这是由于该二聚物的两个结构域之间4FE-4S簇的表面暴露所导致的。
固氮酶的固氮反应具有以下基本特点：1、需要金属离子和ATP；2、对氧敏感，需要在无氧或低氧条件下才能发挥作用。正是由于固氮酶的以上特点，迄今为止发现的所有固氮生物只限于原核微生物(细菌和放线菌)，这些生物的共同特点是厌氧，除了一个特例——蓝细菌。蓝细菌是一类放氧性光合生物，在 光下，会因光合作用放出的氧而使细胞内氧浓度增高，但同时它又有厌氧的固氮系统，它在长期进化过程中形成两种独特的抗氧机制。
1)分化出特殊的还原性异形胞。某些丝状蓝细菌可在细胞链的中间或末端形成异形胞，体积大且有较厚外膜，可防止氧气进入细胞，此外异形胞只有光合系统Ⅰ，因而光合作用不放氧，因此固氮酶在异形胞中可以很好地发挥固氮作用；
2)非异形胞蓝细菌固氮酶的保护多种多样：有的采用将固氮作用与光合作用进行时间上的分隔，黑暗下固氮，光照下进行光合作用，如织线蓝菌属等；有的则形成束状群体，在其中央处于厌氧环境下的细胞失去光合系统Ⅱ，有利于固氮酶在微氧环境下进行固氮作用，如束毛蓝菌属；有的则在固氮酶活性高时，细胞内用以除去有毒过氧化物的过氧化物酶和超过氧化物酶的活力也均提高，如粘球蓝菌属。Lucas J等1985将不产生异型胞的蓝藻Oscilla-toriasp.，无氮培养基上培养，设置不同的光暗循环，结果在黑暗时间超过8小时条件下，只在黑暗时段固氮；如果黑暗时间少于8小时，固氮酶活性在黑暗开始之前就已经有显示。即使是产生异型胞的蓝藻，营养细胞在缺氧的条件下也可以诱导固氮酶，引发固氮反应。这也说明固氮反应不仅局限于厌氧细胞中，确切地说这应该是一个产氧细胞中的固氮需求与高浓度氧反馈抑制的平衡问题。
利用叶绿体具有的与固氮基因相似的原核特性及其它有利因素，程奇提出在叶绿体中表达固氮基因的设想。程奇等2005年，将Klebsiella pneumoniae的固氮酶的铁蛋白基因nifH转化Chlamydomonas reinhardtii叶绿体并替代黑暗条件下在叶绿素合成过程中起作用的DPOR蛋白的chlL，结果转化衣藻在黑暗条件下仍然可以合成叶绿素，说明NIFH在衣藻叶绿体中可以发挥功能，这是叶绿体表达固氮酶设想的成功前期探索。张中林等2001年，成功将固氮酶铁蛋白ni fH基因导入到高等模式植物烟草的叶绿体中进行表达。但至今为止没有关于钼铁蛋白成功在植物中表达的相关报道。
目前，也有一些利用体外合成的方法，对固氮酶基因的组分进行研究，L.M.Rubio等2008年报道，在钼，铁，硫磺和高柠檬酸存在的条件下，仅用Nif B,NifEN,NifH三个基因即可完成钼铁蛋白的体外生物合成。Yilin Hu等，2009年研究表明，NifEN具有乙炔还原活性，却并不能将N2还原成NH 3 +，这说明NifEN具有与钼铁蛋白相似的催化作用，但是它仅仅是钼铁蛋白的一个“骨 架”，它在结构上更简单，功能上也更少，因此，它的反应底物范围更窄，底物的还原活性也更低。
尽管人们在生物固氮上进行了大量的研究，但目前没有任何关于钼铁蛋白在植物中成功表达的报道。
发明内容
本发明的目的是为了解决现有技术的不足，而提供一种在真核细胞中表达固氮酶基因的方法，实现了固氮酶基因在植物中的表达，从而为植物自主固氮奠定基础。
本发明采用如下技术方案：
一种在真核细胞中表达固氮酶基因的方法，包括以下步骤:
步骤一：扩增所述固氮酶基因片段；
步骤二，构建重组载体：将所述固氮酶基因片段连接至病毒转化载体；
步骤三：用步骤二构建的重组载体转染所述真核细胞；
步骤四：筛选表达所述固氮酶基因的植物细胞。
进一步地，步骤一中所述固氮酶基因片段为NifH、NifE、NifB和/或NifN。
更进一步地，步骤一中扩增所述固氮酶基因片段时的引物如SEQ ID No.1、SEQ ID No.2、SEQ ID No.3、SEQ ID No.4、SEQ ID No.5、SEQ ID No.6、SEQ ID No.7及SEQ ID No.8所示。
更进一步地，步骤一中所述扩增时的PCR反应体系为：
50ul
10pmol上游引物，
10pmol下游引物，
2ul模板DNA，
4ul 2.5mM dNTP，
5ul 10倍Ex Taq TM缓冲液，
0.5ul TaKaRa Ex Taq TM
PCR反应条件：
94℃5分钟
94℃30秒，
60℃30秒，
72℃2分钟，35个循环，
72℃7分钟。进一步地，步骤二具体为：将NifH/NifE/NifB/NifN基因，分别通过ClaI/SalI，EagI/SalI，EagI/SalI，ClaI/SalI酶切位点连入载体PVX表达载体PVX-HisG-MF1，得到PVX-HisG-MF1-NifH/NifE/NifB/NifN载体。
