WO2023019693A1 - Artificial non-coding rna module capable of turning off nifl gene expression - Google Patents

Artificial non-coding rna module capable of turning off nifl gene expression Download PDF

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WO2023019693A1
WO2023019693A1 PCT/CN2021/121276 CN2021121276W WO2023019693A1 WO 2023019693 A1 WO2023019693 A1 WO 2023019693A1 CN 2021121276 W CN2021121276 W CN 2021121276W WO 2023019693 A1 WO2023019693 A1 WO 2023019693A1
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artificial non
coding rna
ansr1
nifl
expression
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林敏�
战嵛华
燕永亮
韩月月
柯秀彬
毋少宇
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中国农业科学院生物技术研究所
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/78—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Pseudomonas

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  • the invention relates to the field of biotechnology, in particular to an artificial non-coding RNA module capable of shutting down the expression of nifL, a negative regulation gene for nitrogen fixation.
  • RNA interference (RNAi) technology has developed into the main method for studying expression regulation in eukaryotes, but so far in prokaryotes, the main tools for studying gene expression are still homologous recombination, gene knockout and other technologies.
  • non-coding RNA As a new type of regulatory factor in the bacterial metabolic regulatory network, non-coding RNA has the advantages of rapid response, flexible and precise control, easy recovery, and no metabolic burden. Using the concept of synthetic biology to design artificial non-coding RNA can achieve rapid and high-throughput regulation of gene expression without changing chromosomal genes.
  • artificial non-coding RNA is only used in the metabolic engineering of biological products such as biofuels (butanol, propane, etc.), glutamic acid, and N-acetylglucosamine.
  • NifL is a negative regulator of nitrogen fixation genes. If it is inactivated, it can release its inhibitory effect on the nitrogen fixation positive regulator NifA, thereby affecting the expression of nif genes.
  • Functional artificial non-coding RNA can provide intelligent regulatory elements for the construction of efficient nitrogen fixation system.
  • the purpose of the present invention is to construct artificial non-coding RNA with gene silencing function in the chassis of nitrogen-fixing microorganisms for the study of gene expression regulation.
  • the present invention designs and synthesizes 5 artificial non-coding RNAs (Artificial Nitrogenase activity-Silencing non-coding RNA) with nitrogen-fixing gene silencing function respectively, named respectively as AnsR1, AnsR2, AnsR3, AnsR4, AnsR5, and their nucleotide sequences are respectively Shown as SEQ ID NO:1, NO:2, NO:3, NO:4, NO:5.
  • 5 artificial non-coding RNAs All have the following functional regions and characteristics:
  • the five artificial non-coding RNAs were able to combine with the SD sequence of the target nitrogen fixation regulatory gene nifL mRNA 5'UTR, thereby preventing the mRNA from combining with ribosomal RNA, thereby silencing the expression of the nitrogen fixation gene nifL.
  • the nitrogen-fixing gene silencing function is designed for the nitrogen-fixing gene nifL.
  • NifL can form a complex through the interaction between NifA proteins, leading to the inactivation of the nitrogen-fixing positive regulator NifA, thereby turning off nif Therefore, the construction of artificial non-coding RNA with the function of silencing nifL gene can provide intelligent regulatory elements for the construction of efficient nitrogen fixation system.
  • the present invention also constructs the expression vector pBBR1MCS-AnsR1/2/3/4/5 of the artificial non-coding RNA AnsR1/2/3/4/5, and the expression of the 5 artificial RNA coding sequences is artificially initiated by inducing expression under nitrogen fixation conditions child control.
  • five artificially constructed expression vectors are respectively transformed into nitrogen-fixing Pseudomonas stutzeri A1501 (P. stutzeri A1501), and five nitrogen-fixing recombinant engineering strains A1501 (AnsR1/AnsR2/AnsR3/AnsR4/AnsR5) are respectively obtained.
  • the present invention designs the above-mentioned artificial non-coding RNA through the following specific work, and confirms its function:
  • nifL promoter sequences of Pseudomonas stutzeri, Klebsiella pneumoniae and Azotobacter brownis five genes containing the nifL mRNA 5'UTR of Pseudomonas stutzeri A1501 were synthesized by artificial chemical synthesis
  • the SD sequence binds complementary paired sequences and artificial non-coding RNA AnsR1/2/3/4/5 with different numbers of Hfq binding sites (Table 1).
  • the expression of these five artificial non-coding RNAs is not only controlled by an artificial promoter that specifically responds to nitrogen fixation signals, but also their stability is regulated by the Hfq protein.
  • RNA and ribosomal RNA silences the expression of nitrogen-fixing gene nifL, and its nucleotide sequence is SEQ ID NO: 1, NO: 2, NO: 3, NO: 4, NO: 5.
  • the nitrogenase activity of five nitrogen-fixing engineering strains A1501 was measured by acetylene reduction method. /AnsR3/AnsR4/AnsR5) nitrogenase activities were significantly down-regulated, and the inhibitory effect of the two artificial non-coding RNAs AnsR4/AnsR5 with better stability was the most obvious, and the nitrogenase activities were 38% of the wild-type A1501 nitrogenase activity. and 30%, indicating that the Hfq protein affects the function of the artificial non-coding RNA (Fig. 3).
  • the expression levels of nitrogenase structural gene nifHDK and nitrogenase regulatory gene nifLA were compared between wild-type A1501 and five recombinant strains under nitrogen fixation conditions by qRT-PCR.
  • the results showed that: compared with the wild-type A1501, the expression levels of nifHDK and nifLA mRNA in the recombinant bacteria were lower than those in the wild-type A1501 ( Figure 4), indicating that the artificial non-coding AnsR inhibited the expression of the target gene nifL, and also affected the The expression of nifA in the same transcription unit further affected the expression of nitrogenase gene nifHDK, resulting in a decrease in the nitrogen fixation capacity of A1501.
  • Microscale Thermophoresis (MST) technology was used to analyze the combination of AnsR4, AnsR5 and the SD sequence of nifL mRNA 5'UTR.
  • the thermophoresis fitting curves are all typical "S" curves, indicating that AnsR4, AnsR5 and the SD sequence of nifL mRNA 5'UTR have a good binding tendency ( Figure 5), proving that the artificial non-coding AnsR is indeed By binding to the SD sequence of the target gene nifLmRNA 5'UTR, it prevents the binding of mRNA to ribosomal RNA, thereby silencing the expression of nitrogen-fixing genes.
  • the artificial non-coding RNA AnsR1/2/3/4/5 of the present invention can be stably expressed in chassis microorganisms, and can realize the silencing of the target gene nifL.
  • the artificial non-coding RNA design strategy of the present invention can be applied to the silencing of different genes in the microbial chassis.
  • Figure 1 Construction of artificial non-coding RNA AnsR1/2/3/4/5 expression vector.
  • Figure A shows the schematic diagram of the construction of the artificial non-coding RNA AnsR1/2/3/4/5 expression vector, and the insertion sites are Bam HI and Hind III;
  • Figure B in the figure shows the expression vector pBBR1MCS-AnsR1/2/3/4/ 5 PCR validation.
  • Figure 2 Half-life determination of artificial non-coding RNA AnsR1/2/3/4/5 under nitrogen fixation conditions.
  • Figure 3 Determination of nitrogenase activity of chassis strain A1501 and recombinant engineered strain A1501 (AnsR1/AnsR2/AnsR3/AnsR4/AnsR5).
  • Figure 4 qRT-PCR analysis of the transcription levels of nitrogen fixation-related genes in the recombinant nitrogen fixation engineered strain A1501 (AnsR1/AnsR2/AnsR3/AnsR4/AnsR5) under nitrogen fixation conditions.
  • Figure 5 Determination of the binding ability of artificially encoded RNA AnsR4/AnsR5 to target gene nifL mRNA.
  • SEQ ID NO:1 ⁇ SEQ ID NO:5 are the nucleotide sequences of 5 different artificial non-coding RNA AnsR5 coding genes respectively.
  • RNA AnsR1, AnsR2, AnsR3, AnsR4, and AnsR5 full-length sequences were synthesized by chemical synthesis, and their sizes were 75bp, 88bp, 293bp, 354bp, and 379bp (see Table 1).
