CRISPR system and application thereof in mortierella alpina
阅读说明：本技术 一种crispr系统及其在高山被孢霉中的应用 (CRISPR system and application thereof in mortierella alpina ) 是由 陈海琴 王顺贤 常璐璐 张灏 陈永泉 陈卫 于 2020-12-28 设计创作，主要内容包括：本发明公开了一种CRISPR系统及其在高山被孢霉中的应用,属于生物工程技术领域技术领域。本发明采用的核酸酶为毛螺科菌(Lachnospiraceae bacterium)的LbCpf1,所使用的诱导型启动子为里氏木霉(Trichoderma reesei)中的cbh1,所选靶向基因为尿嘧啶基因ura5,所用的出发菌株为高山被孢霉尿嘧啶营养缺陷型MAU1,本发明提供的基于CRISPR技术的高山被孢霉系统,可实现生物量和产脂含量与野生菌无显著性差异,并可以实现多基因表达,此方法将为构建高工业价值的重组高山被孢霉提供技术手段。(The invention discloses a CRISPR system and application thereof in mortierella alpina, belonging to the technical field of biological engineering. The nuclease adopted by the invention is LbCpf1 of Lachnospiraceae (Lachnospiraceae bacteria), the used inducible promoter is cbh1 in Trichoderma reesei, the selected targeting gene is uracil gene ura5, and the used starting strain is Mortierella alpina uracil auxotroph MAU 1.)
1. A nuclease LbCpf1 encoding gene is characterized by comprising a sequence shown in SEQ ID NO. 1.
2. An expression vector comprising a gene encoding the nuclease LbCpf1 of claim 1.
3. The expression vector of claim 2, which is a constitutive expression vector or an inducible expression vector.
4. The expression vector of claim 3, wherein the inducible expression vector comprises an inducible promoter, Pcbh 1.
5. A recombinant filamentous fungal cell comprising the expression vector of any one of claims 2 to 4, or having a gene encoding the nuclease LbCpf1 of claim 1 integrated into its genome.
6. The recombinant filamentous fungal cell according to claim 5, wherein Mortierella alpina is used as a host.
7. A filamentous fungus system based on CRISPR technology is characterized by comprising an expression vector for generating guide strand CrRNA and Mortierella alpina for expressing a nuclease LbCpf1 gene shown in SEQ ID NO. 1.
8. A method of gene editing, comprising gene editing the recombinant filamentous fungal cell of claim 6 using CRISPR technology; the method comprises the following steps:
(1) constructing a plasmid containing the nuclease LbCpf1 and a screening marker; constructing a guide chain plasmid containing a promoter, a guide chain and a terminator;
(2) transforming the plasmid constructed in the step (1) into a mortierella alpina uracil auxotrophic strain by adopting agrobacterium tumefaciens;
(3) isolating said host cell with edited genome from the culture using a selectable marker.
9. The method according to claim 8, wherein the Agrobacterium tumefaciens of step (2) is Agrobacterium tumefaciens AGL 1.
10. A method for expressing multiple genes in Mortierella alpina, which comprises introducing a plasmid containing a foreign gene into the recombinant Mortierella alpina engineering strain of claim 6, and expressing the foreign gene by integrating the foreign gene into the genome of Mortierella alpina.
Technical Field
The invention relates to a CRISPR system and application thereof in mortierella alpina, belonging to the technical field of biological engineering.
Background
The mortierella alpina is an oil-producing filamentous fungus with high yield of arachidonic acid and industrial value. At present, the lipid production pathway of mortierella alpina is basically clear, and the functions of a plurality of lipid production related genes in vivo are successfully verified. In order to further construct a high-yield strain, researchers need to introduce a lipid-producing key gene with a verified function into a mortierella alpina genome by means of a multigene expression means. Currently, gene expression in mortierella alpina is limited to two gene levels. Due to the limitations of transformation methods and selection markers, traditional co-transformation methods, multi-operon methods and the like cannot be applied to mortierella alpina. The traditional screening marker recovery method comprises Cre/LoxP and FLP/FRT, and the two systems have the problem that the normal gene function of host bacteria is lost or the host bacteria die due to the leaving of a plurality of recognition sites. How to solve the technical problem that the multi-gene expression is the first problem to be solved for constructing high-yield strains subsequently according to the genetic background of mortierella alpina and the existing technical means.
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system is an emerging technology for targeted gene knockout in recent years. The system mainly comprises two parts of nuclease and a guide chain, and the main principle of the function of the system is that the target guide chain guides the nuclease to cut a double-stranded DNA of a target gene. Since the invention, this technology has been widely used in various organisms. By means of an inducible promoter, the CRISPR system is designed into a screening marker recovery system for targeted knockout of a screening marker, and the purpose of carrying out multi-gene expression by means of a single screening marker can be realized in Mortierella alpina.
