CN113817732A - Artificial non-coding RNA with nitrogen fixation gene silencing function and application thereof - Google Patents
Artificial non-coding RNA with nitrogen fixation gene silencing function and application thereof Download PDFInfo
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- 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
Abstract
The invention designs and constructs 5 artificial non-coding RNAs respectively. The artificial noncoding RNA sequence has a complementary pairing region with a target mRNA, a binding site for Hfq protein, and a Rho independent transcriptional terminator. The artificial non-coding RNAs can be combined with the SD sequence of the 5' UTR of the target nitrogen fixation gene nifL mRNA, can respectively prevent the combination of the mRNA and ribosomal RNA, and further silence the expression of a nitrogen fixation gene negative regulator nifL.
Description
The technical field is as follows:
the invention relates to the technical field of biology, in particular to artificial non-coding RNA with a nitrogen fixation gene silencing function and application thereof in microbial gene expression regulation.
Background art:
RNA interference (RNAi) technology has been developed as a main means for studying expression regulation in eukaryotes, but the main tools for studying gene expression in prokaryotes are still homologous recombination, gene knockout and the like.
The non-coding RNA is used as a novel regulation factor in a bacterial metabolism regulation network, and has the advantages of rapid response, flexible and accurate control, easy recovery, no metabolic burden and the like. By utilizing the synthetic biology concept and designing artificial non-coding RNA, the rapid and high-flux gene expression regulation can be realized on the premise of not changing chromosome genes.
However, artificial non-coding RNA is currently used only in metabolic engineering of biological products such as biofuels (butanol, propane, etc.), glutamic acid, and N-acetylglucosamine.
There is much space available for artificial non-coding RNAs in many fields. For example, in the field of biological nitrogen fixation, NifL is known to be a negative regulator of a nitrogen fixation gene, and if it is inactivated, the inhibition effect of NifL on a positive regulator of nitrogen fixation NifA can be released, so that the expression of nif gene is influenced, therefore, artificial non-coding RNA with the function of silencing NifL gene is constructed, and an intelligent regulatory 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-fixing microorganism chassis for gene expression regulation and control research.
The invention designs and synthesizes 5 Artificial non-coding RNAs (Artificial Nitrogen fixing gene activity-Silenting non-coding RNAs) with nitrogen fixing gene Silencing function, which 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:5, respectively. The 5 artificial non-coding RNAs all have the following functional regions and characteristics:
(1) has a complementary pairing region with the SD sequence of 5' UTR of nifL mRNA of a nitrogenase-fixing regulatory gene;
(2) contains an Hfq protein binding site (5 artificial non-coding RNA coding sequences contain different numbers of Hfq protein binding sites, wherein the artificial non-coding RNA shown in SEQ ID NO:5 contains the largest number of Hfq binding sites);
(3) a Rho factor-independent transcription terminator.
The principle that the artificial non-coding RNA constructed by the invention plays a gene silencing function is as follows:
the 5 artificial non-coding RNAs can be respectively combined with the SD sequence of the 5' UTR of the target nitrogen fixation regulation gene nifL mRNA, so that the combination of the mRNA and ribosomal RNA is prevented, and the expression of the nitrogen fixation gene nifL is silenced.
The silencing function of the nitrogen-fixing gene is designed aiming at the nifL, the NifL is used as a negative regulator of the nifL, and a compound can be formed through the interaction between NifA proteins, so that the inactivation of the NifA is caused, and the expression of the nif is closed, therefore, the artificial non-coding RNA with the function of silencing the nifL gene is constructed, and an intelligent regulation element can be provided for the construction of a high-efficiency nitrogen-fixing system.
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 induced expression under the nitrogen-fixing condition.
The invention transfers 5 artificially constructed expression vectors into nitrogen-fixing Pseudomonas stutzeri A1501(P.stutzeri A1501) respectively to obtain 5 nitrogen-fixing recombinant engineering strains A1501(AnsR1/AnsR2/AnsR3/AnsR4/AnsR 5).