  • the broad host plasmid pBBR1MCS was digested with Bam HI and HindIII, and the artificial non-coding RNA AnsR1/2/3/4/5 were respectively connected to the linear vector by the method of seamless cloning to obtain the fusion expression vector pBBR1MCS-AnsR1 /2/3/4/5. And the correct sequence was verified by PCR sequencing.
  • the fusion expression vector pBBR1MCS-AnsR1/2/3/4/5 was transferred into A1501 by the method of triparental combination, and the helper plasmid pRK2013 and A1501 were needed as recipient bacteria in this process, and pBBR1MCS-AnsR1/2/3/4 /5 as the donor bacteria, the detailed steps are as follows:
  • the single colony successfully verified by colony PCR is Pseudomonas stutzeri A1501 (AnsR1/AnsR2/AnsR3/AnsR4/AnsR5).
  • Example 3 Analysis of artificial non-coding RNA expression in recombinant engineering bacteria A1501 (AnsR1/AnsR2/AnsR3/AnsR4/AnsR5) under nitrogen fixation conditions
  • the copy number concentrations ( ⁇ 10 4 copies/ng) of artificial noncoding RNAs AnsR1, AnsR2, AnsR3, AnsR4, AnsR5 in total RNA per ng sample under nitrogen fixation conditions were 3.57 ⁇ 0.18, 8.32 ⁇ 0.63, 5.33 ⁇ 0.36, 3.81, respectively ⁇ 0.36, 3.54 ⁇ 0.47, 1.18 ⁇ 0.17 (see Table 2).
  • the artificial non-coding RNA AnsR1/2/3/4/5 could be stably expressed in the chassis microorganism A1501 under nitrogen fixation conditions.
  • Example 4 Determination of half-life of artificial non-coding RNA in recombinant engineering bacteria A1501 (AnsR1/AnsR2/AnsR3/AnsR4/AnsR5) under nitrogen fixation conditions
  • Inoculation Pick a single colony of recombinant bacteria A1501 (AnsR1/AnsR2/AnsR3/AnsR4/AnsR5) and inoculate them into LB liquid medium containing Km resistance, culture overnight at 30°C on a shaker at 200 rpm.
  • A1501 (AnsR1/AnsR2/AnsR3) was treated with rifampicin for 0, 7, 10, 13 and 16 minutes, 1 mL of the bacterial solution was drawn into a 1.5 mL EP tube, and the bacterial cells were collected by centrifugation at 12000 rpm for 2 minutes; A1501 ( AnsR4/AnsR5) were treated with rifampicin for 0, 10, 15, and 20 minutes respectively, and 1 mL of the bacterial liquid was drawn into a 1.5 mL EP tube, and the bacterial cells were collected by centrifugation at 12,000 rpm for 2 minutes.
  • RNAlater (2 times the volume of rifampicin) to the cells from which the supernatant was removed, suspend the above cells, treat at room temperature for 5 min, then centrifuge rapidly at 12,000 rpm for 2 min, remove the supernatant, and freeze in liquid nitrogen. Store at -80°C for later use.
  • the stability of artificial non-coding RNA under nitrogen fixation is regulated by Hfq protein, and the artificial non-coding RNA with more binding sites of Hfq protein is more stable.
  • nitrogenase activity ethylene peak Calculate the nitrogenase activity of the recombinant bacteria by area ⁇ (total gas phase volume of the flask/sampling volume)/(1 nmol ethylene standard peak area ⁇ reaction time ⁇ total protein total amount of bacteria).
  • A1501 Compared with the wild-type A1501, the nitrogenase activity of the recombinant strain A1501 (AnsR) decreased.
  • the nitrogenase activity of A1501 (AnsR1), A1501 (AnsR2), A1501 (AnsR3), A1501 (AnsR4), and A1501 (AnsR5) were 63%, 52%, 51%, 38%, and 30% of wild-type A1501, respectively ( image 3).
  • the induced expression of artificial non-coding RNA 1/2/3/4/5 under nitrogen fixation conditions can reduce the nitrogen fixation ability of chassis microorganism A1501.
  • qRT-PCR was used to detect the expression levels of nitrogen fixation-related genes nifL, nifA, nifH, nifD, and nifK in recombinant bacteria A1501 (AnsR1/AnsR2/AnsR3/AnsR4/AnsR5)
  • Hai Biological Engineering Co., Ltd. synthesized 20 bp of the sequence complementary to the artificial non-coding RNA in nifL mRNA (including the SD sequence of nifL mRNA), and carried out 5'FAM fluorescent labeling as a probe; the full-length was obtained by in vitro transcription 354bp and 379bp artificial non-coding RNA AnsR4, AnsR5 coding sequence, as a ligand.
  • Kd [A]*[L]/[AL], where [A] is the concentration of free fluorescent molecules, [L] is the concentration of free ligands, and [AL] is the concentration of A and L complexes.
  • microthermophoresis fitting curves between the artificial non-coding RNAs AnsR4, AnsR5 and nifL mRNA are all typical "S" curves, indicating that there is a good binding tendency between the artificial non-coding RNAs AnsR4, AnsR5 and nifL mRNA ( Figure 5).
  • the artificial non-coding RNAs AnsR4 and AnsR5 can respectively interact with the target gene nifL mRNA through complementary base pairing and silence its expression.

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Abstract

The present invention designs and constructs five artificial non-coding RNAs, respectively. The sequence of each artificial non-coding RNA has a complementary pairing region with a target mRNA, a binding site of an Hfq protein, and a Rho independent transcription terminator. The artificial non-coding RNAs can all be combined with an SD sequence of a target nitrogen fixation regulatory gene nifL mRNA 5'UTR, and can respectively prevent the mRNA from being combined with a ribosome RNA, so that the expression of a nitrogen fixation gene negative regulatory factor nifL is silenced.

Description

可关闭nifL基因表达的人工非编码RNA模块An artificial non-coding RNA module that shuts down the expression of the nifL gene 技术领域：Technical field:
本发明涉及生物技术领域，具体涉及一种可关闭固氮负调节基因nifL表达的人工非编码RNA模块。The invention relates to the field of biotechnology, in particular to an artificial non-coding RNA module capable of shutting down the expression of nifL, a negative regulation gene for nitrogen fixation.
背景技术：Background technique:
RNA干扰(RNAi)技术已经发展成为真核生物中研究表达调控的主要手段，但是迄今为止在原核生物中，研究基因表达的主要工具仍然是同源重组、基因敲除等技术。RNA interference (RNAi) technology has developed into the main method for studying expression regulation in eukaryotes, but so far in prokaryotes, the main tools for studying gene expression are still homologous recombination, gene knockout and other technologies.
非编码RNA作为细菌代谢调控网络中的一类新型调控因子，具有反应迅速、控制灵活精确、恢复容易、没有代谢负担等优点。利用合成生物学理念，设计人工非编码RNA，可以在不改变染色体基因的前提下，实现迅速、高通量的基因表达调控。As a new type of regulatory factor in the bacterial metabolic regulatory network, non-coding RNA has the advantages of rapid response, flexible and precise control, easy recovery, and no metabolic burden. Using the concept of synthetic biology to design artificial non-coding RNA can achieve rapid and high-throughput regulation of gene expression without changing chromosomal genes.
但目前人工非编码RNA仅在生物燃料(丁醇、丙烷等)、谷氨酸、N-乙酰氨基葡萄糖等生物制品的代谢工程中应用。However, at present, artificial non-coding RNA is only used in the metabolic engineering of biological products such as biofuels (butanol, propane, etc.), glutamic acid, and N-acetylglucosamine.
在许多领域人工非编码RNA有许多可应用的空间。比如，在生物固氮领域，已知NifL是固氮基因的负调节因子，如果使其失活,可以解除其对固氮正调节因子NifA的抑制作用，从而影响nif基因的表达，因此构建具有沉默nifL基因功能的人工非编码RNA，可以为高效固氮体系的构建提供智能化的调控元件。Artificial non-coding RNA has many applications in many fields. For example, in the field of biological nitrogen fixation, it is known that NifL is a negative regulator of nitrogen fixation genes. If it is inactivated, it can release its inhibitory effect on the nitrogen fixation positive regulator NifA, thereby affecting the expression of nif genes. Functional artificial non-coding RNA can provide intelligent regulatory elements for the construction of efficient nitrogen fixation system.
