CN113817732B - Artificial non-coding RNA with nitrogen-fixing gene silencing function and its application - Google Patents
Artificial non-coding RNA with nitrogen-fixing gene silencing function and its application Download PDFInfo
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Abstract
The invention designs and constructs 5 artificial non-coding RNAs respectively. The artificial non-coding RNA sequence has a complementary pairing region with the target mRNA, a binding site for the Hfq protein, and a Rho-independent transcription terminator. The artificial non-coding RNA can be combined with SD sequences of target nitrogen fixation regulatory gene nifL mRNA5' UTR, and can respectively prevent the combination of mRNA and ribosomal RNA, thereby silencing the expression of nitrogen fixation gene negative regulatory factor nifL.
Description
Technical field:
the invention relates to the technical field of biology, in particular to artificial non-coding RNA with nitrogen fixation gene silencing function and application thereof in microbial gene expression regulation.
The background technology is as follows:
RNA interference (RNAi) technology has been developed as a major means of studying expression regulation in eukaryotes, but so far in prokaryotes, the major means of studying gene expression have remained as techniques of homologous recombination, gene knockout, and the like.
The non-coding RNA is used as a novel regulatory factor in a bacterial metabolism regulation network, and has the advantages of quick response, flexible and accurate control, easy recovery, no metabolic burden and the like. By utilizing the synthetic biology concept, the artificial non-coding RNA is designed, and the rapid and high-flux gene expression regulation and control can be realized on the premise of not changing the chromosome genes.
However, currently, artificial non-coding RNA is only applied to metabolic engineering of biological products such as biofuel (butanol, propane, etc.), glutamic acid, N-acetylglucosamine, etc.
There are many areas where artificial non-coding RNAs have a number of available spaces. For example, in the field of biological nitrogen fixation, nifL is known to be a negative regulator of a nitrogen fixation gene, if the NifL is inactivated, the inhibition effect of the NifL on a nitrogen fixation positive regulator NifA can be relieved, so that the expression of the nif gene is influenced, and therefore, an artificial non-coding RNA with the function of silencing the nifL gene is constructed, and an intelligent regulating element can be provided for the construction of a high-efficiency nitrogen fixation system.
Disclosure of Invention
The invention aims to construct artificial non-coding RNA with a gene silencing function in a nitrogen fixation microorganism chassis for gene expression regulation and control research.
According to the invention, 5 artificial non-coding RNAs (Artificial Nitrogenase activity-cloning non-coding RNAs) with nitrogen fixation gene Silencing functions are respectively designed and synthesized, and are respectively named as AnsR1, ansR2, ansR3, ansR4 and AnsR5, and the nucleotide sequences of the artificial non-coding RNAs are respectively shown as SEQ ID NO:1, no:2, no:3, NO:4, NO: shown at 5. All of these 5 artificial non-coding RNAs have the following functional regions and features:
(1) Has a complementary pairing region with the SD sequence of the nifL mRNA5' UTR of the azotase regulating gene;
(2) Contains Hfq protein binding sites (the number of Hfq protein binding sites contained in 5 artificial non-coding RNA coding sequences is different, wherein the number of Hfq binding sites contained in the artificial non-coding RNA shown in SEQ ID NO:5 is the largest);
(3) Transcription terminators independent of Rho factor.
The principle of the artificial non-coding RNA constructed by the invention for playing the gene silencing function is as follows:
the 5 artificial non-coding RNAs can be respectively combined with SD sequences of target nitrogen fixation regulatory gene nifL mRNA5' UTR, so that the combination of the mRNA and ribosomal RNA is prevented, and the expression of the nitrogen fixation gene nifL is silenced.
The nitrogen fixation gene silencing function is designed aiming at a nitrogen fixation gene nifL, nifL is used as a negative regulation factor of the nitrogen fixation gene, a compound can be formed through interaction between NifA proteins, and the inactivation of a nitrogen fixation positive regulation factor NifA is caused, so that the expression of the nif gene is closed, and therefore, an intelligent regulation element can be provided for the construction of a high-efficiency nitrogen fixation system by constructing artificial non-coding RNA with the function of silencing the nifL gene.
The invention also constructs an expression vector pBBR1MCS-AnsR1/2/3/4/5 of the artificial non-coding RNA AnsR1/2/3/4/5, and the expression of 5 artificial RNA coding sequences is controlled by an artificial promoter for inducing expression under the condition of nitrogen fixation.