Experiments prove that the 5 artificial non-coding RNAs AnsR1/2/3/4/5 can silence the expression of the target gene nifL mRNA under the nitrogen fixation condition, and the AnsR5 with the largest number of Hfq binding sites has the strongest inhibition capacity, which indicates that the Hfq protein is involved in the silencing of the nitrogen fixation gene nifL by influencing the stability of the artificial non-coding RNA.
The invention designs the artificial non-coding RNA through the following specific work and confirms the function of the artificial non-coding RNA:
1. design and Synthesis of Artificial non-coding RNA AnsR1/2/3/4/5
Through bioinformatics analysis of promoter sequences of pseudomonas stutzeri, klebsiella pneumoniae and azotobacter vinelandii, 5 artificial noncoding RNAs AnsR1/2/3/4/5 containing sequences binding complementary pairs with SD sequences of pseudomonas stutzeri a1501 nifL mRNA5' UTR and different numbers of Hfq binding sites were synthesized by artificial chemical synthesis (table 1). The expression of the 5 artificial non-coding RNAs is controlled by an artificial promoter which specifically responds to a nitrogen fixation signal, the stability of the artificial non-coding RNAs is regulated and controlled by Hfq protein, the artificial non-coding RNAs are combined with an SD sequence of a5' UTR of a target nitrogen fixation regulatory gene nifL mRNA to prevent the combination of the mRNA and ribosomal RNA, and further the expression of the nitrogen fixation gene nifL is silenced, and the nucleotide sequence of the artificial non-coding RNA is SEQ ID NO:1, NO: 2, NO: 3, NO: 4, NO: 5.
2. constructing fusion expression vector of artificial non-coding RNA, transferring the fusion expression vector into nitrogen-fixing Pseudomonas stutzeri to obtain 5 nitrogen-fixing recombinant engineering strains
(1) The broad host expression vector pBBR1MCS is subjected to double enzyme digestion of Bam HI and Hind III, and the artificially synthesized non-coding RNA AnsR1/2/3/4/5 fragments are respectively inserted into the polyclone sites of the broad host expression vector pBBR1MCS-AnsR1/2/3/4/5 by a seamless cloning technology to obtain the artificial non-coding RNA fusion expression vector pBBR1MCS-AnsR1/2/3/4/5 (figure 1).
(2) The fusion expression vector pBBR1MCS-AnsR1/2/3/4/5 is respectively transferred into a microorganism chassis nitrogen-fixing Pseudomonas stutzeri A1501 to obtain a recombinant engineering strain A1501(AnsR1/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 artificial non-coding RNA AnsR1/2/3/4/5 in A1501 by digital PCR under nitrogen fixation conditions. The results show that the artificial non-coding RNAs 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 RNAs AnsR1, AnsR2, AnsR3, AnsR4 and AnsR5 in each ng of total sample RNA under the nitrogen fixation condition4copies/ng) of 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 in A1501 of artificial non-encoded AnsR1/2/3/4/5 under nitrogen fixation conditions by qRT-PCR. The results show that the stability of the artificial non-coding RNA AnsR1/2/3/4/5 in a1501 is regulated by the Hfq protein under nitrogen fixation conditions, 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 (fig. 2), respectively.
(3) Determination of nitrogenase activity of recombinant nitrogen-fixing engineering bacteria
The azotase activity of 5 nitrogen-fixing engineering strains A1501(AnsR1/AnsR2/AnsR3/AnsR4/AnsR5) under the nitrogen-fixing condition was measured by an acetylene reduction method, and the results show that: compared with the wild type A1501, the nitrogenase activities of 5 strains of the engineering bacteria A1501(AnsR1/AnsR2/AnsR3/AnsR4/AnsR5) are all remarkably reduced, wherein the inhibition effects of two artificial non-coding RNAs AnsR4/AnsR5 with good stability are most obvious, and the nitrogenase activities are respectively 38% and 30% of the nitrogenase activity of the wild type A1501, which indicates that the Hfq protein influences the exertion of the functions of the artificial non-coding RNAs (figure 3).