发明内容Contents of the invention
本发明的目的是在固氮微生物底盘中构建具有基因沉默功能的人工非编码RNA，用于基因表达调控研究。The purpose of the present invention is to construct artificial non-coding RNA with gene silencing function in the chassis of nitrogen-fixing microorganisms for the study of gene expression regulation.
本发明分别设计合成了5个具有固氮基因沉默功能的人工非编码RNA(Artificial Nitrogenase activity-Silencing non-coding RNA)，分别命名为AnsR1、AnsR2、AnsR3、AnsR4、AnsR5，它们的核苷酸序列分别如SEQ ID NO：1，NO：2，NO：3，NO：4，NO：5所示。这5个人工非编码RNA均具有以下功能区与特征：The present invention designs and synthesizes 5 artificial non-coding RNAs (Artificial Nitrogenase activity-Silencing non-coding RNA) with nitrogen-fixing gene silencing function respectively, named respectively as AnsR1, AnsR2, AnsR3, AnsR4, AnsR5, and their nucleotide sequences are respectively Shown as SEQ ID NO:1, NO:2, NO:3, NO:4, NO:5. These five artificial non-coding RNAs all have the following functional regions and characteristics:
(1)具有与固氮酶调节基因nifL mRNA 5'UTR的SD序列互补配对区；(1) It has a complementary pairing region with the SD sequence of the nitrogenase regulatory gene nifL mRNA 5'UTR;
(2)含有Hfq蛋白结合位点(5个人工非编码RNA编码序列含有的Hfq蛋白结合位 点数量不同，其中SEQ ID NO：5所示的人工非编码RNA含的有Hfq结合位点数量最多)；(2) Containing Hfq protein binding sites (the number of Hfq protein binding sites contained in the five artificial non-coding RNA coding sequences is different, and the artificial non-coding RNA shown in SEQ ID NO: 5 contains the largest number of Hfq binding sites );
(3)不依赖于Rho因子的转录终止子。(3) Transcription terminator independent of Rho factor.
本发明构建的人工非编码RNA发挥基因沉默功能的原理是：The principle that the artificial non-coding RNA constructed in the present invention exerts the function of gene silencing is:
5个人工非编码RNA分别能够与靶标固氮调节基因nifL mRNA 5'UTR的SD序列结合，因而阻止该mRNA与核糖体RNA的结合，进而沉默了固氮基因nifL的表达。The five artificial non-coding RNAs were able to combine with the SD sequence of the target nitrogen fixation regulatory gene nifL mRNA 5'UTR, thereby preventing the mRNA from combining with ribosomal RNA, thereby silencing the expression of the nitrogen fixation gene nifL.
所述固氮基因沉默功能是针对固氮基因nifL设计的，NifL作为固氮基因的负调节因子,可通过NifA蛋白之间的相互作用,形成复合物,导致固氮正调节因子NifA的失活,从而关闭nif基因的表达，因此构建具有沉默nifL基因功能的人工非编码RNA可以为高效固氮体系的构建提供智能化的调控元件。The nitrogen-fixing gene silencing function is designed for the nitrogen-fixing gene nifL. As a negative regulator of the nitrogen-fixing gene, NifL can form a complex through the interaction between NifA proteins, leading to the inactivation of the nitrogen-fixing positive regulator NifA, thereby turning off nif Therefore, the construction of artificial non-coding RNA with the function of silencing nifL gene can provide intelligent regulatory elements for the construction of efficient nitrogen fixation system.
本发明还构建了人工非编码RNA AnsR1/2/3/4/5的表达载体pBBR1MCS-AnsR1/2/3/4/5，5个人工RNA编码序列的表达受固氮条件下诱导表达的人工启动子控制。The present invention also constructs the expression vector pBBR1MCS-AnsR1/2/3/4/5 of the artificial non-coding RNA AnsR1/2/3/4/5, and the expression of the 5 artificial RNA coding sequences is artificially initiated by inducing expression under nitrogen fixation conditions child control.
本发明将人工构建的5个表达载体分别转入固氮施氏假单胞菌A1501(P.stutzeri A1501)，分别获得5个固氮重组工程菌株A1501(AnsR1/AnsR2/AnsR3/AnsR4/AnsR5)。In the present invention, five artificially constructed expression vectors are respectively transformed into nitrogen-fixing Pseudomonas stutzeri A1501 (P. stutzeri A1501), and five nitrogen-fixing recombinant engineering strains A1501 (AnsR1/AnsR2/AnsR3/AnsR4/AnsR5) are respectively obtained.
实验证实，在固氮条件下本发明的5个人工非编码RNA AnsR1/2/3/4/5均能够沉默靶标基因nifL mRNA的表达，并且含有Hfq结合位点数量最多的AnsR5的抑制能力最强，表明Hfq蛋白通过影响人工非编码RNA的稳定性进而参与固氮基因nifL的沉默。Experiments have confirmed that the five artificial non-coding RNAs AnsR1/2/3/4/5 of the present invention can all silence the expression of the target gene nifL mRNA under nitrogen fixation conditions, and AnsR5, which contains the largest number of Hfq binding sites, has the strongest inhibitory ability , indicating that Hfq protein participates in the silencing of nitrogen fixation gene nifL by affecting the stability of artificial non-coding RNA.
本发明是通过以下具体工作设计上述人工非编码RNA，并证实其功能：The present invention designs the above-mentioned artificial non-coding RNA through the following specific work, and confirms its function:
1.人工非编码RNA AnsR1/2/3/4/5的设计与合成1. Design and synthesis of artificial non-coding RNA AnsR1/2/3/4/5
通过固氮施氏假单胞菌、肺炎克氏杆菌以及棕色固氮菌nifL启动子序列的生物信息学分析，用人工化学合成的方法合成了5个含有与施氏假单胞菌A1501 nifL mRNA5'UTR的SD序列结合互补配对的序列和不同数量Hfq结合位点的人工非编码RNA AnsR1/2/3/4/5(表1)。这5个人工非编码RNA的表达不但受一个由特异响应固氮信号的人工启动子控制，而且稳定性受Hfq蛋白的调控，它们通过与靶标固氮调节基因nifL mRNA 5'UTR的SD序列结合，阻止mRNA与核糖体RNA的结合，进而沉默了固氮基因nifL的表达，其核苷酸序列是SEQ ID NO：1，NO：2，NO：3，NO：4，NO：5。Through the bioinformatics analysis of the nifL promoter sequences of Pseudomonas stutzeri, Klebsiella pneumoniae and Azotobacter brownis, five genes containing the nifL mRNA 5'UTR of Pseudomonas stutzeri A1501 were synthesized by artificial chemical synthesis The SD sequence binds complementary paired sequences and artificial non-coding RNA AnsR1/2/3/4/5 with different numbers of Hfq binding sites (Table 1). The expression of these five artificial non-coding RNAs is not only controlled by an artificial promoter that specifically responds to nitrogen fixation signals, but also their stability is regulated by the Hfq protein. They bind to the SD sequence of the target nitrogen fixation regulatory gene nifL mRNA 5'UTR to prevent The combination of mRNA and ribosomal RNA silences the expression of nitrogen-fixing gene nifL, and its nucleotide sequence is SEQ ID NO: 1, NO: 2, NO: 3, NO: 4, NO: 5.
2.构建人工非编码RNA的融合表达载体，将其转入固氮施氏假单胞菌中，获得5株固氮重组工程菌株2. Construct the fusion expression vector of artificial non-coding RNA, transfer it into nitrogen-fixing Pseudomonas stutzeri, and obtain 5 strains of nitrogen-fixing recombinant engineering strains
(1)将广宿主表达载体pBBR1MCS进行Bam HI和Hind III双酶切，通过无缝克隆技术将人工合成的非编码RNA AnsR1/2/3/4/5片段分别插入其多克隆位点处，获得本发明 人工非编码RNA的融合表达载体pBBR1MCS-AnsR1/2/3/4/5(图1)。(1) Carry out Bam HI and Hind III double digestion of the broad host expression vector pBBR1MCS, and insert artificially synthesized non-coding RNA AnsR1/2/3/4/5 fragments into its multiple cloning sites through seamless cloning technology, The fusion expression vector pBBR1MCS-AnsR1/2/3/4/5 of the artificial non-coding RNA of the present invention was obtained ( FIG. 1 ).