The invention respectively transfers 5 artificially constructed expression vectors into Pseudomonas stutzeri A1501 (P. Stutzeri A1501) to respectively obtain 5 nitrogen fixation recombinant engineering strains A1501 (AnsR 1/AnsR2/AnsR3/AnsR4/AnsR 5).
Experiments prove that 5 artificial non-coding RNAs AnsR1/2/3/4/5 can silence target gene nifL mRNA expression under nitrogen fixation conditions, and the inhibition capability of AnsR5 with the largest number of Hfq binding sites is strongest, which indicates that the Hfq protein participates in silencing of nitrogen fixation genes nifL by affecting the stability of the artificial non-coding RNAs.
The invention designs the artificial non-coding RNA through the following specific works, and confirms the functions of the artificial non-coding RNA:
1. design and synthesis of artificial non-coding RNA AnsR1/2/3/4/5
By bioinformatic analysis of the pseudomonas azotobacter schneider, klebsiella pneumoniae and azotobacter vinelandii promoter sequences, 5 artificial non-coding RNAs AnsR1/2/3/4/5 (table 1) containing sequences complementary to the SD sequence binding pair of pseudomonas schneider a1501 nifL mRNA5' UTR and a different number of Hfq binding sites were synthesized by artificial chemical synthesis. The expression of the 5 artificial non-coding RNAs is not only controlled by an artificial promoter specifically responding to the nitrogen fixation signal, but also the stability is regulated by Hfq proteins, and the expression of the nitrogen fixation gene nifL is silenced by preventing the combination of mRNA and ribosomal RNA by combining with the SD sequence of the 5' UTR of the target nitrogen fixation regulating gene nifL, wherein the nucleotide sequence is SEQ ID NO:1, no:2, no:3, NO:4, NO:5.
2. constructing a fusion expression vector of artificial non-coding RNA, and transferring the fusion expression vector into Pseudomonas stutzeri to obtain 5 nitrogen fixation recombinant engineering strains
(1) The broad host expression vector pBBR1MCS is subjected to BamHI and HindIII double digestion, and the artificially synthesized non-coding RNA AnsR1/2/3/4/5 fragments are respectively inserted into the multiple cloning sites of the non-coding RNA AnsR1/2/3/4/5 fragments by a seamless cloning technology, so that the fusion expression vector pBBR1MCS-AnsR1/2/3/4/5 (figure 1) of the artificial non-coding RNA is obtained.
(2) The fusion expression vector pBBR1MCS-AnsR1/2/3/4/5 is respectively transferred into a microorganism chassis nitrogen fixation Pseudomonas stutzeri A1501 to obtain a recombinant engineering strain A1501 (AnsR 1/AnsR2/AnsR3/AnsR4/AnsR 5).
3. Functional analysis of artificial non-coding RNA
(1) Expression analysis of artificial non-coding RNA
We examined the expression level of the artificial non-coding RNA AnsR1/2/3/4/5 in A1501 by digital PCR under nitrogen fixation. The results show that the artificial non-coding RNA AnsR1/2/3/4/5 can be stably expressed in A1501 under the nitrogen fixation condition, and the copy number concentration (x 10) of the artificial non-coding RNA AnsR1, ansR2, ansR3, ansR4 and AnsR5 in each ng total sample RNA under the nitrogen fixation condition 4 cobies/ng) was 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) Stability analysis of artificial non-coding RNA
We examined the half-life of artificial non-coding AnsR1/2/3/4/5 in a1501 under nitrogen fixation conditions by qRT-PCR. The results show that the stability of the artificial non-coding RNAs AnsR1/2/3/4/5 in A1501 under nitrogen fixation conditions is regulated by the Hfq protein, and the half-lives (min) of the artificial non-coding RNAs AnsR1, ansR2, ansR3, ansR4, ansR5 under nitrogen fixation conditions are 16, 17, 16, 20, 18, respectively (FIG. 2).
(3) Determination of activity of azotase of recombinant azotometer
The activity of the nitrogen fixation enzyme of 5 nitrogen fixation engineering strains A1501 (AnsR 1/AnsR2/AnsR3/AnsR4/AnsR 5) under the nitrogen fixation condition is measured by using an acetylene reduction method, and the result shows that: compared with the wild type A1501, the activity of the azotase of the 5 engineering bacteria A1501 (AnsR 1/AnsR2/AnsR3/AnsR4/AnsR 5) is obviously down-regulated, wherein the inhibition effect of two artificial non-coding RNAs AnsR4/AnsR5 with better stability is most obvious, and the azotase activity is 38 percent and 30 percent of that of the wild type A1501, respectively, which indicates that the Hfq protein influences the function of the artificial non-coding RNAs (figure 3).