More commonly used nucleases in CRISPR include both Cas9 and Cpf 1. Compared with Cas9, Cpf1 has a series of advantages, such as that the guide strand does not need trans-activated CRISPR RNA (trans-acting CRISPR RNA, tracrRNA), DNA and RNA can be sheared, double-stranded DNA is sheared by Cpf1 to generate a sticky end, and the like. Currently, the Cpf1 which are widely used include three cpfs respectively derived from the amino acid coccus sp, Lachnospiraceae bacteria (LbCpf1) and Francisella novicida, but the efficiency of knocking out genes by the CRISPR system of filamentous fungi in the prior art is relatively low, and the CRISPR knocking out system for mortierella alpina is absent.
Disclosure of Invention
The invention aims to solve the technical problem of providing a CRISPR technology-based gene knockout method and a polygene expression technology for filamentous fungi Mortierella alpina.
The first purpose of the invention is to provide a nuclease LbCpf1 encoding gene, which comprises a sequence shown in SEQ ID NO. 1.
The second purpose of the invention is to provide an expression vector containing the gene coding the nuclease LbCpf 1.
In one embodiment, the expression vector is a constitutive expression vector or an inducible expression vector.
In one embodiment, the inducible expression vector contains the inducible promoter, Pcbh 1.
In one embodiment, the inducible promoter, Pcbh1, has the nucleotide sequence shown in SEQ ID No. 2.
The third purpose of the invention is to provide a recombinant microbial cell containing the expression vector or integrating a gene encoding the nuclease LbCpf1 shown in SEQ ID NO.1 on the genome.
In one embodiment, the recombinant microbial cell is a host Mortierella alpina.
In one embodiment, the recombinant microbial cell is a recombinant bacterium MA-pcb 1-LbCpf1 useful for targeted knockout of uracil.
A fourth object of the present invention is to provide a CRISPR technology based filamentous fungal system comprising mortierella alpina expressing the nuclease LbCpf1, and an expression vector for the production of guide strand CrRNA.
In one embodiment, the 3' end of the sequence encoding the nuclease LbCpf1 incorporates the nuclear localization sequence SV 40; the nucleotide sequence of the nuclease LbCpf1 is shown as SEQ ID NO. 1.
The fifth purpose of the invention is to provide a gene editing method, which is to use CRISPR technology to edit the gene of the recombinant microorganism cell.
In one embodiment, the gene editing includes, but is not limited to, knock-out, or expression of one or more genes.
In one embodiment, the genome editing method comprises:
(1) constructing a plasmid containing the nuclease LbCpf1 and a screening marker; constructing a guide chain plasmid containing a promoter, a guide chain and a terminator;
(2) transforming the plasmid constructed in the step (1) into a mortierella alpina uracil auxotrophic strain by adopting agrobacterium tumefaciens;
(3) isolating said host cell with edited genome from the culture using a selectable marker.
In one embodiment, the selectable marker is the ura5 gene.
In one embodiment, the method comprises the specific steps of:
(1) constructing a plasmid containing the nuclease LbCpf1 and a screening marker; constructing a guide chain plasmid containing a promoter, a guide chain and a terminator;
(2) transforming the plasmid constructed in the step (1) into a mortierella alpina uracil auxotrophic strain by adopting agrobacterium tumefaciens;
(3) culturing the recombinant mortierella alpina strain obtained in the step (2) in a screening culture medium containing 5-fluoroorotic acid (5-fluroic acid, 5-FOA).
In one embodiment, the promoter used to guide the strand is SNR52p and the terminator is Sup4 t.
In one embodiment, the plasmid used in step (1) is the universal binary vector His550-ITS-TrpCT in Mortierella alpina, wherein the His550 promoter on the vector is replaced by the inducible promoter Pcbh 1.
In one embodiment, the agrobacterium tumefaciens described in step (2) is agrobacterium tumefaciens AGL 1.
In one embodiment, the Mortierella alpina uracil auxotrophic strain is Mortierella alpina MAU1, which is disclosed in publication No. CN 103468581B.
The sixth object of the present invention is to provide a method for expressing multiple genes in Mortierella alpina, wherein a plasmid containing a foreign gene is introduced into the recombinant Mortierella alpina MA-Pcbh1-LbCpf1, and the foreign gene is expressed in the recombinant Mortierella alpina MA-Pcbh1-LbCpf 1.
In one embodiment, the exogenous gene is the omega-3 desaturase gene oPpFADS17 from Phytophthora parasitica.
Has the advantages that: on the basis of the existing Mortierella alpina transformation system, the invention adopts an Agrobacterium tumefaciens mediated transformation method to construct a CRISPR-based recombinant Mortierella alpina engineering strain capable of performing multi-gene expression. The genetic engineering strain of mortierella alpina obtained by the method has no obvious difference in growth and lipid production characteristics compared with wild strains.