(2)将融合表达载体pBBR1MCS-AnsR1/2/3/4/5分别转入微生物底盘固氮施氏假单胞菌A1501中，获得重组工程菌株A1501(AnsR1/AnsR2/AnsR3/AnsR4/AnsR5)。(2) Transform the fusion expression vector pBBR1MCS-AnsR1/2/3/4/5 into the microbial chassis nitrogen-fixing Pseudomonas stutzeri A1501 to obtain the recombinant engineering strain A1501 (AnsR1/AnsR2/AnsR3/AnsR4/AnsR5).
3.人工非编码RNA的功能分析3. Functional analysis of artificial non-coding RNA
(1)人工非编码RNA的表达分析(1) Expression analysis of artificial non-coding RNA
我们通过数字PCR检测了固氮条件下人工非编码RNA AnsR1/2/3/4/5在A1501中的表达量。结果显示固氮条件下人工非编码RNA AnsR1/2/3/4/5均能在A1501中稳定表达，在固氮条件下每ng总样品RNA中人工非编码RNA AnsR1、AnsR2、AnsR3、AnsR4、AnsR5的拷贝数浓度(×10 4copies/ng)分别为3.57±0.18、8.32±0.63、5.33±0.36、3.81±0.36、3.54±0.47、1.18±0.17(表2)。 We detected the expression levels of artificial non-coding RNA AnsR1/2/3/4/5 in A1501 under nitrogen fixation conditions by digital PCR. The results showed that the artificial non-coding RNA AnsR1/2/3/4/5 could be stably expressed in A1501 under nitrogen-fixing conditions. The copy number concentrations (×10 4 copies/ng) were 3.57±0.18, 8.32±0.63, 5.33±0.36, 3.81±0.36, 3.54±0.47, 1.18±0.17, respectively (Table 2).
(2)人工非编码RNA的稳定性分析(2) Stability analysis of artificial non-coding RNA
我们通过qRT-PCR检测了固氮条件下人工非编码AnsR 1/2/3/4/5在A1501中的半衰期。结果显示固氮条件下人工非编码RNA AnsR1/2/3/4/5在A1501中的稳定性受Hfq蛋白调控，固氮条件下人工非编码RNA AnsR1、AnsR2、AnsR3、AnsR4、AnsR5的半衰期(min)分别为16、17、16、20、18(图2)。We examined the half-life of artificial non-coding AnsR 1/2/3/4/5 in A1501 under nitrogen fixation conditions by qRT-PCR. The results show that the stability of artificial non-coding RNA AnsR1/2/3/4/5 in A1501 under nitrogen fixation is regulated by Hfq protein, and the half-lives of artificial non-coding RNA AnsR1, AnsR2, AnsR3, AnsR4, AnsR5 under nitrogen fixation (min) 16, 17, 16, 20, 18, respectively (Figure 2).
(3)重组固氮工程菌的固氮酶活性测定(3) Determination of nitrogenase activity of recombinant nitrogen-fixing engineering bacteria
利用乙炔还原法测定了固氮条件下5株固氮工程菌株A1501(AnsR1/AnsR2/AnsR3/AnsR4/AnsR5)的固氮酶活性，结果显示：与野生型A1501相比，5株工程菌A1501(AnsR1/AnsR2/AnsR3/AnsR4/AnsR5)的固氮酶活性均显著下调，其中稳定性较好的两个人工非编码RNA AnsR4/AnsR5的抑制效果最为明显，固氮酶活性分别是野生型A1501固氮酶活的38％和30％，表明Hfq蛋白影响了人工非编码RNA功能的发挥(图3)。The nitrogenase activity of five nitrogen-fixing engineering strains A1501 (AnsR1/AnsR2/AnsR3/AnsR4/AnsR5) was measured by acetylene reduction method. /AnsR3/AnsR4/AnsR5) nitrogenase activities were significantly down-regulated, and the inhibitory effect of the two artificial non-coding RNAs AnsR4/AnsR5 with better stability was the most obvious, and the nitrogenase activities were 38% of the wild-type A1501 nitrogenase activity. and 30%, indicating that the Hfq protein affects the function of the artificial non-coding RNA (Fig. 3).
(4)重组固氮工程菌中固氮相关基因的表达量分析(4) Analysis of expression levels of nitrogen fixation-related genes in recombinant nitrogen-fixing engineering bacteria
利用qRT-PCR技术比较了固氮条件下野生型A1501和5株重组菌中固氮酶结构基因nifHDK，固氮酶调节基因nifLA的表达量。结果显示：与野生型A1501相比，重组菌中nifHDK，nifLA mRNA的表达量均比野生型A1501中的低(图4)，说明人工非编码AnsR通过抑制靶标基因nifL的表达，同时也影响了同一个转录单元的nifA的表达，进一步影响了固氮酶基因nifHDK的表达，从而导致了A1501固氮能力下降。The expression levels of nitrogenase structural gene nifHDK and nitrogenase regulatory gene nifLA were compared between wild-type A1501 and five recombinant strains under nitrogen fixation conditions by qRT-PCR. The results showed that: compared with the wild-type A1501, the expression levels of nifHDK and nifLA mRNA in the recombinant bacteria were lower than those in the wild-type A1501 (Figure 4), indicating that the artificial non-coding AnsR inhibited the expression of the target gene nifL, and also affected the The expression of nifA in the same transcription unit further affected the expression of nitrogenase gene nifHDK, resulting in a decrease in the nitrogen fixation capacity of A1501.
4.人工非编码RNA与靶标基因nifL mRNA结合能力的检测4. Detection of binding ability between artificial non-coding RNA and target gene nifL mRNA
利用微量热泳动(Microscale Thermophoresis,MST)技术分析AnsR4、AnsR5与nifL mRNA 5'UTR的SD序列之间的结合情况，结果显示：AnsR4、AnsR5与nifL mRNA 5'UTR 的SD序列之间的微量热泳动拟合曲线均为典型的“S”型曲线，说明AnsR4，AnsR5与nifL mRNA 5'UTR的SD序列之间均有很好的结合趋势(图5)，证明人工非编码AnsR确实是通过与靶标基因nifLmRNA 5'UTR的SD序列结合，阻止mRNA与核糖体RNA的结合，进而沉默了固氮基因的表达。Microscale Thermophoresis (MST) technology was used to analyze the combination of AnsR4, AnsR5 and the SD sequence of nifL mRNA 5'UTR. The thermophoresis fitting curves are all typical "S" curves, indicating that AnsR4, AnsR5 and the SD sequence of nifL mRNA 5'UTR have a good binding tendency (Figure 5), proving that the artificial non-coding AnsR is indeed By binding to the SD sequence of the target gene nifLmRNA 5'UTR, it prevents the binding of mRNA to ribosomal RNA, thereby silencing the expression of nitrogen-fixing genes.
本发明的有益成果：Beneficial results of the present invention:
实验证实，在固氮条件下，本发明的人工非编码RNA AnsR1/2/3/4/5能够在底盘微生物中稳定表达，并且能够实现靶标基因nifL的沉默。Experiments have confirmed that under nitrogen fixation conditions, the artificial non-coding RNA AnsR1/2/3/4/5 of the present invention can be stably expressed in chassis microorganisms, and can realize the silencing of the target gene nifL.
本发明的人工非编码RNA设计策略，可以应用于微生物底盘中不同基因的沉默。The artificial non-coding RNA design strategy of the present invention can be applied to the silencing of different genes in the microbial chassis.
附图说明Description of drawings
图1：人工非编码RNA AnsR1/2/3/4/5表达载体的构建。其中A图表示人工非编码RNA AnsR1/2/3/4/5表达载体的构建示意图，插入位点为Bam HI和Hind Ⅲ；图中B图表示表达载体pBBR1MCS-AnsR1/2/3/4/5的PCR验证。Figure 1: Construction of artificial non-coding RNA AnsR1/2/3/4/5 expression vector. Figure A shows the schematic diagram of the construction of the artificial non-coding RNA AnsR1/2/3/4/5 expression vector, and the insertion sites are Bam HI and Hind III; Figure B in the figure shows the expression vector pBBR1MCS-AnsR1/2/3/4/ 5 PCR validation.
图2：固氮条件下人工非编码RNA AnsR1/2/3/4/5的半衰期测定。Figure 2: Half-life determination of artificial non-coding RNA AnsR1/2/3/4/5 under nitrogen fixation conditions.