(4) Analysis of expression level of nitrogen fixation related genes in recombinant nitrogen fixation engineering bacteria
The qRT-PCR technology is used for comparing the expression quantity of the azotase structural gene nifHDK and the azotase regulating gene nifLA in the wild A1501 and 5 recombinant bacteria under the nitrogen fixation condition. The results show that: compared with the wild type A1501, the expression quantity of nifHDK and nifLA mRNA in the recombinant bacteria is lower than that in the wild type A1501 (figure 4), which shows that the artificial non-coding AnsR further influences the expression of the nifA of the same transcription unit by inhibiting the expression of a target gene nifL and further influences the expression of a nitrogen fixation enzyme gene nifHDK, thereby leading to the reduction of the nitrogen fixation capacity of the A1501.
4. Detection of binding ability of artificial non-coding RNA to target gene nifL mRNA
Binding between the SD sequences of AnsR4, ansR5 and nifL mRNA5' UTR was analyzed using microphoresis (Microscale Thermophoresis, MST) techniques and the results showed that: the trace thermophoresis fit curves between the SD sequences of AnsR4, ansR5 and nifL mRNA5' UTR are typical "S" type curves, which indicate that AnsR4, ansR5 and nifL mRNA5' UTR all have good binding tendencies (FIG. 5), and it is proved that artificial non-coding AnsR does prevent mRNA from binding to ribosomal RNA by binding to the SD sequence of target gene nifL mRNA5' UTR, thereby silencing the expression of nitrogen fixation genes.
The invention has the beneficial effects that:
experiments prove that under the condition of nitrogen fixation, the artificial non-coding RNA AnsR1/2/3/4/5 can be stably expressed in chassis microorganisms, and can realize silencing of a target gene nifL.
The artificial non-coding RNA design strategy can be applied to silencing of different genes in a microorganism chassis.
Drawings
Fig. 1: construction of an artificial non-coding RNA AnsR1/2/3/4/5 expression vector. Wherein A is a schematic diagram of the construction of an artificial non-coding RNA AnsR1/2/3/4/5 expression vector, and insertion sites are BamHI and HindIII; panel B shows the PCR verification of the expression vector pBBR1MCS-AnsR1/2/3/4/5.
Fig. 2: half-life determination of artificial non-coding RNA AnsR1/2/3/4/5 under nitrogen fixation conditions.
Fig. 3: determination of the azotase Activity of Chassis Strain A1501 and recombinant engineering Strain A1501 (AnsR 1/AnsR2/AnsR3/AnsR4/AnsR 5).
Fig. 4: the transcription level of the nitrogen fixation related genes in the recombinant nitrogen fixation engineering strain A1501 (AnsR 1/AnsR2/AnsR3/AnsR4/AnsR 5) under the nitrogen fixation condition is analyzed by qRT-PCR.
Fig. 5: determination of the binding ability of the artificially encoded RNA AnsR4/AnsR5 to the target gene nifL mRNA.
Sequence information
SEQ ID NO. 1-SEQ ID NO. 5 are the nucleotide sequences of 5 different artificial non-coding RNA AnsR5 coding genes respectively.
Detailed Description
The invention will be further illustrated with reference to specific examples. It should be understood that these examples are merely illustrative of the method of the present invention and are not intended to limit the scope of the present invention. The specific experimental conditions are not specified and are conventional conditions well known to those skilled in the art, such as molecular cloning by Sambrook et al: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer.
EXAMPLE 1 construction of fusion expression vector pBBR1MCS-AnsR1/2/3/4/5
Experimental methods
1. The designed artificial non-coding RNA AnsR1, ansR2, ansR3, ansR4 and AnsR5 full-length sequences are synthesized by a chemical synthesis method, and the sizes of the sequences are 75bp,88bp,293bp,354bp and 379bp (see Table 1).
2. The broad host plasmid pBBR1MCS is subjected to BamHI and Hin dIII double digestion, and artificial non-coding RNA AnsR1/2/3/4/5 is respectively connected to a linear vector by a seamless cloning method to obtain a fusion expression vector pBBR1MCS-AnsR1/2/3/4/5. And the correct sequence was verified by PCR sequencing.
(II) results of experiments
The full-length nucleic acid sequences of the artificial non-coding RNAs AnsR1, ansR2, ansR3, ansR4 and AnsR5 are obtained by utilizing an artificial chemical synthesis method, a fusion expression vector pBBR1MCS-AnsR1/2/3/4/5 for expressing the artificial non-coding RNAs is successfully constructed, and the sequence of the artificial non-coding RNA AnsR1/2/3/4/5 in the fusion expression vector is verified to be correct by PCR sequencing (figure 1).