Drawings
FIG. 1 is a schematic diagram of the construction of binary expression vector pBIG2-ura5-Pcbh1-LbCpf1-TrpCT-SNR52 p-CrRNA.
FIG. 2 is an agarose gel electrophoresis picture of identification of a recombinant Mortierella alpina MA-Pcbh1-LbCpf 1; wherein M, marker; the numbers 1 to 6 are respectively the transformation binary expression vector pBIG2-ura5-Pcbh1-LbCpf1-TrpCT-SNR52p-CrRNA Mortierella alpina recombinant strain MA-Pcbh1-LbCpf 1.
FIG. 3 shows targeted knockout strain verification of Mortierella alpina recombinant strain MA-Pcbh1-LbCpf1 uracil (ura 5). Wherein M, marker; accession numbers 1-18 are targeted knockout and phenotypically correct uracil-deficient strain MA-Pcbh1-LbCpf-ura5-。
FIG. 4 shows the sequencing result of uracil ura5 gene knockout strain in Mortierella alpina recombinant strain MA-Pcbh1-LbCpf 1.
FIG. 5 is a diagram of agarose gel electrophoresis for verifying transformants of Mortierella alpina MA-Pcbh1-LbCpf1-oPpFADS17 based on a multigene operating system. M, Marker; n1, mortierella alpina wild type strain ATCC 32222; n2, Mortierella alpina uracil auxotrophic strain MA-Pcbh1-LbCpf1-ura5-(original strain); the codes are 1 to 7, and 7 transformants of Mortierella alpina are MA-Pcbh1-LbCpf1-oPpFADS17 based on a polygene operating system.
Detailed Description
SOC recovery culture medium: 20g/L tryptone, 5g/L yeast powder, 0.5g/L sodium chloride, 2.5mM potassium chloride, 10mM magnesium chloride, 20mM glucose and the balance of water.
LB solid medium: 10g/L tryptone, 5g/L yeast powder, 10g/L sodium chloride, 20g/L agar and the balance of water.
MM solid medium: 1.74g/L dipotassium hydrogen phosphate, 1.37g/L potassium dihydrogen phosphate, 0.1468g/L sodium chloride, 0.49g/L magnesium sulfate heptahydrate, 0.01g/L calcium chloride, 0.005g/L ferrous sulfate heptahydrate, 0.53g/L ammonium sulfate, 1.8g/L glucose, 0.5 wt% glycerol, 20g/L agar, and the balance of water, and the pH is 6.8.
IM medium: reducing the glucose content to 0.9g/L on the basis of MM medium, and adding 200 μ M Acetosyringone (AS).
SC medium: 20g/L glucose, 5g yeast nitrogen source without amino acid and ammonium sulfate, 1.7g/L ammonium sulfate, 60mg/L isoleucine, 60mg/L leucine, 60mg/L phenylalanine, 50mg L threonine, 40mg/L lysine, 30mg/L tyrosine, 20mg/L adenine, 20mg/L arginine, 20mg/L histidine, 10mg/L methionine, 20g/L agar, the balance water, pH 6.8.
GK solid medium: 20g/L glucose, 10g/L yeast extract, 2g/L potassium nitrate, 1g/L sodium dihydrogen phosphate, 3g/L magnesium sulfate heptahydrate, 20g/L agar, and the balance water, and the pH is 6.8.
YEP medium: 10g/L of yeast extract, 10g/L of trypsin, 5g/L of sodium chloride and the balance of water. When the solid medium is concerned, 20g/L agar is added.
Example 1 construction of the expression vector pBIG2-Pcbh1-LbCpf1
The selected nuclease is LbCpf1 from Lapiromyces. SV40 nuclear localization sequence (CCCAAGAAGAAGCGCAAGGTCC) was added to the end of the nuclease sequence, and the entire fragment was codon-optimized and then biosynthesized by Kinsley, the nucleotide sequence being shown in SEQ ID NO. 1. The synthesized LbCpf1 nuclease fragment is subjected to NheI and XhoI double enzyme digestion treatment and purification to obtain a high-purity high-concentration gene fragment, and the gene fragment is integrated into a binary vector pBIG2-ura5-Pcbh1-ITs-TrpCT in the Mortierella alpina which is subjected to enzyme digestion treatment through T4 ligase to obtain a plasmid pBIG2-Pcbh1-LbCpf 1.