图3：底盘菌株A1501和重组工程菌株A1501(AnsR1/AnsR2/AnsR3/AnsR4/AnsR5)的固氮酶活性测定。Figure 3: Determination of nitrogenase activity of chassis strain A1501 and recombinant engineered strain A1501 (AnsR1/AnsR2/AnsR3/AnsR4/AnsR5).
图4：qRT-PCR分析固氮条件下重组固氮工程菌株A1501(AnsR1/AnsR2/AnsR3/AnsR4/AnsR5)中固氮相关基因的转录水平。Figure 4: qRT-PCR analysis of the transcription levels of nitrogen fixation-related genes in the recombinant nitrogen fixation engineered strain A1501 (AnsR1/AnsR2/AnsR3/AnsR4/AnsR5) under nitrogen fixation conditions.
图5：人工编码RNA AnsR4/AnsR5与靶标基因nifL mRNA结合能力测定。Figure 5: Determination of the binding ability of artificially encoded RNA AnsR4/AnsR5 to target gene nifL mRNA.
序列信息sequence information
SEQ ID NO:1～SEQ ID NO:5分别是5个不同的人工非编码RNA AnsR5编码基因的核苷酸序列。SEQ ID NO:1～SEQ ID NO:5 are the nucleotide sequences of 5 different artificial non-coding RNA AnsR5 coding genes respectively.
具体实施方式Detailed ways
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于举例说明本发明的方法，而不用于限制本发明的范围。凡未注明具体实验条件的，均为按照本领域技术人员熟知的常规条件，例如Sambrook等分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the method of the present invention, and are not intended to limit the scope of the present invention. Where the specific experimental conditions are not indicated, all are according to the conventional conditions well known to those skilled in the art, such as molecular cloning such as Sambrook: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to Conditions recommended by the manufacturer.
实施例1 构建融合表达载体pBBR1MCS-AnsR1/2/3/4/5Example 1 Construction of fusion expression vector pBBR1MCS-AnsR1/2/3/4/5
(一)实验方法(1) Experimental method
1.通过化学合成的方法将设计的人工非编码RNA AnsR1、AnsR2、AnsR3、AnsR4、 AnsR5全长序列合成，他们的大小分别为75bp，88bp，293bp，354bp，379bp(见表1)。1. The artificial non-coding RNA AnsR1, AnsR2, AnsR3, AnsR4, and AnsR5 full-length sequences were synthesized by chemical synthesis, and their sizes were 75bp, 88bp, 293bp, 354bp, and 379bp (see Table 1).
2.将广宿主质粒pBBR1MCS进行Bam HI和Hin dIII双酶切，通过无缝克隆的方法将人工非编码RNA AnsR1/2/3/4/5分别连接至线性载体上获得融合表达载体pBBR1MCS-AnsR1/2/3/4/5。并且通过PCR测序验证序列正确。2. The broad host plasmid pBBR1MCS was digested with Bam HI and HindIII, and the artificial non-coding RNA AnsR1/2/3/4/5 were respectively connected to the linear vector by the method of seamless cloning to obtain the fusion expression vector pBBR1MCS-AnsR1 /2/3/4/5. And the correct sequence was verified by PCR sequencing.
(二)实验结果(2) Experimental results
利用人工化学合成的方法获得了人工非编码RNA AnsR1、AnsR2、AnsR3、AnsR4、AnsR5的全长核酸序列，成功构建了表达人工非编码RNA的融合表达载体pBBR1MCS-AnsR1/2/3/4/5，经PCR测序验证融合表达载体中人工非编码RNA AnsR1/2/3/4/5的序列正确(图1)。The full-length nucleic acid sequences of artificial non-coding RNA AnsR1, AnsR2, AnsR3, AnsR4, AnsR5 were obtained by artificial chemical synthesis, and the fusion expression vector pBBR1MCS-AnsR1/2/3/4/5 expressing artificial non-coding RNA was successfully constructed , the sequence of the artificial non-coding RNA AnsR1/2/3/4/5 in the fusion expression vector was verified by PCR sequencing to be correct (Figure 1).
(三)实验结论(3) Experimental conclusion
完成了人工非编码RNA的融合表达载体pBBR1MCS-AnsR1/2/3/4/5的构建。The construction of the artificial non-coding RNA fusion expression vector pBBR1MCS-AnsR1/2/3/4/5 was completed.
表1 人工非编码RNA的信息Table 1 Information of artificial non-coding RNA
Figure PCTCN2021121276-appb-000001
Figure PCTCN2021121276-appb-000001
实施例2 重组工程菌株A1501(AnsR1/AnsR2/AnsR3/AnsR4/AnsR5)的构建Example 2 Construction of recombinant engineering strain A1501 (AnsR1/AnsR2/AnsR3/AnsR4/AnsR5)
(一)实验方法(1) Experimental method
通过三亲结合的方法将融合表达载体pBBR1MCS-AnsR1/2/3/4/5分别转入A1501，在此过程中需要辅助质粒pRK2013，A1501作为受体菌，pBBR1MCS-AnsR1/2/3/4/5作为供体菌，详细步骤如下：The fusion expression vector pBBR1MCS-AnsR1/2/3/4/5 was transferred into A1501 by the method of triparental combination, and the helper plasmid pRK2013 and A1501 were needed as recipient bacteria in this process, and pBBR1MCS-AnsR1/2/3/4 /5 as the donor bacteria, the detailed steps are as follows:
1.分别挑取平板上pBBR1MCS-AnsR1/2/3/4/5，A1501，pRK2013菌株的单菌落接种到加有相应抗性的LB液体培养基中，摇床过夜培养。A1501：无抗性，30℃，200rpm；pBBR1MCS-AnsR1/2/3/4/5，pRK2013：Km抗性，37℃，220rpm。1. Pick single colonies of pBBR1MCS-AnsR1/2/3/4/5, A1501, and pRK2013 strains on the plate and inoculate them into LB liquid medium with corresponding resistance, and culture overnight on a shaking table. A1501: no resistance, 30°C, 200rpm; pBBR1MCS-AnsR1/2/3/4/5, pRK2013: Km resistance, 37°C, 220rpm.
2.将过夜培养好的菌液分别按2％的接菌量再次转接至新鲜的无抗LB液体培养基中，继续在合适温度下的摇床中培养，直至OD 600＝0.6或0.8 2. Retransfer the overnight cultured bacterial solution into fresh anti-antibiotic-free LB liquid medium according to 2% inoculum amount, and continue to cultivate in a shaker at a suitable temperature until OD 600 =0.6 or 0.8
3.将上步培养好的菌液按照一定比例(受体菌1mL：供体菌2mL：助质粒600μL)加到1.5mL离心管中，5500rpm，4℃离心10min。3. Add the bacteria solution cultivated in the previous step into a 1.5mL centrifuge tube according to a certain ratio (1mL of recipient bacteria: 2mL of donor bacteria: 600μL of helper plasmid), centrifuge at 5500rpm, 4°C for 10min.
4.倒掉上清，分别用1mL 0.85％的生理盐水重悬菌体，5500rpm，4℃离心10min。4. Pour off the supernatant, resuspend the bacteria with 1mL 0.85% normal saline, centrifuge at 5500rpm, 4°C for 10min.
5.倒掉上清，取1ml 0.85％的生理盐水将受体菌，供体菌，助质粒菌重悬混在一起，5500rpm，4℃离心10min。5. Pour off the supernatant, take 1ml of 0.85% normal saline to resuspend the recipient bacteria, donor bacteria, and helper plasmid bacteria, and centrifuge at 5500rpm at 4°C for 10min.
6.倒掉上清，留下少量液体混匀菌体，每次吸取20μL点在无抗性的LB平板上，30℃培养箱，正置培养4-5d。6. Pour off the supernatant, leave a small amount of liquid to mix the cells, pipette 20 μL each time and spot on a non-resistant LB plate, in a 30°C incubator, and culture it upright for 4-5 days.
7.用移液枪轻轻将上步培养好的菌落刮下，用1mL 0.85％的生理盐水重悬，蘸取菌液在含有Km抗性(50μg/mL)和Cm抗性(17μg/mL)的LB固体培养基上划线，30℃培养箱培养。7. Gently scrape off the colonies cultivated in the previous step with a pipette gun, resuspend them with 1mL 0.85% normal saline, and dip the bacterial solution in a medium containing Km resistance (50μg/mL) and Cm resistance (17μg/mL) ) on the LB solid medium, cultured in a 30°C incubator.