更进一步地，步骤二中所述PVX-HisG-MF1-NifH、PVX-HisG-MF1-NifE、P VX-HisG-MF1-NifN、PVX-HisG-MF1-NifB载体的序列如SEQ ID No.13、SEQ ID No.14、SEQ ID No.15、SEQ ID No.16所示，克隆得到的NifH、NifE、Ni fB、NifN基因序列如SEQ ID No.9、SEQ ID No.10、SEQ ID No.11、SEQ I D No.12所示。
本发明与现有技术相比，其有益效果为：实现了固氮酶结构基因在植物中的表达：本发明实现了固氮酶结构基因在植物中的表达，从而为植物自主固氮奠定基础。固氮酶结构基因仅存在于原核生物中，而原核基因在真核生物中的表达并不是特别容易的，目前为止，已报道的在植物中成功表达的固氮酶结构基因仅有NifH。原核生物基因分为编码区与非编码区。非编码区上的基因决定某些性状是否表达，表达多少次以及何时开始表达。编码区能转录为相应的信使RNA，进而指导蛋白质的合成。非编码区则相反，但是非编码区对遗传信息的表达是必不可少的，因为在非编码区上有调控遗传信息表达的核苷酸序列。非编码区位于编码区的上游及下游。在调控遗传信息表达的核苷酸序列中最重要的是位于编码区上游的RNA聚合酶结合位点。RNA聚合酶是催化DNA转录为RNA，能识别调控序列中的结合位点并与其结合。
T7启动子，是来自于T7噬菌体的能够对T7RNA聚合酶有特异性反应的强启动子，是启动T7噬菌体基因转录的一段序列。T7RNA聚合酶能选择性的激活T7噬菌体启动子的转录，其合成mRNA的速度比普通大肠杆菌的RNA聚合酶快5倍左右。
植物病毒载体(PVX载体)表达外源蛋白表达水平高、表达速度快、宿主范围广。本发明利用T7启动的PVX载体成功在植物细胞中表达了NifH/NifE/NifB/NifN。
附图说明
图1为Heliobacteriumchlorum中nifH,nifE,nifB,nifN基因的PCR克隆。
1.Marker 1000,2.nifH,3.Marker 1000,4.nifE,5.Marker 2000,6.ni  fB,7.Marker2000,8.nifN。
图2为PVX/HisG-nifH,PVX/HisG-nifE,PVX/HisG-nifB和PVX/HisG-nif N载体的PCR检测。
A1.Marker 2000,A2.nifH阳性对照,A3-8.nifH 1-6单克隆检测.3号克隆测序结果与NCBI上公布的nifH序列同源性达 100％..A9.阴性对照；
B1.Marker 2000,B2.nifE阳性对照,B3-8.nifE1-6单克隆检测.4号克隆测序结果与NCBI上公布的nifE序列同源性达 100％.B9.阴性对照；
C1.Marker 2000,C2.nifB阳性对照,C3-8.nifB 1-6单克隆检测.3号克隆测序结果与NCBI上公布的nifB序列同源性达 100％.C9.阴性对照；
D1.Marker 2000,D2.nifN阳性对照,D3-8.nifN 1-6单克隆检测.3号克隆测序结果与NCBI上公布的nifN序列同源性达 100％.D9.阴性对照；
图3 NIFH/E/N/B蛋白在烟草中表达的Western检测。
A1.蛋白Marker；A2.转染PVX-HisG-MF1-NifH载体的烟草叶片总蛋白；A3.对照烟草叶片总蛋白；B4.NIFH-His Western杂交检测；B5.对照烟草叶片蛋白的Western杂交检测；
C6.转染PVX-HisG-MF1-NifE载体的烟草叶片总蛋白；C7.对照烟草叶片总蛋白；D8.NIFE-His Western杂交检测；D9.对照烟草叶片蛋白的Western杂交检测；
E1.蛋白Marker；E2.对照烟草叶片总蛋白；E3.转染PVX-HisG-MF1-NifB载体的烟草叶片总蛋白；F4.对照烟草叶片蛋白的Western杂交检测；F5.NIFB-His Western杂交检测；
G6.转染PVX-HisG-MF1-NifN载体的烟草叶片总蛋白；G7.对照烟草叶片总蛋白；H8.NIFN-His Western杂交检测；H9.对照烟草叶片蛋白的Western杂交检测。
具体实施方式
下面结合实施例对本发明作进一步的详细描述。
本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。
Heliobacteriumchlorum，厌氧菌，用HELIOBACTERIUM CHLORUM MED IUM 370培养基培养。
HELIOBACTERIUM CHLORUM MEDIUM 370培养基：K2HPO4 1.0g，
MgSO 4x 7H 2O 1.0g，Yeast extract 10.0g，Distilled water 1000.0m l，调节pH7.0。充入氮气，加入sodium ascorbate(0.5g)继续充入氮气。调节pH 6.8.将45ml培养基倒入50ml容量瓶中，用胶塞旋紧。121℃灭菌15分钟。低光照条件培养Heliobacteriumchlorum。