Example 2 construction of Mortierella alpina binary expression vector pBIG2-ura5-Pcbh1-ITs-TrpCT
The selected inducible promoter Pcbh1 (shown in SEQ ID NO. 2) is derived from Trichoderma reesei, and the sequence is synthesized by Kinry organism company, and is constructed into pBIG2-ura5 plasmid (see the study on transcriptional level regulation and reducing power of Mortierella alpina fatty acid synthesis process published in 2015 for details) through XbaI enzyme digestion and laboratory preliminary construction into pBIG2-ura5-Pcbh 1-ITS-TrpCT. The specific digestion reaction and ligation reaction are as follows:
1.1 enzyme digestion reaction: on the basis of the plasmid pBIG2-ura5-ITS, the promoter H550 of the H550-ITS-TrpCT fragment is replaced by Pcbh1 to obtain Pcbh1-IT-TrpCT (for the construction of ITS-TrpCT, see the doctor's paper research on the transcriptional level regulation and reducing power of the synthesis process of Mortierella alpina fatty acid). The fragment Pcbh1-ITs-TrpCT and the vector pBIG2-ura5 were first digested with the restriction enzyme XbaI at 37 ℃. The 100. mu.L XbaI digestion system is: 2 mu L of XbaI-FD, 30 mu L of plasmid or PCR product, 10 mu L of cutmarst Buffer, 58 mu L of deionized water, and carrying out water bath digestion at 37 ℃ for 2h, and carrying out purification operation after the digestion product is recovered.
1.2 ligation: and (3) connecting the fragment Pcbh1-ITS-TrpCT subjected to enzyme cutting and purification in the step 1.1 with the vector pBIG2-ura5 subjected to enzyme cutting and purification by using T4 ligase, and connecting for 12 hours at 4 ℃ to obtain a recombinant expression vector pBIG2-ura5-Pcbh 1-ITS-TrpCT. Linker system (10 μ L): mu.L of the fragment after the enzyme digestion of the target gene Pcbh1-ITS-TrpCT, 3 mu.L of the fragment after the enzyme digestion of the vector pBIG2-ura5, 1uL of ligase buffer, 1 mu L T4 of ligase, 3 mu.L of sterile water and 12h of ligation reaction at 4 ℃ to obtain a ligation product.
The ligation product obtained in step 1.2 was transformed into competent cells of E.coli T0P10 by the following method:
(1) mu.L of competent cells were taken aseptically and 1-2. mu.L of the ligation product obtained in step 1.2 was added.
(2) Moving the mixed competent cells into the pre-cooled electrorotating cup avoids the creation of air bubbles.
(3) And (3) putting the electric rotating handle into a Bio-Rad electric rotating instrument, adjusting to a proper preset program gear, and performing electric rotation under the voltage condition of 1.8 kv.
(4) Add 1mL of SOC recovery medium to the transduced competent cells, mix well and transfer to a 1.5mL centrifuge tube, incubate at 37 ℃ and 150rpm for 1 hour.
(5) Take 200. mu.L of LB solid medium plate plated with 100. mu.g/mL kanamycin. The cells were inverted and cultured overnight at 37 ℃.
(6) And (3) selecting a positive transformant, extracting a plasmid, and obtaining a binary expression vector pBIG2-ura5-Pcbh1-ITs-TrpCT as a result of sequencing verification showing successful connection.
Example 3 construction of the expression vector pBIG2-ura5-Pcbh1-LbCpf1-TrpCT-SNR52p-CrRNA
Guide strand CrRNA design for nuclease LbCpf1 for the targeted gene ura5, in casofFinder the relevant parameters for the guide strand were set as: the operating microorganism was mortierella alpina ATCC 32222: PAM sequence 5 '-3' TTTN of LbCpf 1: the targeting gene is ura5 fragment; the length of the leader strand fragment is 23 bp. For the conserved region of core function of ura5 gene, two higher scoring guide strands were selected that cover its area to the greatest extent. The specific information is as follows: the off-target scores of the guide strands were all 62.9, with the CrRNA corresponding to LbCpf1 covering 207bp to 231bp of the ura5 fragment. The nucleotide sequence of CrRNA is shown as SEQ ID NO. 4.
The guide strand of the CRISPR system needs to be guided by RNA polymerase III (pol III) promoter for its function, and there is no relevant research on the functional verification of such promoter in mortierella alpina. The small SNR52p fragment was selected as the promoter, and the Sup4t (the nucleotide sequence of Sup4t is shown in SEQ ID NO. 5) used in combination with SNR52p shown in SEQ ID NO.3 was selected as the terminator. The SNR52p-CrRNA-Sup4t fragment formed by forward ligation of SEQ ID NO.3 to SEQ ID NO.5 was synthesized by Kinry. The synthetic fragment sequence and the vector are subjected to XbaI enzyme digestion and ligation treatment (the specific enzyme digestion reaction and the ligation reaction refer to example 2), and the final binary vector pBIG2-ura5-Pcbh1-LbCpf1-TrpCT-SNR52p-CrRNA is obtained.