8.待上步平板上培养出单菌落后，挑取单菌落进行菌落PCR，分别用质粒pBBR1MCS的通用引物M13和受体菌A1501中nifH基因的特异引物验证连接有人工非编码RNA序列的融合表达载体pBBR1MCS-AnsR1/2/3/4/5是否成功转化到A1501野生型菌中。8. After a single colony was cultured on the plate in the previous step, pick a single colony for colony PCR, and use the general primer M13 of the plasmid pBBR1MCS and the specific primer of the nifH gene in the recipient strain A1501 to verify the fusion of the artificial non-coding RNA sequence Whether the expression vector pBBR1MCS-AnsR1/2/3/4/5 was successfully transformed into A1501 wild-type bacteria.
9.菌落PCR验证成功的单菌落即为施氏假单胞菌A1501(AnsR1/AnsR2/AnsR3/AnsR4/AnsR5)。9. The single colony successfully verified by colony PCR is Pseudomonas stutzeri A1501 (AnsR1/AnsR2/AnsR3/AnsR4/AnsR5).
(二)实验结果(2) Experimental results
重组菌株A1501(AnsR1/AnsR2/AnsR3/AnsR4/AnsR5)的抗性筛选和PCR验证结果正确，成功将融合表达载体pBBR1MCS-AnsR1/2/3/4/5转入了A1501(图1)。The results of resistance screening and PCR verification of the recombinant strain A1501 (AnsR1/AnsR2/AnsR3/AnsR4/AnsR5) were correct, and the fusion expression vector pBBR1MCS-AnsR1/2/3/4/5 was successfully transferred into A1501 (Figure 1).
(三)实验结论(3) Experimental conclusion
完成了重组工程菌A1501(AnsR1/AnsR2/AnsR3/AnsR4/AnsR5)的构建。The construction of recombinant engineering bacteria A1501 (AnsR1/AnsR2/AnsR3/AnsR4/AnsR5) was completed.
实施例3 固氮条件下重组工程菌A1501(AnsR1/AnsR2/AnsR3/AnsR4/AnsR5)中人工非编码RNA表达分析Example 3 Analysis of artificial non-coding RNA expression in recombinant engineering bacteria A1501 (AnsR1/AnsR2/AnsR3/AnsR4/AnsR5) under nitrogen fixation conditions
(一)实验方法(1) Experimental method
1.固氮条件下收集重组菌的菌体1. Collect the cells of the recombinant bacteria under the condition of nitrogen fixation
(1)分别挑取重组菌A1501(AnsR1/AnsR2/AnsR3/AnsR4/AnsR5)的单菌落接种至含有Km抗性的LB液体培养基中，30℃，200rpm摇床过夜培养。(1) Single colonies of recombinant bacteria A1501 (AnsR1/AnsR2/AnsR3/AnsR4/AnsR5) were picked and inoculated into LB liquid medium containing Km resistance, cultured overnight at 30°C on a shaker at 200 rpm.
(2)将过夜培养好的菌液分装至50ml离心管中，5500rpm，4℃离心十分钟，倒掉上清后用0.85％的生理盐水重悬菌体清洗两次，然后用K无氮的培养基重悬菌体调OD 600＝1。 (2) Divide the overnight cultured bacterial solution into 50ml centrifuge tubes, centrifuge at 5500rpm, 4°C for ten minutes, pour off the supernatant and wash twice with 0.85% normal saline to resuspend the bacterial cells, and then use K nitrogen-free The culture medium resuspended the bacteria to adjust OD 600 =1.
(3)向测固氮酶活的小瓶中分别加入9ml的K无氮培养基，再加入1ml OD 600＝1 的菌液。使得固氮酶活小瓶中菌液的起始OD 600＝0.1。 (3) Add 9 ml of K nitrogen-free medium to the vials for measuring nitrogenase activity, and then add 1 ml of bacterial solution with OD 600 =1. Make the initial OD 600 of the bacterial solution in the vial of nitrogenase activity = 0.1.
(4)用酒精灯灼烧过的镊子夹取灭过菌的胶塞封住小瓶，并加盖固封。(4) Use tweezers burned by an alcohol lamp to pick up the sterilized rubber stopper to seal the vial, and seal it with a cap.
(5)在小瓶中注入5分钟氩气以排尽瓶内空气，然后注入0.1％的氧气和10％的乙炔。(5) Inject argon gas into the vial for 5 minutes to exhaust the air in the vial, and then inject 0.1% oxygen and 10% acetylene.
(6)将小瓶置于30℃，200rpm摇床培养6h后，8000rpm，离心10min收集菌体。(6) Place the vial at 30° C., culture on a shaker at 200 rpm for 6 hours, then centrifuge at 8000 rpm for 10 minutes to collect the bacteria.
2.采用analytik jenagon公司的innuPREPMini Kit2.0试剂盒提取菌体总RNA。2. The innuPREPMini Kit 2.0 kit from analytik jenagon company was used to extract the total RNA of the bacteria.
3.采用Vazyme公司HiScript III 1st Strand Cdna Synthesis Kit(+gDNAwiper)试剂盒将等量的样品RNA进行单链DNA(cDNA)反转。3. Use the Vazyme HiScript III 1st Strand Cdna Synthesis Kit (+gDNAwiper) kit to reverse the same amount of sample RNA to single-stranded DNA (cDNA).
4.采用STILLA公司的Naica Geode扩增系统和Naica Prism3扫描系统检测固氮条件下人工非编码RNA的绝对表达量。4. Using STILLA's Naica Geode amplification system and Naica Prism3 scanning system to detect the absolute expression of artificial non-coding RNA under nitrogen fixation conditions.
(二)实验结果(2) Experimental results
在固氮条件下每ng样品总RNA中人工非编码RNA AnsR1、AnsR2、AnsR3、AnsR4、AnsR5的拷贝数浓度(×10 4copies/ng)分别为3.57±0.18、8.32±0.63、5.33±0.36、3.81±0.36、3.54±0.47、1.18±0.17(见表2)。 The copy number concentrations (×10 4 copies/ng) of artificial noncoding RNAs AnsR1, AnsR2, AnsR3, AnsR4, AnsR5 in total RNA per ng sample under nitrogen fixation conditions were 3.57±0.18, 8.32±0.63, 5.33±0.36, 3.81, respectively ±0.36, 3.54±0.47, 1.18±0.17 (see Table 2).
(三)实验结论(3) Experimental conclusion
在固氮条件下人工非编码RNA AnsR1/2/3/4/5能够在底盘微生物A1501中稳定表达。The artificial non-coding RNA AnsR1/2/3/4/5 could be stably expressed in the chassis microorganism A1501 under nitrogen fixation conditions.
表2 固氮条件下人工非编码RNA的绝对表达量Table 2 Absolute expression of artificial non-coding RNA under nitrogen fixation conditions
Figure PCTCN2021121276-appb-000002
Figure PCTCN2021121276-appb-000002
实施例4 固氮条件下重组工程菌A1501(AnsR1/AnsR2/AnsR3/AnsR4/AnsR5)中人工非编码RNA半衰期测定Example 4 Determination of half-life of artificial non-coding RNA in recombinant engineering bacteria A1501 (AnsR1/AnsR2/AnsR3/AnsR4/AnsR5) under nitrogen fixation conditions
(一)实验方法(1) Experimental method
1.固氮条件下收集重组菌的菌体1. Collect the cells of the recombinant bacteria under the condition of nitrogen fixation
(1)接菌：分别挑取重组菌A1501(AnsR1/AnsR2/AnsR3/AnsR4/AnsR5)的单菌落接种至含有Km抗性的LB液体培养基中，30℃，200rpm摇床过夜培养。(1) Inoculation: Pick a single colony of recombinant bacteria A1501 (AnsR1/AnsR2/AnsR3/AnsR4/AnsR5) and inoculate them into LB liquid medium containing Km resistance, culture overnight at 30°C on a shaker at 200 rpm.