实施例1
钼铁蛋白结构基因NifH/NifE/NifB/NifN的克隆
依据Ausubel et al.(1995)的方法提取Heliobacteriumchlorum基因组DNA。
(Ausubel FM,Brent R,Kingston RE,Moore DD,Seidman JG,Smith J A,Struhl K(1995)Preparation of genomic DNA from bacteria.In:Current protocolsin molecular biology.Wiley,New York)
在NCBI网站National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/)搜索Heliobacteriumchlorum中的NifH/NifE/NifB/NifN的同源序列片段，利用Primer5软件设计引物。引物序列见表一。克隆得到了857/1388/831/1306bp的NifH/NifE/NifB/NifN基因序列，测序结果显示与NCBI网上公布的序列同源性100％。
PCR反应体系：50ul
10pmol上游引物，
10pmol下游引物，
2ul模板DNA，
4ul 2.5mM dNTP，
5ul 10倍Ex Taq TM缓冲液，
0.5ul TaKaRa Ex Taq TM
PCR反应条件：
94℃5分钟
94℃30秒，
60℃30秒，
72℃2分钟，35个循环，
72℃7分钟。
表1 HeliobacteriumchlorumNifH/NifE/NifN/NifB基因引物序列表
引物 序列 SEQ ID No.
NifH N cacggaatcgatcgtcagatagccatttacggaaaaggtg 1
NifH C gtctcagtcgacgattagcccagtttggccatc 2
NifE N acgcgtcggccgtttgtcaatcttaccaaactggacttaaaag 3
NifE C ctgtcagtcgaccgttggtgctgccatataggttgattg 4
NifN N cacggaatcgatactggtccggtagctgaacctcgcag 5
NifN C gactcagtcgacttaagtccttatcccgccatggcgtttaag 6
NifB N acgcgtcggccgggttgcactggttcttgcggaggaaattc 7
NifB C gtctcagtcgacctatatgatgccgatggcgtcagctc 8
实施例2
构建NifH/NifE/NifB/NifN病毒转化载体及转染
NifH/NifE/NifB/NifN基因，分别通过ClaI/SalI，EagI/SalI,EagI/SalI,ClaI/SalI酶切位点连入载体Potato X pot virus(PVX)表达载体PVX-HisG-MF1。PVX-HisG-MF1-NifH/NifE/NifB/NifN载体经测序验证，已成功将基因克隆到表达载体。PVX-HisG-MF1-NifH/NifE/NifB/NifN经体外转录试剂盒作用后，用于烟草Nicotianabenthamiana的转染实验。转染之后，在20-26度，12小时光照，12小时黑暗条件下培养烟草2-3周，筛选具有病毒侵染斑点症状的烟草叶片进行蛋白质SDS-PAGE及WESTERN杂交检测。
实施例3
转基因烟草SDS-PAGE检测及western blot分析
转基因及非转基因烟草叶片总蛋白用植物蛋白提取试剂盒进行提取。得到的转基因及非转基因烟草叶片总蛋白进行SDS-PAGE电泳检测。利用Anti-his antibody(Invitrogen)抗体及horseradish peroxidase-conjugated secondary antib ody(Invitrogen)二抗进行WESTERN杂交检测。结果显示NifH/NifE/NifB/Nif N蛋白均在烟草中获得了成功表达。
本发明中涉及的其他序列如下：
NifH序列(SEQ ID No.9)：
Figure PCTCN2018089745-appb-000001
NifE序列(SEQ ID No.10)：
Figure PCTCN2018089745-appb-000002
Figure PCTCN2018089745-appb-000003
NifB序列(SEQ ID No.11)：
Figure PCTCN2018089745-appb-000004
NifN序列(SEQ ID No.12)：
Figure PCTCN2018089745-appb-000005
Figure PCTCN2018089745-appb-000006
PVX-HisG-MF1-NifH序列(SEQ ID No.13)：