(4) Analysis of expression quantity of nitrogen fixation related gene in recombinant nitrogen fixation engineering bacteria
The expression quantity of a structural gene nifHDK of the azotobacter and a regulatory gene nifLA of the azotobacter in wild type A1501 and 5 recombinant bacteria under the azotobacter condition is compared by utilizing a qRT-PCR technology. The results show that: compared with wild type A1501, the expression level of nifHDK and nifLA mRNA in the recombinant strain is lower than that in wild type A1501 (figure 4), which shows that artificial non-coding AnsR inhibits the expression of target gene nifL, simultaneously influences the expression of nifA of the same transcription unit, further influences the expression of nifHDK gene, and further reduces the nitrogen fixing capability of A1501.
4. Detection of binding capacity of artificial non-coding RNA and target gene nifL mRNA
The binding between the SD sequences of AnsR4, AnsR5 and nifL mRNA5' UTR was analyzed using microcalorie thermolysis (MST) technique and the results showed: the microcalorimetric swimming fit curves between the AnsR4, AnsR5 and the SD sequence of nifL mRNA5' UTR are all typical "S" type curves, which shows that there is a good binding tendency between the SD sequences of AnsR4, AnsR5 and nifL mRNA5' UTR (fig. 5), and it is proved that artificial non-coding AnsR is indeed bound with the SD sequence of target gene nifLmRNA 5' UTR to prevent the binding of mRNA and ribosomal RNA, and further silence the expression of nitrogen-fixing gene.
The invention has the following beneficial results:
experiments prove that the artificial non-coding RNA AnsR1/2/3/4/5 can be stably expressed in the chassis microorganism under the nitrogen fixation condition, and can realize the silencing of a target gene nifL.
The artificial non-coding RNA design strategy can be applied to silencing of different genes in a microbial chassis.
Drawings
FIG. 1: construction of an artificial non-coding RNA AnsR1/2/3/4/5 expression vector. Wherein A represents a construction schematic diagram of an artificial non-coding RNA AnsR1/2/3/4/5 expression vector, and insertion sites are Bam HI and Hind III; FIG. B shows the PCR validation of the expression vector pBBR1MCS-AnsR 1/2/3/4/5.
FIG. 2: half-life assay of the artificial non-coding RNA AnsR1/2/3/4/5 under nitrogen fixation conditions.
FIG. 3: and (3) measuring the azotase activity of the chassis strain A1501 and the recombinant engineering strain A1501(AnsR1/AnsR2/AnsR3/AnsR4/AnsR 5).
FIG. 4: and analyzing the transcription level of the nitrogen fixation related genes in the recombinant nitrogen fixation engineering strain A1501(AnsR1/AnsR2/AnsR3/AnsR4/AnsR5) under the nitrogen fixation condition by qRT-PCR.
FIG. 5: the binding capacity of the artificially encoded RNA AnsR4/AnsR5 to the nifL mRNA as the target gene was determined.
Sequence information
SEQ ID NO 1-5 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 the following specific examples. It should be understood that these examples are only intended to illustrate the process of the present invention and are not intended to limit the scope of the present invention. Where no specific experimental conditions are indicated, the molecular cloning is carried out according to conventional conditions well known to the person skilled in the art, for example Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations.
Example 1 construction of fusion expression vector pBBR1MCS-AnsR1/2/3/4/5
(I) Experimental method
1. The designed artificial non-coding RNAs AnsR1, AnsR2, AnsR3, AnsR4 and AnsR5 are synthesized by a chemical synthesis method, and the sizes of the artificial non-coding RNAs are respectively 75bp, 88bp, 293bp, 354bp and 379bp (see Table 1).
2. The broad host plasmid pBBR1MCS is subjected to Bam HI and HindIII double enzyme digestion, and the 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-AnsR 1/2/3/4/5. And the correct sequence was verified by PCR sequencing.