(III) conclusion of experiments
The construction of the fusion expression vector pBBR1MCS-AnsR1/2/3/4/5 of the artificial non-coding RNA is completed.
TABLE 1 information on artificial non-coding RNAs
EXAMPLE 2 construction of recombinant engineering Strain A1501 (AnsR 1/AnsR2/AnsR3/AnsR4/AnsR 5)
Experimental methods
The fusion expression vector pBBR1MCS-AnsR1/2/3/4/5 is respectively transferred into A1501 by a method of three-parent binding, in the process, auxiliary plasmid pRK2013 is needed, A1501 is taken as a receptor bacterium, pBBR1MCS-AnsR1/2/3/4/5 is taken as a donor bacterium, and the detailed steps are as follows:
1. single colonies of pBBR1MCS-AnsR1/2/3/4/5, A1501 and pRK2013 strains on the plates were picked up and inoculated into LB liquid medium with the corresponding resistances, and shake cultured overnight. A1501: no resistance, 30 ℃,200rpm; pBBR1MCS-AnsR1/2/3/4/5, pRK2013: km resistance, 37 ℃,220rpm.
2. Transferring the cultured bacterial solutions to fresh LB-free liquid culture medium according to 2% bacterial inoculation amount, and culturing in shaking table at appropriate temperature until OD 600 =0.6 or 0.8
3. The bacterial liquid cultured in the previous step is added into a 1.5mL centrifuge tube according to a certain proportion (1 mL of recipient bacteria: 2mL of donor bacteria: 600 mu L of auxiliary plasmid), and the mixture is centrifuged at 5500rpm and 4 ℃ for 10min.
4. The supernatant was removed, and the cells were resuspended in 1mL of 0.85% physiological saline, respectively, and centrifuged at 5500rpm at 4℃for 10min.
5. The supernatant was removed, 1ml of 0.85% physiological saline was used to resuspend the recipient, donor and helper bacteria together, and the mixture was centrifuged at 5500rpm at 4℃for 10min.
6. The supernatant was removed, a small amount of liquid was left to mix, 20. Mu.L of each liquid was pipetted onto a non-resistant LB plate, an incubator at 30℃and incubated for 4-5 days.
7. The colonies cultured in the above step were scraped off gently by a pipette, resuspended in 1mL of 0.85% physiological saline, and the bacterial suspension was streaked on LB solid medium containing Km resistance (50. Mu.g/mL) and Cm resistance (17. Mu.g/mL), and cultured in an incubator at 30 ℃.
8. After single colony is cultured on the upper plate, single colony is selected for colony PCR, and a universal primer M13 of plasmid pBBR1MCS and a specific primer of nifH gene in recipient bacterium A1501 are respectively used for verifying whether fusion expression vector pBBR1MCS-AnsR1/2/3/4/5 connected with artificial non-coding RNA sequence is successfully transformed into A1501 wild type bacterium.
9. The single colony with successful colony PCR verification is Pseudomonas stutzeri A1501 (AnsR 1/AnsR2/AnsR3/AnsR4/AnsR 5).
(II) results of experiments
The results of resistance screening and PCR of recombinant strain A1501 (AnsR 1/AnsR2/AnsR3/AnsR4/AnsR 5) were correct, and the fusion expression vector pBBR1MCS-AnsR1/2/3/4/5 was successfully transferred into A1501 (FIG. 1).
(III) conclusion of experiments
The construction of recombinant engineering bacteria A1501 (AnsR 1/AnsR2/AnsR3/AnsR4/AnsR 5) is completed.
Example 3 analysis of expression of Artificial non-coding RNA in recombinant engineering bacterium A1501 (AnsR 1/AnsR2/AnsR3/AnsR4/AnsR 5) under Nitrogen fixation conditions
Experimental methods
1. Collecting recombinant bacteria under nitrogen fixation condition
(1) Single colonies of recombinant bacteria A1501 (AnsR 1/AnsR2/AnsR3/AnsR4/AnsR 5) were picked separately and inoculated into LB liquid medium containing Km resistance, and shake cultured at 30℃and 200rpm overnight.