Example 4 construction of Agrobacterium tumefaciens AGL1
The binary expression vector pBIG2-ura5-Pcbh1-LbCpf1-TrpCT-SNR52p-CrRNA is transformed into competent cells of Agrobacterium tumefaciens by electric shock, and the Agrobacterium tumefaciens AGL1 containing the plasmid pBIG2-ura5-Pcbh1-LbCpf1-TrpCT-SNR52p-CrRNA is obtained.
Example 5 Agrobacterium tumefaciens-mediated transformation of Mortierella alpina MAU1
The method is characterized by carrying out appropriate optimization and adjustment aiming at the agrobacterium tumefaciens transformation method, and specifically comprising the following steps:
(1) agrobacterium tumefaciens AGL1 containing plasmid pBIG2-ura5-Pcbh1-LbCpf1-TrpCT-SNR52p-CrRNA, constructed in example 4, stored at-80 ℃ was streaked on YEP solid medium plates containing 100. mu.g/mL rifampicin and 100. mu.g/mL kanamycin, and cultured for 48 hours at 28 ℃ in an inverted dark state;
(2) selecting a single clone, inoculating the single clone into 20mL liquid YEP culture medium containing 100 mu g/mL rifampicin and 100 mu g/mL kanamycin, and culturing the single clone at 28 ℃ and 200rpm in the dark for 24-48 hours;
(3) transferring 200 μ L into MM culture medium, culturing at 28 deg.C and 200rpm for 24-48 hr;
(4) adjusting the concentration of the bacterial liquid to 0D by using an IM (instant Messaging) culture medium600Is 0.3. Culturing at 28 deg.C and 200rpm in dark to OD600To 1.0.
(5) Washing Mortierella alpina uracil auxotrophic strain MAU1 (disclosed in patent publication No. CN 103468581B) cultured on GY-U slant for 1 month or more with 5mL of sterilized normal saline, collecting spores, counting with a hemocytometer, and adjusting the concentration of the spores to 107one/mL.
(6) 100 mu L of agrobacterium tumefaciens and 100 mu L of spore are uniformly mixed and coated on an IM solid culture medium paved with glass paper, and are cultured for 36 to 48 hours in a dark place at 23 ℃.
(7) The cellophane was transferred to SC screening medium (SC-CS medium) containing 100. mu.g/mL spectinomycin and 100. mu.g/mL cefotaxime sodium. Incubated at 16 ℃ in the dark for 12h, followed by transfer to 23 ℃.
(8) And continuously observing the growth condition of the colonies on the SC-CS plate, if obvious colonies grow, picking out the outer edge of the colonies (about 2mm) by using a pointed forceps in time, inoculating the colonies on a new SC-CS plate, and continuously and normally culturing the colonies in an incubator at 28 ℃.
(9) After the transformants on the SC-CS plate grew, the mycelia were picked and transferred to the SC-CS plate, and repeated screening was carried out for 3 times to exclude negative transformants.
(10) Liquid culture and activation are carried out on the bacterial colony which can still stably grow after being screened for 3 times, then the bacterial colony is inoculated to a GY slant culture medium, the culture medium is placed at 28 ℃ for 10 to 14 days until hyphae grow out, and the culture medium is transferred to a refrigerator at 4 ℃ for sporulation for standby.
Taking the suspected transformant preserved in the GY slant culture medium, scraping spores or picking hyphae, inoculating into a broth liquid activation culture medium, and culturing for 2d at 28 ℃ to obtain a bacterial liquid; extracting fungus genome DNA from a bacterial liquid, carrying out PCR verification, and carrying out PCR verification on the LbCpf1 fragment in the obtained genome by using Taq enzyme, wherein the used primer sequence is as follows:
LbCpf1F(sense):AAGCTTGGTACCGCTAGCATG；
LbCpf1R(antisense):GACTCGAGGACCTTGCGCTTC；
the result of agarose gel electrophoresis analysis for recombinant strain identification is shown in FIG. 2, a product band of nearly 3737bp can be amplified by using specific primers of LbCpf1 and the genome of a positive transformant, and the electrophoresis result shows that the binary expression vector is successfully integrated into the genome of Mortierella alpina MA-Pcbh1-LbCpf 1.