(II) results of the experiment
The full-length nucleic acid sequences of artificial non-coding RNAs AnsR1, AnsR2, AnsR3, AnsR4 and AnsR5 are obtained by 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 RNAs AnsR1/2/3/4/5 in the fusion expression vector is verified to be correct through PCR sequencing (figure 1).
(III) conclusion of the experiment
The construction of the artificial non-coding RNA fusion expression vector pBBR1MCS-AnsR1/2/3/4/5 was completed.
TABLE 1 information on artificial non-coding RNAs
Example 2 construction of recombinant engineered Strain A1501(AnsR1/AnsR2/AnsR3/AnsR4/AnsR5)
(I) Experimental method
The fusion expression vector pBBR1MCS-AnsR1/2/3/4/5 is respectively transferred into A1501 by a triparental combination method, in the process, a helper plasmid pRK2013 is needed, A1501 is used as a recipient bacterium, pBBR1MCS-AnsR1/2/3/4/5 is used 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 are respectively picked and inoculated into LB liquid culture medium with corresponding resistance, and the liquid culture medium is cultured by a shaking table overnight. A1501: no resistance, 30 ℃, 200 rpm; pBBR1MCS-AnsR1/2/3/4/5, pRK 2013: km resistance, 37 ℃, 220 rpm.
2. Will have passedRespectively transferring the liquid cultured at night into a fresh LB liquid culture medium without antibiotics according to the inoculum size of 2%, and continuously culturing in a shaking table at a proper temperature until OD6000.6 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 bacterium: 2mL of donor bacterium: 600 mu L of helper plasmid), and centrifuged at 5500rpm for 10min at 4 ℃.
4. The supernatant was decanted, and the cells were resuspended in 1mL of 0.85% physiological saline, and centrifuged at 5500rpm at 4 ℃ for 10 min.
5. The supernatant was decanted, and 1ml of 0.85% saline was used to resuspend the recipient, donor, and helper plasmid together, followed by centrifugation at 5500rpm and 4 ℃ for 10 min.
6. The supernatant was decanted, a small amount of liquid was left to mix the cells, 20. mu.L of each was pipetted onto a nonresistant LB plate, and the cells were cultured in an incubator at 30 ℃ for 4-5 days.
7. The colonies cultured in the above step were gently scraped off with a pipette gun, resuspended in 1mL of 0.85% physiological saline, and the dipped cells were 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 colonies are cultured on the plate in the previous step, selecting the single colonies for colony PCR, and respectively verifying whether the fusion expression vector pBBR1MCS-AnsR1/2/3/4/5 connected with the artificial non-coding RNA sequence is successfully transformed into the A1501 wild type strain by using a universal primer M13 of the plasmid pBBR1MCS and a specific primer of nifH genes in the recipient strain A1501.
9. The single colony successfully verified by colony PCR is Pseudomonas stutzeri A1501(AnsR1/AnsR2/AnsR3/AnsR4/AnsR 5).
(II) results of the experiment
The results of resistance selection 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 (FIG. 1).
(III) conclusion of the experiment
The construction of the recombinant engineered bacterium A1501(AnsR1/AnsR2/AnsR3/AnsR4/AnsR5) was completed.
Example 3 analysis of the expression of Artificial non-coding RNA in the recombinant engineered bacterium A1501(AnsR1/AnsR2/AnsR3/AnsR4/AnsR5) under Nitrogen fixation
(I) Experimental method
1. Collecting thallus of recombinant bacteria under nitrogen fixation condition
(1) Single colonies of the recombinant strain A1501(AnsR1/AnsR2/AnsR3/AnsR4/AnsR5) were picked and inoculated into a Km-resistant LB liquid medium, followed by shaking overnight at 30 ℃ and 200 rpm.