Figure PCTCN2018089745-appb-000007
Figure PCTCN2018089745-appb-000008
Figure PCTCN2018089745-appb-000009
Figure PCTCN2018089745-appb-000010
Figure PCTCN2018089745-appb-000011
PVX-HisG-MF1-NifE序列(SEQ ID No.14)：
Figure PCTCN2018089745-appb-000012
Figure PCTCN2018089745-appb-000013
Figure PCTCN2018089745-appb-000014
Figure PCTCN2018089745-appb-000015
Figure PCTCN2018089745-appb-000016
PVX-HisG-MF1-NifN序列(SEQ ID No.15)：
Figure PCTCN2018089745-appb-000017
Figure PCTCN2018089745-appb-000018
Figure PCTCN2018089745-appb-000019
Figure PCTCN2018089745-appb-000020
Figure PCTCN2018089745-appb-000021
Figure PCTCN2018089745-appb-000022
PVX-HisG-MF1-NifB序列(SEQ ID No.16)：
Figure PCTCN2018089745-appb-000023
Figure PCTCN2018089745-appb-000024
Figure PCTCN2018089745-appb-000025
Figure PCTCN2018089745-appb-000026
Figure PCTCN2018089745-appb-000027
本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下，本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。

Claims (5)

  1. 一种在真核细胞中表达固氮酶基因的方法，其特征在于，包括以下步骤:
    步骤一：扩增所述固氮酶基因片段；
    步骤二，构建重组载体：将所述固氮酶基因片段连接至病毒转化载体；
    步骤三：用步骤二构建的重组载体转染所述真核细胞；
    步骤四：筛选表达所述固氮酶基因的植物细胞。
  2. 根据权利要求1所述的在真核细胞中表达固氮酶基因的方法，其特征在于，步骤一中所述固氮酶基因片段为NifH、NifE、NifB和/或NifN。
  3. 根据权利要求2所述的在真核细胞中表达固氮酶基因的方法，其特征在于，步骤一中扩增所述固氮酶基因片段时的引物如SEQ ID No.1、SEQ ID No.2、SEQ ID No.3、SEQ ID No.4、SEQ ID No.5、SEQ ID No.6、SEQ ID No.7及SEQ ID No.8所示。
  4. 根据权利要求1所述的在真核细胞中表达固氮酶基因的方法，其特征在于，步骤一中所述扩增时的PCR反应体系为：
    50ul
    10pmol上游引物，
    10pmol下游引物，
    2ul模板DNA，
    4ul 2.5mM dNTP，
    5ul 10倍Ex Taq TM缓冲液，
    0.5ul TaKaRa Ex Taq TM
    PCR反应条件：
    94℃ 5分钟
    94℃ 30秒，
    60℃ 30秒，
    72℃ 2分钟，35个循环，
    72℃ 7分钟。5、根据权利要求2所述的在真核细胞中表达固氮酶基因的方法，其特征在于，步骤二具体为：将NifH/NifE/NifB/NifN基因，分别通过ClaI/SalI，EagI/SalI，EagI/SalI，ClaI/SalI酶切位点连入载体PVX表达载体PVX-Hi sG-MF1，得到PVX-HisG-MF1-NifH/NifE/NifB/NifN载体。
  5. 根据权利要求5所述的在真核细胞中表达固氮酶基因的方法，其特征在 于，步骤二中所述PVX-HisG-MF 1-NifH、PVX-HisG-MF 1-NifE、PVX-HisG-MF1-NifN、PVX-HisG-MF1-NifB载体的序列如SEQ ID No.13、SEQ ID No.14、SEQ ID No.15、SEQ ID No.16所示，克隆得到的NifH、NifE、NifB、NifN基因序列如SEQ ID No.9、SEQ ID No.10、SEQ ID No.11、SEQ ID No.12所示。
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