(2) Packaging the cultured fungus solution overnight into 50ml centrifuge tube, centrifuging at 5500rpm and 4deg.C for ten minutes, pouring out supernatant, and cleaning with 0.85% physiological saline to obtain two-component suspensionNext, the cells were resuspended in K nitrogen-free medium to adjust OD 600 ＝1。
(3) To the vial for measuring the activity of the immobilized enzyme, 9ml of K nitrogen-free medium and 1ml of OD were added respectively 600 Bacterial liquid of=1. So that the initial OD of bacterial liquid in the immobilized enzyme activity vial 600 ＝0.1。
(4) The forceps burned by the alcohol lamp are used for clamping the sterilized rubber plug to seal the small bottle, and the small bottle is covered and sealed.
(5) Argon was injected into the vial for 5 minutes to exhaust the air from the vial, and then 0.1% oxygen and 10% acetylene were injected.
(6) The vials were placed at 30℃and shaking at 200rpm for 6 hours, and centrifuged at 8000rpm for 10min to collect the cells.
2. Total RNA from the cells was extracted using the innuPREP Mini kit2.0 kit from analytical jenagon.
3. An equivalent amount of sample RNA was subjected to single-stranded DNA (cDNA) inversion using the Vazyme HiScript III 1st Strand Cdna Synthesis Kit (+gDNAwind) kit.
4. The absolute expression level of the artificial non-coding RNA under the nitrogen fixation condition is detected by adopting a Naica Geode amplification system and a Naica Prism3 scanning system of the STILLA company.
(II) results of experiments
Copy number concentrations of artificial non-coding RNAs AnsR1, ansR2, ansR3, ansR4, ansR5 per ng total sample RNA under nitrogen fixation conditions (. Times.10) 4 cobies/ng) was 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 (see Table 2).
(III) conclusion of experiments
The artificial non-coding RNA AnsR1/2/3/4/5 can be stably expressed in the chassis microorganism A1501 under the nitrogen fixation condition.
TABLE 2 absolute expression level of artificial non-coding RNA under Nitrogen fixation conditions
EXAMPLE 4 determination of half-life of Artificial non-coding RNA in recombinant engineering bacterium A1501 (AnsR 1/AnsR2/AnsR3/AnsR4/AnsR 5) under Nitrogen fixation conditions
Experimental methods
1. Collecting recombinant bacteria under nitrogen fixation condition
(1) Inoculating: single colonies of recombinant bacteria A1501 (AnsR 1/AnsR2/AnsR3/AnsR4/AnsR 5) were picked separately and inoculated into LB liquid medium containing Km resistance, and shake cultured at 30℃and 200rpm overnight.
(2) OD (optical density) adjustment 600 : packaging the cultured bacterial liquid into 50ml centrifuge tube, centrifuging at 5500rpm and 4deg.C for ten minutes, pouring out supernatant, washing with 0.85% physiological saline to resuspend the bacterial liquid twice, and adjusting OD with K nitrogen-free culture medium 600 ＝1。
(3) To the vials for measuring the activity of the immobilized enzyme, 6ml of K nitrogen-free medium and 4ml of OD were added, respectively 600 Bacterial liquid of=1. So that the initial OD of bacterial liquid in the immobilized enzyme activity vial 600 ＝0.4。
(4) The forceps burned by the alcohol lamp are used for clamping the sterilized rubber plug to seal the small bottle, and the small bottle is covered and sealed.
(5) Argon was injected into the vial for 5 minutes to exhaust the air from the vial, and then 0.1% oxygen and 10% acetylene were injected.
(6) After shaking culture at 200rpm for 8 hours at 30℃the vials were placed, 200. Mu.L of 40mg/mL of rifampicin mother liquor was added simultaneously to the bacterial solution and mixed well. A1501 After the bacterial solutions of (AnsR 1/AnsR2/AnsR 3) are treated for 0, 7, 10, 13 and 16min respectively, 1mL of bacterial solution is sucked into a 1.5mL EP tube, and bacterial cells are collected by rapid centrifugation at 12000rpm for 2 min; a1501 After the bacterial solutions of (AnsR 4/AnsR 5) were treated with rifampicin for 0, 10, 15 and 20min, 1mL of the bacterial solution was aspirated into a 1.5mL EP tube, and the bacterial cells were collected by rapid centrifugation at 12000rpm for 2 min.
(7) To the cells from which the supernatant had been removed, 400. Mu.L of RNA later (2 times the volume of rifampicin) was added, the cells were suspended, and after 5 minutes of treatment at room temperature, the cells were rapidly centrifuged at 12000rpm for 2 minutes, the supernatant was removed, and the cells were quickly frozen with liquid nitrogen. Placing at-80deg.C, and storing.