(2) Subpackaging the overnight cultured bacterial liquid into 50ml centrifuge tubes, centrifuging at 5500rpm and 4 ℃ for ten minutes, pouring out the supernatant, then re-suspending the bacteria with 0.85% physiological saline for two times, then re-suspending the bacteria with K nitrogen-free culture medium for adjusting OD600＝1。
(3) Adding 9ml of K nitrogen-free culture medium into the small bottles for measuring the activity of the azotobacter, and then adding 1ml of OD600 Bacterial solution 1. Initial OD of bacteria liquid in azotase activity small bottle600＝0.1。
(4) The sterilized rubber plug is clamped by tweezers burned by an alcohol lamp to seal the small bottle, and the bottle is covered and sealed.
(5) The vial was filled with argon for 5 minutes to exhaust the air from the vial, and then with 0.1% oxygen and 10% acetylene.
(6) The vial was incubated at 30 ℃ for 6 hours at 200rpm with a shaker, and then centrifuged at 8000rpm for 10min to collect the cells.
2. Total RNA of the cells was extracted using innuPREP Mini Kit2.0 kit from Analytik jengagon.
3. An equal amount of sample RNA was subjected to single-stranded DNA (cDNA) inversion using the Vazyme HiScript III 1st Strand CdnaSynthesis Kit (+ gDNAwiper) Kit.
4. The absolute expression level of the artificial noncoding RNA under nitrogen fixation conditions was determined using a Naica Geode amplification system and a Naica Prism3 scanning system from STILLA.
(II) results of the experiment
Copy number concentrations (× 10) of the artificial non-coding RNAs AnsR1, AnsR2, AnsR3, AnsR4, AnsR5 per ng of total RNA in the sample under nitrogen fixation4copies/ng) of 3.57 + -0.18, 8.32 + -0.63, 5.33 + -0.36, 3.81 + -0.36, 3.54 + -0.47, 1.18 + -0.17 (see Table 2).
(III) conclusion of the experiment
The artificial non-coding RNA AnsR1/2/3/4/5 was able to be stably expressed in Chassis microorganism A1501 under nitrogen fixation conditions.
TABLE 2 Absolute expression of Artificial non-coding RNA under Nitrogen fixation
Example 4 determination of half-Life of Artificial non-coding RNA in recombinant engineered bacterium A1501(AnsR1/AnsR2/AnsR3/AnsR4/AnsR5) under Nitrogen fixation
(I) Experimental method
1. Collecting thallus of recombinant bacteria under nitrogen fixation condition
(1) Inoculating bacteria: single colonies of the recombinant strain A1501(AnsR1/AnsR2/AnsR3/AnsR4/AnsR5) were picked and inoculated into a Km-resistant LB liquid medium, followed by shaking overnight at 30 ℃ and 200 rpm.
(2) Adjusting OD600: subpackaging the overnight cultured bacterial liquid into 50ml centrifuge tubes, centrifuging at 5500rpm and 4 ℃ for ten minutes, pouring out the supernatant, then re-suspending the bacteria with 0.85% physiological saline for two times, then re-suspending the bacteria with K nitrogen-free culture medium for adjusting OD600＝1。
(3) Adding 6ml of K nitrogen-free culture medium into the small bottle for measuring the azotase activity, and adding 4ml of OD600 Bacterial solution 1. Initial OD of bacteria liquid in azotase activity small bottle600＝0.4。
(4) The sterilized rubber plug is clamped by tweezers burned by an alcohol lamp to seal the small bottle, and the bottle is covered and sealed.
(5) The vial was filled with argon for 5 minutes to exhaust the air from the vial, and then with 0.1% oxygen and 10% acetylene.
(6) After the vial was incubated at 30 ℃ for 8 hours with shaking at 200rpm, 200. mu.L of 40mg/mL rifampicin stock solution was added to the culture broth and mixed well. A1501(AnsR1/AnsR2/AnsR3) bacteria liquid is respectively treated by rifampicin for 0, 7, 10, 13 and 16min, then 1mL of bacteria liquid is sucked into a 1.5mL EP tube, and the bacteria is rapidly collected by centrifugation at 12000rpm for 2 min; a1501(AnsR4/AnsR5) was treated with rifampicin for 0, 10, 15, and 20min, and then 1mL of the broth was aspirated into a 1.5mL EP tube, and the cells were collected by rapid centrifugation at 12000rpm for 2 min.