(2)调OD 600：将过夜培养好的菌液分装至50ml离心管中，5500rpm，4℃离心十 分钟，倒掉上清后用0.85％的生理盐水重悬菌体清洗两次，然后用K无氮的培养基重悬菌体调OD 600＝1。 (2) Adjust OD 600 : Dispense the overnight cultured bacterial solution into 50ml centrifuge tubes, centrifuge at 5500rpm at 4°C for ten minutes, pour off the supernatant and wash twice with 0.85% normal saline to resuspend the bacterial cells, then Resuspend the bacteria with K nitrogen-free medium to adjust OD 600 =1.
(3)向测固氮酶活的小瓶中分别加入6ml的K无氮培养基，再加入4ml OD 600＝1的菌液。使得固氮酶活小瓶中菌液的起始OD 600＝0.4。 (3) Add 6 ml of K nitrogen-free medium to the vials for measuring nitrogenase activity, and then add 4 ml of bacterial solution with OD 600 =1. Make the initial OD 600 of the bacterial solution in the vial of nitrogenase activity = 0.4.
(4)用酒精灯灼烧过的镊子夹取灭过菌的胶塞封住小瓶，并加盖固封。(4) Use tweezers burned by an alcohol lamp to pick up the sterilized rubber stopper to seal the vial, and seal it with a cap.
(5)在小瓶中注入5分钟氩气以排尽瓶内空气，然后注入0.1％的氧气和10％的乙炔。(5) Inject argon gas into the vial for 5 minutes to exhaust the air in the vial, and then inject 0.1% oxygen and 10% acetylene.
(6)将小瓶置于30℃，200rpm摇床培养8h后，向菌液中同时加入40mg/mL的利福平母液200μL，混匀。A1501(AnsR1/AnsR2/AnsR3)的菌液分别在利福平处理0、7、10、13、16min后，吸取1mL菌液于1.5mL EP管中，12000rpm，2min迅速离心收集菌体；A1501(AnsR4/AnsR5)的菌液分别在利福平处理0、10、15、20min后，吸取1mL菌液于1.5mL EP管中，12000rpm，2min迅速离心收集菌体。(6) Place the vial at 30° C. and incubate on a shaker at 200 rpm for 8 hours, then add 200 μL of 40 mg/mL rifampicin mother solution to the bacterial solution at the same time, and mix well. After the bacterial solution of A1501 (AnsR1/AnsR2/AnsR3) was treated with rifampicin for 0, 7, 10, 13 and 16 minutes, 1 mL of the bacterial solution was drawn into a 1.5 mL EP tube, and the bacterial cells were collected by centrifugation at 12000 rpm for 2 minutes; A1501 ( AnsR4/AnsR5) were treated with rifampicin for 0, 10, 15, and 20 minutes respectively, and 1 mL of the bacterial liquid was drawn into a 1.5 mL EP tube, and the bacterial cells were collected by centrifugation at 12,000 rpm for 2 minutes.
(7)向去除上清的菌体中加入400μL的RNAlater(2倍利福平体积)，悬浮上述菌体，室温条件下处理5min后，12000rpm，2min迅速离心，去除上清，液氮速冻。置于-80℃保存备用。(7) Add 400 μL of RNAlater (2 times the volume of rifampicin) to the cells from which the supernatant was removed, suspend the above cells, treat at room temperature for 5 min, then centrifuge rapidly at 12,000 rpm for 2 min, remove the supernatant, and freeze in liquid nitrogen. Store at -80°C for later use.
2.采用analytik jenagon公司的innuPREP Mini Kit2.0试剂盒提取菌体总RNA2. Use the innuPREP Mini Kit 2.0 kit from analytik jenagon to extract the total RNA of the bacteria
3.采用Vazyme公司HiScript III 1st Strand Cdna Synthesis Kit(+gDNAwiper)试剂盒将等量的样品RNA进行单链DNA(cDNA)反转。3. Use the Vazyme HiScript III 1st Strand Cdna Synthesis Kit (+gDNAwiper) kit to reverse the same amount of sample RNA to single-stranded DNA (cDNA).
4.采用ABI公司的7500 Real-time PCR系统，SYBR染料法，qRT-PCR检测样品cDNA中人工非编码RNA的表达量。4. Use ABI's 7500 Real-time PCR system, SYBR dye method, and qRT-PCR to detect the expression of artificial non-coding RNA in cDNA of the sample.
(二)实验结果(2) Experimental results
固氮条件下重组菌A1501(AnsR1/AnsR2/AnsR3/AnsR4/AnsR5)经利福平处理后，人工非编码RNA AnsR1、AnsR2、AnsR3、AnsR4、AnsR5的半衰期(min)分别为16、17、16、20、18(图2)。After the recombinant strain A1501 (AnsR1/AnsR2/AnsR3/AnsR4/AnsR5) was treated with rifampicin under nitrogen-fixing conditions, the half-lives (min) of artificial non-coding RNAs AnsR1, AnsR2, AnsR3, AnsR4, AnsR5 were 16, 17, 16, 20, 18 (Fig. 2).
(三)实验结论(3) Experimental conclusion
固氮条件下人工非编码RNA的稳定性受Hfq蛋白调控，Hfq蛋白结合位点越多的人工非编码RNA越稳定。The stability of artificial non-coding RNA under nitrogen fixation is regulated by Hfq protein, and the artificial non-coding RNA with more binding sites of Hfq protein is more stable.
实施例5 重组菌A1501(AnsR1/AnsR2/AnsR3/AnsR4/AnsR5)的固氮酶活测定Example 5 Determination of Nitrogenase Activity of Recombinant Bacteria A1501 (AnsR1/AnsR2/AnsR3/AnsR4/AnsR5)
(一)实验方法(1) Experimental method
1.挑取重组菌A1501(AnsR)和野生型A1501的单菌落分别接种LB液体培养基中(重组菌卡那抗性，野生型无抗)，30℃，200rpm摇床过夜培养。1. Pick single colonies of recombinant bacteria A1501 (AnsR) and wild-type A1501 and inoculate them in LB liquid medium (recombinant bacteria canard resistance, wild-type no resistance), and cultivate overnight at 30°C on a shaker at 200 rpm.
2.将过夜培养好的菌液分装至50ml离心管中，55000rpm，4℃离心十分钟，倒掉上清后用0.85％的生理盐水重悬菌体清洗两次，然后用K无氮的培养基重悬菌体调OD 600＝1。 2. Dispense the overnight cultured bacterial solution into 50ml centrifuge tubes, centrifuge at 55000rpm, 4°C for ten minutes, pour off the supernatant and wash twice with 0.85% normal saline to resuspend the bacterial cells, and then use K nitrogen-free The medium was resuspended to adjust the OD 600 to 1.
3.向测固氮酶活的小瓶中分别加入9ml的K无氮培养基，再加入1ml OD 600＝1的菌液。使得固氮酶活小瓶中菌液的起始OD 600＝0.1。 3. Add 9 ml of K nitrogen-free medium to the vials for measuring nitrogenase activity, and then add 1 ml of bacterial solution with OD 600 =1. Make the initial OD 600 of the bacterial solution in the vial of nitrogenase activity = 0.1.
4.用酒精灯灼烧过的镊子夹取灭过菌的胶塞封住小瓶，并加盖固封。4. Use tweezers burned by an alcohol lamp to pick up the sterilized rubber stopper to seal the vial, and seal it with a cap.
5.在小瓶中注入5分钟氩气以排尽瓶内空气，然后注入1mL氧气和10mL乙炔。5. Inject argon gas into the vial for 5 minutes to exhaust the air in the vial, then inject 1 mL of oxygen and 10 mL of acetylene.
6.将小瓶置于30℃，200rpm摇床培养，分别在4小时后，6小时后，8小时后，10小时后吸取瓶内2.5mL气体检测乙烯峰面积，利用公式固氮酶活＝乙烯峰面积×(三角瓶的气相总体积/取样体积)/(1nmol乙烯标准峰面积×反应时间×菌体全蛋白总量)计算重组菌的固氮酶活。6. Place the vial at 30°C and incubate on a shaker at 200 rpm. After 4 hours, 6 hours, 8 hours, and 10 hours, draw 2.5 mL of gas in the bottle to detect the ethylene peak area. Use the formula nitrogenase activity = ethylene peak Calculate the nitrogenase activity of the recombinant bacteria by area × (total gas phase volume of the flask/sampling volume)/(1 nmol ethylene standard peak area × reaction time × total protein total amount of bacteria).