2. Total RNA from cells was extracted using the innuPREP Mini kit2.0 kit from analytical ik jenagon
3. An equivalent amount of sample RNA was subjected to single-stranded DNA (cDNA) inversion using the Vazyme HiScript III 1st Strand Cdna Synthesis Kit (+gDNA wind) kit.
4. The expression level of the artificial non-coding RNA in the cDNA of the sample was detected by using 7500Real-time PCR system of ABI, SYBR dye method, qRT-PCR.
(II) results of experiments
Recombinant bacteria A1501 (AnsR 1/AnsR2/AnsR3/AnsR4/AnsR 5) were treated with rifampicin under nitrogen fixation conditions, and half-lives (min) of the artificial non-coding RNAs AnsR1, ansR2, ansR3, ansR4, and AnsR5 were 16, 17, 16, 20, and 18, respectively (FIG. 2).
(III) conclusion of experiments
The stability of the artificial non-coding RNA under the nitrogen fixation condition is regulated and controlled by the Hfq protein, and the more the binding sites of the Hfq protein, the more stable the artificial non-coding RNA.
EXAMPLE 5 determination of Nitrogen-fixing enzyme Activity of recombinant bacterium A1501 (AnsR 1/AnsR2/AnsR3/AnsR4/AnsR 5)
Experimental methods
1. Single colonies of recombinant bacteria A1501 (AnsR) and wild type A1501 are selected and inoculated into LB liquid culture medium (recombinant bacteria Carna resistance, wild type no resistance) respectively, and cultured overnight at 30 ℃ with a shaking table at 200 rpm.
2. Packaging the cultured bacterial liquid into 50ml centrifuge tube, centrifuging at 5500rpm and 4deg.C for ten minutes, pouring out supernatant, washing with 0.85% physiological saline to resuspend the bacterial liquid twice, and adjusting OD with K nitrogen-free culture medium 600 ＝1。
3. To the vial for measuring the activity of the immobilized enzyme, 9ml of K nitrogen-free medium and 1ml of OD were added respectively 600 Bacterial liquid of=1. So that the initial OD of bacterial liquid in the immobilized enzyme activity vial 600 ＝0.1。
4. The forceps burned by the alcohol lamp are used for clamping the sterilized rubber plug to seal the small bottle, and the small bottle is covered and sealed.
5. The vial was purged with argon for 5 minutes to purge the vial of air, then 1mL of oxygen and 10mL of acetylene.
6. The vials were placed at 30℃and shake-cultured at 200rpm, after 4 hours, 6 hours, 8 hours, and 10 hours, 2.5mL of gas in the vials was taken to detect the ethylene peak area, and the nitrogen fixation enzyme activity of the recombinant bacteria was calculated by using the formula nitrogen fixation enzyme activity=ethylene peak area× (gas phase total volume/sample volume of triangular flask)/(1 nmol ethylene standard peak area×reaction time×total protein of the bacterial cells).
(II) results of experiments
Compared with the wild type A1501, the activity of the nitrogen fixation enzyme of the recombinant bacterium A1501 (AnsR) is reduced. A1501 (AnsR 1), A1501 (AnsR 2), A1501 (AnsR 3), A1501 (AnsR 4), A1501 (AnsR 5) were found to have nitrogen fixation enzyme activities of 63%, 52%, 51%, 38%, 30% of wild type A1501, respectively (FIG. 3).
(III) conclusion of experiments
The artificial non-coding RNA 1/2/3/4/5 expressed under the nitrogen fixation condition can reduce the nitrogen fixation capacity of the chassis microorganism A1501.
EXAMPLE 6 analysis of expression of Nitrogen fixation-related Gene in recombinant bacterium A1501 (AnsR 1/AnsR2/AnsR3/AnsR4/AnsR 5)
Experimental methods
1. Recombinant bacteria A1501 (AnsR 1/AnsR2/AnsR3/AnsR4/AnsR 5) were collected under nitrogen fixation conditions.
2. Total RNA from the cells was extracted using the innuPREP Mini kit2.0 kit from analytical jenagon.
3. Single-stranded DNA (cDNA) inversion was performed on the same amount of sample RNA using the Vazyme HiScript III 1st Strand Cdna Synthesis Kit (+gDNA wind) kit.
qRT-PCR for detecting the expression level of the nitrogen fixation-related Gene nifL, nifA, nifH, nifD, nifK in recombinant bacterium A1501 (AnsR 1/AnsR2/AnsR3/AnsR4/AnsR 5), respectively
(II) results of experiments
The expression level of nifL, nifA, nifH, nifD, nifK was lower in recombinant A1501 (AnsR 1/AnsR2/AnsR3/AnsR4/AnsR 5) than in wild type A1501 (FIG. 4).