Example 6 extraction and detection of fatty acid from Mortierella alpina recombinant bacterium MA-Pcbh1-LbCpf1
(1) The wild strain of Mortierella alpina and the recombinant Mortierella alpina MA-Pcbh1-LbCpf1 transferred into the binary expression vector pBIG2-ura5-Pcbh1-LbCpf1-TrpCT-SNR52p-CrRNA T-DNA region and obtained by screening in example 5 are inoculated into broth culture medium and subjected to shake culture at 28 ℃ and 200r/min for 7 days;
(2) collecting thalli, freezing and drying the thalli in vacuum to constant weight, weighing the thalli, and calculating biomass;
(3) grinding the thalli into powder, weighing 50mg, adding 2mL of 4mol/L hydrochloric acid, and adding 100mL of 2mg/L fatty acid internal standard (C15: 0);
(4) standing in 80 deg.C water bath for 1h (-80 deg.C) for 15 min for freeze thawing to break wall, repeating for three times, and then water bath at 80 deg.C for 1 h;
(5) cooling to room temperature, adding 1mL of methanol, and uniformly mixing;
(6) adding 1mL of chloroform, mixing uniformly, and shaking for 10 min. Centrifuging at 6000g for 3min, and collecting chloroform layer to a new bottle;
(7) repeating the step (6) twice;
(8) the chloroform layer was dried by nitrogen blowing, 1mL of 10% (w/w) methanol hydrochloride was added, and the mixture was heated in a water bath at 60 ℃ for 3 hours, shaken every 30min, and then cooled at room temperature.
(9) Adding 1mL saturated sodium chloride solution and 1mL n-hexane, mixing well, shaking for 1min, centrifuging at 6000g for 3min, absorbing the n-hexane layer to obtain fatty acid methyl ester, diluting properly, and storing in a gas phase bottle for subsequent detection.
(10) Fatty acid methyl ester analysis was performed using GC2010(Shimadzu Co., Japan) with a column of DB-Waxetr (30 m.times.0.32 m, 0.22 μm); detecting by a hydrogen flame ion detector, wherein the temperatures of a vaporization chamber and the detector are 240 ℃ and 260 ℃, the sample injection is carried out in a split-flow mode by 1uL, the split-flow ratio is 10:1, and the carrier gas is nitrogen; temperature programming: maintaining the initial temperature at 120 deg.C for 3min, increasing to 190 deg.C at 5 deg.C/min, increasing to 220 deg.C at 4 deg.C/min, and maintaining for 20 min; the fatty acid components in the samples were qualitatively and quantitatively analyzed by mass comparison with a commercial fatty acid methyl ester standard (37 fatty acid methyl ester mixed standard, Supelco, USA) and the addition of an internal standard C15:0, the total fatty acid content being expressed as the mass of total fatty acids per cell.
TABLE 1 analysis of growth and lipid production of Mortierella alpina recombinant strain MA-Pcbh1-LbCpf1
Note: the fatty acid content (TFA%) of each type is the ratio of the corresponding fatty acid content to the total fatty acids, and the total lipid content (dry wt%) is the ratio of the total fatty acid mass to the dry cell weight.
The results in Table 1 show that the biomass and fatty acid content of each transformant are not significantly different from those of wild-type Mortierella alpina, indicating that the introduction of the binary plasmid does not adversely affect the lipid production content and biomass of Mortierella alpina.
Example 7: targeted induction knockout of mortierella alpina recombinant strain MA-Pcbh1-LbCpf1 uracil selection marker (ura5)
The liquid induction medium is 20mL MM medium, 0.1% (w/v) uracil and 1% (w/v) microcrystalline cellulose (avicel) are added, induction is carried out at 28 deg.C and 200rpm for 7d, hyphae are collected and broken by a dispersion machine, 400. mu.L of broken hyphae are taken and spread on GYU-F solid screening medium.
The transformants obtained by screening were subjected to genome extraction, and then subjected to PCR verification using primers histrof 1 and TrpCT 1. The primer sequences used were:
HisproF1(sense):GTGTTCACTCGCATCCCGC；
TrpCT1(antisense):AGGCACTCTTTGCTGCTTGG；
in the PCR validation of the genome, the PCR nucleic acid gel results (FIG. 3) showed that a band of the correct size appeared for a total of 18 transformants; sequencing results show (figure 4), and the sequences of the ura5 expression units of the transformants No. 10 and No. 13 have base deletion at the target position, and prove that the ura5 genes of the two strains are successfully knocked out on the molecular layer surface.
In order to screen uracil auxotrophic strains for subsequent gene transfer, recombinant bacteria No. 10 and No. 13, which were verified to be correct in both phenotype and genotype, were subjected to fermentation culture and analyzed for growth and lipid production.
The results of fatty acid analysis (table 2) showed no significant change in biomass and total lipid content of the uracil auxotrophic recombinant bacteria nos. 10 and 13, compared to the wild-type strain and transformant No. 6, MA-pcb 1-LbCpf1, which is the starting strain in which the uracil ura5 gene was knocked out by induction.
TABLE 2 recombinant strain MA-Pcbh1-LbCpf1-ura5 of Mortierella alpina uracil deficient strain-Growth and adipogenic status
Note: the content of various fatty acids (TFA%) is the ratio of the content of corresponding fatty acids to the total fatty acids, the total content of fatty acids (dry weight%) is the ratio of the mass of the total fatty acids to the dry weight of cells, the starting bacterium 6 represents a recombinant bacterium MA-Pcbh1-LbCpf1-6 for uracil-induced knockout, and the recombinant bacterium No. 10 and No. 13 represent the MA-Pcbh1-LbCpf1-ura5 which carry out uracil-targeted knockout on the basis of the starting bacterium and are proved to be correct by genome-And (4) recombining bacteria.