(7) To the cells from which the supernatant was removed, 400. mu.L of RNA later (2 times the volume of rifampicin) was added, the cells were suspended, treated at room temperature for 5min, then rapidly centrifuged at 12000rpm for 2min, the supernatant was removed, and the cells were frozen in liquid nitrogen. Storing at-80 deg.C for use.
2. Total RNA of the cells was extracted using innuPREP Mini Kit2.0 kit from Analytik jengagon
3. An equal amount of sample RNA was subjected to single-stranded DNA (cDNA) inversion using the Vazyme HiScript III 1st Strand CdnaSynthesis Kit (+ gDNA wiper) Kit.
4. The 7500Real-time PCR system, SYBR dye method and qRT-PCR of ABI company are adopted to detect the expression quantity of the artificial non-coding RNA in the sample cDNA.
(II) results of the experiment
After recombinant bacteria A1501(AnsR1/AnsR2/AnsR3/AnsR4/AnsR5) is treated by rifampicin, the half-lives (min) of artificial non-coding RNAs AnsR1, AnsR2, AnsR3, AnsR4 and AnsR5 are respectively 16, 17, 16, 20 and 18 (FIG. 2).
(III) conclusion of the experiment
The stability of the artificial non-coding RNA under the nitrogen fixation condition is regulated and controlled by the Hfq protein, and the artificial non-coding RNA with more Hfq protein binding sites is more stable.
Example 5 measurement of Azotoxin Activity of recombinant bacterium A1501(AnsR1/AnsR2/AnsR3/AnsR4/AnsR5)
(I) Experimental method
1. Single colonies of the recombinant strain A1501(AnsR) and the wild type A1501 are selected and respectively inoculated into an LB liquid medium (the recombinant strain is kanamycin-resistant, the wild type is non-resistant), and shaking tables are used for overnight culture at 30 ℃ and 200 rpm.
2. Subpackaging the overnight cultured bacteria liquid into 50ml centrifuge tube, centrifuging at 55000rpm and 4 ℃ for ten minutes, pouring out the supernatant, then re-suspending the bacteria with 0.85% physiological saline for two times, then re-suspending the bacteria with K nitrogen-free culture medium for adjusting OD600＝1。
3. Adding 9ml of K nitrogen-free culture medium into the small bottle for measuring the azotobacter activity, and then adding 1ml OD600 Bacterial solution 1. Initial OD of bacteria liquid in azotase activity small bottle600＝0.1。
4. The sterilized rubber plug is clamped by tweezers burned by an alcohol lamp to seal the small bottle, and the bottle is covered and sealed.
5. The vial was filled with 5 minutes of argon to exhaust the air from the vial, and then with 1mL of oxygen and 10mL of acetylene.
6. And (3) placing the small bottle at 30 ℃, carrying out shake cultivation at 200rpm, respectively, after 4 hours, 6 hours and 8 hours, absorbing 2.5mL of gas in the bottle after 10 hours to detect the ethylene peak area, and calculating the azotobacter activity of the recombinant bacteria by using a formula of azotobacter activity, namely ethylene peak area x (total gas phase volume/sampling volume of a triangular flask)/(standard ethylene peak area x reaction time x total mycoprotein amount).
(II) results of the experiment
Compared with the wild type A1501, the recombinant strain A1501(AnsR) has reduced azotase activity. The azotase activities of a1501(AnsR1), a1501(AnsR2), a1501(AnsR3), a1501(AnsR4) and a1501(AnsR5) were 63%, 52%, 51%, 38% and 30% of wild-type a1501, respectively (fig. 3).
(III) conclusion of the experiment
The artificial non-coding RNA 1/2/3/4/5 induced and expressed under the nitrogen fixing condition can reduce the nitrogen fixing capacity of the Chassis microorganism A1501.