(III) conclusion of experiments
The artificial non-coding AnsR1/2/3/4/5 can inhibit the expression of a target gene nifL, and as nifL and a nitrogen fixation positive regulating gene nifA are transcribed together, the expression of nifA is also influenced, the expression of a nitrogen fixation enzyme coding gene nifHDK is further influenced, and thus the nitrogen fixation capacity of A1501 is reduced.
EXAMPLE 7 identification of the binding Capacity of Artificial non-coding RNA AnsR4/AnsR5 to target Gene nifL mRNA
Experimental methods
Synthesis and labelling of RNA
The complementary pairing sequence of nifL mRNA and artificial non-coding RNA is synthesized by the sea bioengineering limited company to be 20bp (including SD sequence of nifL mRNA), and 5' FAM fluorescent labeling is carried out as a probe; artificial non-coding RNA AnsR4 and AnsR5 coding sequences with the total length of 354bp and 379bp are obtained by an in vitro transcription method and used as ligands.
2. Mixing reaction of probes with ligands
100nM of labeled probe nifL mRNA and 3. Mu.M of unlabeled ligand AnsR4, ansR5 were added to 16 standard treated capillaries, respectively, and allowed to stand for 5min.
3. Micro thermal surge measurement and data analysis
The binding capacity between AnsR4, ansR5 and nifL mRNA was analyzed and dissociation constants Kd calculated using an NT.115 instrument (NanoTemper Technologies GmbH), respectively. Kd= [ a ] [ L ]/[ AL ], where [ a ] is the concentration of free fluorescent molecules, [ L ] is the concentration of free ligands, [ AL ] is the concentration of a and L complexes.
(II) results of experiments
The microphoresis fitted curves between the artificial non-coding RNAs AnsR4, ansR5 and nifL mRNA were all typical "S" shaped curves, demonstrating a good binding trend between the artificial non-coding RNAs AnsR4, ansR5 and nifL mRNA (fig. 5).
(III) conclusion of experiments
The artificial non-coding RNAs AnsR4 and AnsR5 can respectively interact with target gene nifL mRNA by means of base complementary pairing to silence the expression.
Sequence listing
<110> institute of biotechnology of national academy of agricultural sciences
<120> Artificial non-coding RNA having Nitrogen-fixing Gene silencing function and use thereof
<160> 5
<170> PatentIn version 3.1
<210> 1
<211> 75
<212> DNA
<213> artificial sequence
<400> 1
CATACGGTTAGGGGGCTGTTGCACAACAACAATAACAAATGTAACGGCTCCAGCAAGCTA 60
CACGCGCAGCTTTTT 75
<210> 2
<211> 88
<212> DNA
<213> artificial sequence
<400> 2
CATACGGTTAGGGGGCTGTTGCACAACAACAATAACAACAATAAGAACAAGACGTAACGG 60
CTCCAGCAAGCTACACGCGCAGCTTTTT 88
<210> 3
<211> 293
<212> DNA
<213> artificial sequence
<400> 3
CATACGGTTAGGGGGCTGTTGCACAACAACAATAACAAGCAGATCCCACAATAAGAACAA 60
GACGTAACGGCTCCAGCAAAACAACGACAAGAAGGCGGAGGCGCAGCTAACTGATTCTTT 120
TGGAGAGGACATGCCACGGGGTTCGCCCCACGACCAGGCCGAGAACAACAAAAACTGCAT 180
CGAGCAGGCCCTGCACTGGTTGGATCGAAGATCAAGGCAACGTCAGCGACCAAAGAAATC 240
CGTTTGCTATTGGCTCCCACTGTGGGAGCTACGTGCTACACGCGCAGCTTTTT 293
<210> 4
<211> 354
<212> DNA
<213> artificial sequence
<400> 4
CATACGGTTAGGGGGCTGTTGCACAACAACAATAACAAGCAGATCCCACAATAAGAACAA 60
GACGTAACGGCTCCAGCAAAACAACGACAAGAAGGCGGAGGCGCAGCTAACTGATTCTTT 120
TGGAGAGGACATGCCACGGGGTTCGCCCCACGACCAGGCCGAGAACAACAAAAACTGCAT 180
CGAGCAGGCCCTGCACTGGTTGGATCGAAGATCAAGGCAACGTCAGCGACCAAAGAAATC 240
CGTTTGCTATTGGCTCCCACTGTGGGAGCGTTCCCGACGGCCACGGCCCGAAGGACAGGC 300
GAACAACAAGAACAGCAACGCCTGAGAAGTTAAATGCTACACGCGCAGCTTTTT 354
<210> 5
<211> 379
<212> DNA
<213> artificial sequence
<400> 5