(二)实验结果(2) Experimental results
与野生型A1501相比，重组菌A1501(AnsR)的固氮酶活下降。A1501(AnsR1)，A1501(AnsR2)、A1501(AnsR3)、A1501(AnsR4)、A1501(AnsR5)的固氮酶活则分别是野生型A1501的63％、52％、51％、38％、30％(图3)。Compared with the wild-type A1501, the nitrogenase activity of the recombinant strain A1501 (AnsR) decreased. The nitrogenase activity of A1501 (AnsR1), A1501 (AnsR2), A1501 (AnsR3), A1501 (AnsR4), and A1501 (AnsR5) were 63%, 52%, 51%, 38%, and 30% of wild-type A1501, respectively ( image 3).
(三)实验结论(3) Experimental conclusion
固氮条件下诱导表达的人工非编码RNA 1/2/3/4/5均能够降低底盘微生物A1501的固氮能力。The induced expression of artificial non-coding RNA 1/2/3/4/5 under nitrogen fixation conditions can reduce the nitrogen fixation ability of chassis microorganism A1501.
实施例6 重组菌A1501(AnsR1/AnsR2/AnsR3/AnsR4/AnsR5)中固氮相关基因的表达分析Example 6 Expression Analysis of Nitrogen Fixation Related Genes in Recombinant Bacteria A1501 (AnsR1/AnsR2/AnsR3/AnsR4/AnsR5)
(一)实验方法(1) Experimental method
1.固氮条件下收集重组菌A1501(AnsR1/AnsR2/AnsR3/AnsR4/AnsR5)的菌体。1. Collect the cells of recombinant bacteria A1501 (AnsR1/AnsR2/AnsR3/AnsR4/AnsR5) under nitrogen-fixing conditions.
2.采用analytik jenagon公司的innuPREP Mini Kit2.0试剂盒提取菌体总RNA。2. Use the innuPREP Mini Kit 2.0 kit from analytik jenagon company to extract the total RNA of the bacteria.
3.采用Vazyme公司HiScript III 1st Strand Cdna Synthesis Kit(+gDNA wiper)试剂盒将量相同的样品RNA进行单链DNA(cDNA)反转。3. Use Vazyme HiScript III 1st Strand Cdna Synthesis Kit (+gDNA wiper) kit to invert the same amount of sample RNA into single-stranded DNA (cDNA).
4.qRT-PCR分别检测重组菌A1501(AnsR1/AnsR2/AnsR3/AnsR4/AnsR5)中固氮相关基因nifL、nifA、nifH、nifD、nifK的表达量4. qRT-PCR was used to detect the expression levels of nitrogen fixation-related genes nifL, nifA, nifH, nifD, and nifK in recombinant bacteria A1501 (AnsR1/AnsR2/AnsR3/AnsR4/AnsR5)
(二)实验结果(2) Experimental results
重组菌A1501(AnsR1/AnsR2/AnsR3/AnsR4/AnsR5)中nifL、nifA、nifH、nifD、nifK的表达量均比野生型A1501中的表达量低(图4)。The expression levels of nifL, nifA, nifH, nifD, and nifK in recombinant strain A1501 (AnsR1/AnsR2/AnsR3/AnsR4/AnsR5) were lower than those in wild-type A1501 (Figure 4).
(三)实验结论(3) Experimental conclusion
人工非编码AnsR1/2/3/4/5通过抑制靶标基因nifL的表达，由于nifL与固氮正调节基因nifA共转录，因此也影响了nifA的表达，进一步影响了固氮酶编码基因nifHDK的表达，从而导致了A1501固氮能力下降。Artificial non-coding AnsR1/2/3/4/5 suppresses the expression of the target gene nifL, and since nifL is co-transcribed with the nitrogen fixation positive regulator gene nifA, it also affects the expression of nifA, further affecting the expression of the nitrogenase-encoding gene nifHDK, As a result, the nitrogen fixation ability of A1501 decreased.
实施例7 人工非编码RNA AnsR4/AnsR5与靶基因nifL mRNA结合能力鉴定Example 7 Identification of binding ability of artificial non-coding RNA AnsR4/AnsR5 to target gene nifL mRNA
(一)实验方法(1) Experimental method
1.RNA的合成与标记1. RNA synthesis and labeling
由海生物工程有限公司合成了nifL mRNA中与人工非编码RNA互补配对的序列20bp(包含了nifL mRNA的SD序列)，并进行5'FAM荧光标记作为探针；通过体外转录的方法获得全长354bp和379bp的人工非编码RNA AnsR4、AnsR5编码序列，作为配体。Hai Biological Engineering Co., Ltd. synthesized 20 bp of the sequence complementary to the artificial non-coding RNA in nifL mRNA (including the SD sequence of nifL mRNA), and carried out 5'FAM fluorescent labeling as a probe; the full-length was obtained by in vitro transcription 354bp and 379bp artificial non-coding RNA AnsR4, AnsR5 coding sequence, as a ligand.
2.探针与配体的混合反应2. Mixed reaction of probe and ligand
将100nM的标记探针nifL mRNA和3μM的非标记配体AnsR4、AnsR5分别加入到16个标准处理的毛细管中，静置5min。Add 100 nM of labeled probe nifL mRNA and 3 μM of unlabeled ligands AnsR4 and AnsR5 to 16 standard-treated capillaries, and let stand for 5 minutes.
3.微量热涌动测量和数据分析3. Microcalorimetric thermal surge measurement and data analysis
利用NT.115仪器(NanoTemper Technologies GmbH)分别对AnsR4、AnsR5与nifL mRNA之间的结合能力进行分析并计算出解离常数Kd。Kd＝[A]*[L]/[AL]，其中[A]是自由荧光分子的浓度，[L]是自由配体的浓度，[AL]是A和L复合物的浓度。NT.115 instrument (NanoTemper Technologies GmbH) was used to analyze the binding ability between AnsR4, AnsR5 and nifL mRNA and calculate the dissociation constant Kd. Kd = [A]*[L]/[AL], where [A] is the concentration of free fluorescent molecules, [L] is the concentration of free ligands, and [AL] is the concentration of A and L complexes.
(二)实验结果(2) Experimental results
人工非编码RNA AnsR4、AnsR5与nifL mRNA之间的微量热泳动拟合曲线均为典型的“S”型曲线，说明人工非编码RNA AnsR4、AnsR5与nifL mRNA之间均有很好的结合趋势(图5)。The microthermophoresis fitting curves between the artificial non-coding RNAs AnsR4, AnsR5 and nifL mRNA are all typical "S" curves, indicating that there is a good binding tendency between the artificial non-coding RNAs AnsR4, AnsR5 and nifL mRNA (Figure 5).
(三)实验结论(3) Experimental conclusion
人工非编码RNA AnsR4、AnsR5能够通过碱基互补配对的方式分别与靶标基因nifL mRNA相互作用，沉默其表达。The artificial non-coding RNAs AnsR4 and AnsR5 can respectively interact with the target gene nifL mRNA through complementary base pairing and silence its expression.

Claims (7)

  1. 核苷酸序列如SEQ ID NO：1、NO：2、NO：3、NO：4和NO：5所示的人工非编码RNA。The nucleotide sequence is artificial non-coding RNA as shown in SEQ ID NO:1, NO:2, NO:3, NO:4 and NO:5.
  2. 权利要求1所述的人工非编码RNA在微生物基因表达调控中的应用。The application of the artificial non-coding RNA described in claim 1 in the regulation of microbial gene expression.
  3. 权利要求2所述的应用，是所述的人工非编码RNA在固氮基因表达中的应用。The application according to claim 2 is the application of the artificial non-coding RNA in the expression of nitrogen fixation genes.
  4. 权利要求3所述的应用，是所述的人工非编码RNA与靶标固氮调节基因nifL mRNA相互作用的应用。The application according to claim 3 is an application in which the artificial non-coding RNA interacts with the target nitrogen fixation regulatory gene nifL mRNA.
  5. 权利要求4所述的应用，是沉默固氮调节基因nifL mRNA的表达。The application according to claim 4 is to silence the expression of nitrogen fixation regulation gene nifL mRNA.
  6. 含权利要求1所述的人工非编码RNA的表达质粒。An expression plasmid containing the artificial non-coding RNA according to claim 1.
  7. 含权利要求1所述的人工非编码RNA的重组工程菌株。A recombinant engineering strain containing the artificial non-coding RNA according to claim 1.
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