Example 6 analysis of expression of Nitrogen fixation-related genes in recombinant bacterium A1501(AnsR1/AnsR2/AnsR3/AnsR4/AnsR5)
(I) Experimental method
1. The cells of the recombinant strain A1501(AnsR1/AnsR2/AnsR3/AnsR4/AnsR5) were collected under nitrogen fixation.
2. Total RNA of the cells was extracted using innuPREP Mini Kit2.0 kit from Analytik jengagon.
3. The same amount of sample RNA was subjected to single-stranded DNA (cDNA) inversion using the Vazyme HiScript III 1st Strand CdnaSynthesis Kit (+ gDNA wiper) Kit.
qRT-PCR (quantitative reverse transcription-polymerase chain reaction) for respectively detecting expression quantities of nitrogen fixation related genes nifL, nifA, nifH, nifD and nifK in recombinant bacteria A1501(AnsR1/AnsR2/AnsR3/AnsR4/AnsR5)
(II) results of the experiment
The expression levels of nifL, nifA, nifH, nifD and nifK in the recombinant strain A1501(AnsR1/AnsR2/AnsR3/AnsR4/AnsR5) were all lower than those in the wild-type A1501 (FIG. 4).
(III) conclusion of the experiment
The artificial non-coding AnsR1/2/3/4/5 inhibits the expression of a target gene nifL, and since nifL is co-transcribed with a nitrogen fixation positive regulatory gene nifA, the expression of nifA is influenced, the expression of a nitrogen fixation enzyme coding gene nifHDK is further influenced, and the nitrogen fixation capability of A1501 is reduced.
Example 7 identification of the binding Capacity of the Artificial non-coding RNA AnsR4/AnsR5 to the NifL mRNA of the target Gene
(I) Experimental method
RNA Synthesis and labeling
A sequence 20bp (including an SD sequence of nifL mRNA) which is complementary and matched with the artificial non-coding RNA in nifL mRNA is synthesized by marine bioengineering limited company, and 5' FAM fluorescent label is used 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 are used as ligands.
2. Mixed reaction of probe and ligand
100nM of labeled probe nifL mRNA and 3. mu.M of unlabeled ligands AnsR4, AnsR5 were added to 16 standard-treated capillaries, respectively, and allowed to stand for 5 min.
3. Microcalorimetric surge measurement and data analysis
The binding capacity between AnsR4, AnsR5 and nifL mRNA was analyzed using NT.115 instrument (NanoTemper Technologies GmbH) and the dissociation constant Kd was calculated. 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.
(II) results of the experiment
The microcalorimetric swimming fit curves between the artificial non-coding RNAs AnsR4, AnsR5 and nifL mRNA are typical 'S' -shaped curves, which shows that the artificial non-coding RNAs AnsR4, AnsR5 and nifL mRNA have good combination tendency (FIG. 5).
(III) conclusion of the experiment
The artificial non-coding RNAs AnsR4 and AnsR5 can interact with the nifL mRNA of the target gene respectively in a base complementary pairing mode, and the expression of the genes is silenced.
Sequence listing
<110> institute of biotechnology of Chinese academy of agricultural sciences
<120> artificial non-coding RNA with nitrogen fixation gene silencing function and application thereof
<160> 5
<170> PatentIn version 3.1
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Claims (7)
1. The nucleotide sequence is shown as SEQ ID NO: 1. NO: 2. NO: 3. NO: 4 and NO:5, artificial non-coding RNA.
2. Use of the artificial non-coding RNA of claim 1 for the regulation of gene expression in a microorganism.
3. The use of claim 2, wherein the artificial non-coding RNA is used for nitrogen fixation gene expression.
4. The use of claim 3, wherein said artificial non-coding RNA interacts with the target nifL mRNA.
5. The use according to claim 4 for silencing the expression of nifL mRNA, a nitrogen fixation regulatory gene.
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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| WO2013132518A1 (en) * | 2012-03-03 | 2013-09-12 | Department Of Biotechnology Ministry Of Science & Technology | Recombinant nitrogen fixing microorganism and uses thereof |
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| 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 |
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