CATACGGTTAGGGGGCTGTTGCACAACAACAATAACAAGCAGATCCCACAATAAGAACAA 60
GACGTAACGCAAGGAGCAAAACAACGACAAGAAGGCGGAGGCGCAGCTAACTGATTCTTT 120
TGGAGAGGACATGCCACGGGGTTCGCCCCACGACCAGGCCGAGAACAACAAAAACTGCAT 180
CGAGCAGGCCCTGCACTGGTTGGATCGAAGATCAAGGCAACGTCAGCGACCAAAGAAATC 240
CGTTTGCTATTGGCTCCCACTGTGGGAGCGTTCCCGACGGCCACGGCCCGAAGGACAGGC 300
GAACAACAAGAACAGCAACGCCTGAGAAAACAAACAACAATAAATAAGCACGCGACATTA 360
GCTACACGCGCAGCTTTTT 379
Claims (7)
1. The nucleotide sequence is shown in SEQ ID NO: 1. NO: 2. NO: 3. NO:4 and NO:5, and a human non-coding RNA shown in FIG. 5.
2. Use of the artificial non-coding RNA of claim 1 for microbial gene expression regulation.
3. The use of claim 2, wherein said artificial non-coding RNA is used for nitrogen fixation gene expression.
4. The use of claim 3, wherein the artificial non-coding RNA interacts with the target nitrogen fixation regulatory gene nifL mRNA.
5. The use according to claim 4, for silencing the expression of the nitrogen fixation regulatory gene nifL mRNA.
6. An expression plasmid comprising the artificial non-coding RNA of claim 1.
7. A recombinant engineered strain comprising the artificial non-coding RNA of claim 1.
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| PCT/CN2021/121276 WO2023019693A1 (en) | 2021-08-18 | 2021-09-28 | Artificial non-coding rna module capable of turning off nifl gene expression |
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| CN1746304A (en) * | 2004-09-10 | 2006-03-15 | 中国农业科学院生物技术研究所 | The structure of secreting the ammonium engineering bacteria and the application of the sudden change of fixed nitrogen negative regulator gene |
| CN102690808A (en) * | 2011-03-23 | 2012-09-26 | 北京大学 | Construction of prokaryotic gene expression island for purpose of eukaryotic expression |
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| WO2013105807A2 (en) * | 2012-01-11 | 2013-07-18 | 한국과학기술원 | Novel synthesis-regulating srna and method for preparing same |
| CN104204211B (en) * | 2012-03-03 | 2017-07-04 | 科技部生物技术局 | Restructuring nitrogen-fixing microorganism and application thereof |
| US10329560B2 (en) * | 2013-09-23 | 2019-06-25 | Georgia Tech Research Corporation | Targeting non-coding RNA for RNA interference |
| WO2015190627A1 (en) * | 2014-06-11 | 2015-12-17 | 한국과학기술원 | Method for minutely regulating gene expression using synthesis-regulating srna |
| CN104593361B (en) * | 2015-01-07 | 2017-12-15 | 中国农业科学院生物技术研究所 | A kind of artificial synthesized sRNA of anti-oxidant and resistance to high-salt stress and application thereof |
| US20180223283A1 (en) * | 2017-02-03 | 2018-08-09 | Ohio State Innovation Foundation | Antisense fingerloop rnas and uses thereof |
| AU2018354221B2 (en) * | 2017-10-25 | 2025-04-17 | Pivot Bio, Inc. | Methods and compositions for improving engineered microbes that fix nitrogen |
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| CN102690808A (en) * | 2011-03-23 | 2012-09-26 | 北京大学 | Construction of prokaryotic gene expression island for purpose of eukaryotic expression |
| CN108291219A (en) * | 2015-10-05 | 2018-07-17 | 麻省理工学院 | It is fixed using the nitrogen of reconstruct nif clusters |
| CN112739202A (en) * | 2018-07-11 | 2021-04-30 | 皮沃特生物股份有限公司 | Temporally and spatially targeted dynamic nitrogen delivery by remodeling microbes |
| CN111926008A (en) * | 2020-06-16 | 2020-11-13 | 中国农业科学院生物技术研究所 | Artificial non-coding RNA module for enhancing nitrogen fixation capacity of microorganisms |
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