Example 8 application of Mortierella alpina CRISPR System in expressing Gene
Taking the previously constructed plasmid pBIG2-ura5s-oPpFADS17 containing the omega-3 fatty acid desaturase gene oPpFADS17 (the construction method is disclosed in the patent with the publication number CN 105647822B) as an example, the desaturase gene can catalyze Arachidonic Acid (AA) in mortierella alpina to generate docosapentaenoic acid (EPA), so the oPpFADS17 is an ideal reporter gene for investigating whether a foreign gene can be correctly expressed and exert functions in the recombinant bacterium MA-Pcbh1-LbCpf1-ura 5-.
Using the method for transformation and selection of Mortierella alpina in examples 6 and 7, the strain MA-Pcbh1-LbCpf1-ura5 was recovered as a uracil selection marker obtained in example 7-(strain 13) was genetically transformed as a recipient. And (3) continuously passaging a batch of Mortierella alpina transformants obtained by screening on an SC-CS screening culture medium, selecting 7 transformants capable of stably growing, carrying out amplification culture, extracting genome DNA, carrying out PCR verification by using a universal primer HisproF1/TrpCR1, and carrying out gel electrophoresis, wherein the gel electrophoresis result is shown in FIG. 5. The genome verification result shows that the obtained 7 mortierella alpina transformants can amplify a uracil expression unit (818bp) and a target fragment oPpFADS17(1331bp), which indicates that the transformants are verified correctly at the genome level, and also indicates that the 7 mortierella alpina recombinant strains based on the CRISPR system successfully realize the anaplerosis of uracil and successfully introduce a target gene oPpFADS 17. The obtained recombinant strain is named as MA-Pcbh1-LbCpf1-oPpFADS17, and each strain is respectively assigned with the number of 1-7.
In order to analyze the function of the foreign gene, the 7 Mortierella alpina transformants were further subjected to fermentation culture and recombinant bacteria growth and fatty acid component analysis in the same manner as in example 6, and the results are shown in Table 3. Compared with wild Mortierella alpina, the Mortierella alpina CRISPR system constructed by the invention has the advantages that the function of the oPpFADS17 in the 2, 4, 5 and 6 transformants of the MA-Pcbh1-LbCpf1-oPpFADS17 recombinant strain is realized, AA can be converted into EPA, and the conversion rates are respectively 21.1%, 3.8%, 2.2% and 44.1%, so that the Mortierella alpina CRISPR system constructed by the invention can realize the introduction of functional genes. Except that AA can be converted into EPA, biomass, fatty acid content and fatty acid of each recombinant strain have no obvious change compared with the original strain, which shows that homologous fragments of pBIG plasmid are introduced into the Mortierella alpina engineering strain based on the CRISPR system again, and have no obvious influence on the growth and lipid production performance of host bacteria.
TABLE 3 analysis of growth and lipid production levels of Mortierella alpina MA-Cpf1-Pcbh1-oPpFADS17 transformants
Note: the content of each type of fatty acid (TFA%) is the ratio of the corresponding fatty acid content to the total fatty acids, the total content (dry weight%) is the ratio of the total fatty acid mass to the dry weight of the cell, and the starting bacterium 13 represents a recombinant bacterium MA-Pcbh1-LbCpf1-ura5 for uracil targeted knockout-(number 13), numbers 1-7 are in the starting bacterium MA-Pcbh1-LbCpf1-ura5-And 7 transformants obtained after overexpression of the oPpFADS 17.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> CRISPR system and application thereof in mortierella alpina
<130> BAA201495A
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 3737
<212> DNA
<213> Artificial sequence
<400> 1
cgctagcatg tcgaagctgg agaagtttac caactgctac tccctgagca agaccctccg 60
cttcaaggcc atccctgtcg gaaagaccca ggagaacatc gacaacaagc gcctgctcgt 120
cgaggacgag aagagggccg aggattacaa gggcgtcaag aagctgctcg atcgctacta 180
cctgagcttt atcaacgacg tcctccactc gatcaagctg aagaacctca acaactacat 240
ctcgctgttc cgcaagaaga cccgcaccga gaaggagaac aaggagctgg agaacctgga 300
gatcaacctg cgcaaggaga tcgccaaggc cttcaagggc aacgagggat acaagtccct 360
ctttaagaag gacatcatcg agaccatcct gccagagttc ctcgacgata aggacgagat 420
cgccctggtc aactcgttca acggctttac caccgccttc accggattct ttgacaaccg 480
cgagaacatg ttttccgagg aggccaagtc cacctcgatc gccttccgct gcatcaacga 540
gaacctcacc cgctacatca gcaacatgga catcttcgag aaggtcgatg ccatctttga 600
taagcatgag gtccaggaga tcaaggagaa gatcctgaac tcggactacg atgtcgagga 660
tttctttgag ggcgagttct ttaactttgt cctcacccag gagggaatcg acgtctacaa 720
cgccatcatc ggcggattcg tcaccgagag cggcgagaag atcaagggac tgaacgagta 780
catcaacctc tacaaccaga agaccaagca gaagctgcct aagtttaagc cactgtacaa 840
gcaggtcctc tcggatcgcg agagcctctc gttctacggc gagggataca cctccgacga 900
ggaggtcctg gaggtctttc gcaacaccct caacaagaac tcggagatct tctcgtccat 960
caagaagctg gagaagctct tcaagaactt tgacgagtac agctcggccg gcatctttgt 1020
caagaacgga cccgccatct cgaccatctc caaggacatc ttcggcgagt ggaacgtcat 1080
ccgcgacaag tggaacgccg agtacgacga catccacctg aagaagaagg ccgtcgtcac 1140
cgagaagtac gaggacgatc gccgcaagtc cttcaagaag atcggctcgt tcagcctgga 1200
gcagctccag gagtacgccg acgccgatct gagcgtcgtc gagaagctca aggagatcat 1260
catccagaag gtcgatgaga tctacaaggt ctacggctcc agcgagaagc tcttcgacgc 1320
cgattttgtc ctggagaagt cgctcaagaa gaacgacgcc gtcgtcgcca tcatgaagga 1380
cctgctcgat agcgtcaagt cgtttgagaa ctacatcaag gccttctttg gcgagggaaa 1440
ggagaccaac cgcgacgagt ccttctacgg agattttgtc ctggcctacg acatcctgct 1500
caaggtcgat cacatctacg atgccatccg caactacgtc acccagaagc cctacagcaa 1560
ggataagttc aagctctact ttcagaaccc tcagttcatg ggcggatggg acaaggataa 1620
ggagaccgac taccgcgcca ccatcctgcg ctacggctcg aagtactacc tcgccatcat 1680
ggataagaag tacgccaagt gcctccagaa gatcgacaag gacgatgtca acggcaacta 1740
cgagaagatc aactacaagc tgctccccgg acctaacaag atgctcccaa aggtcttctt 1800
tagcaagaag tggatggcct actacaaccc ctcggaggac atccagaaga tctacaagaa 1860
cggcaccttc aagaagggag atatgtttaa cctgaacgac tgccataagc tcatcgactt 1920
ctttaaggat tcgatctccc gctacccaaa gtggtccaac gcctacgatt tcaactttag 1980
cgagaccgag aagtacaagg acatcgccgg cttttaccgc gaggtcgagg agcagggata 2040
caaggtctcc ttcgagagcg cctcgaagaa ggaggtcgat aagctggtcg aggagggcaa 2100
gctctacatg ttccagatct acaacaagga cttttccgat aagagccacg gaacccccaa 2160
cctgcatacc atgtacttca agctgctctt tgacgagaac aaccacggac agatccgcct 2220
gtcgggagga gcagagctct tcatgcgccg cgcctccctg aagaaggagg agctcgtcgt 2280
ccatcccgcc aactcgccta tcgccaacaa gaacccagat aaccccaaga agaccaccac 2340
cctgtcctac gacgtctaca aggataagcg ctttagcgag gaccagtacg agctccacat 2400
cccaatcgcc atcaacaagt gccccaagaa catcttcaag atcaacaccg aggtccgcgt 2460
cctgctcaag catgacgata acccctacgt catcggcatc gaccgcggag agcgcaacct 2520
gctctacatc gtcgtcgtcg atggcaaggg aaacatcgtc gagcagtact ccctgaacga 2580
gatcatcaac aactttaacg gcatccgcat caagaccgat taccacagcc tgctcgacaa 2640
gaaggagaag gagcgcttcg aggcccgcca gaactggacc tccatcgaga acatcaagga 2700
gctgaaggcc ggatacatca gccaggtcgt ccataagatc tgcgagctcg tcgagaagta 2760
cgatgccgtc atcgccctgg aggacctcaa cagcggcttt aagaactcgc gcgtcaaggt 2820
cgagaagcag gtctaccaga agttcgagaa gatgctgatc gacaagctca actacatggt 2880
cgataagaag agcaaccctt gcgccaccgg aggagcactg aagggctacc agatcaccaa 2940
caagttcgag tcgtttaagt ccatgagcac ccagaacgga ttcatctttt acatccctgc 3000
ctggctcacc tccaagatcg acccaagcac cggctttgtc aacctgctca agaccaagta 3060
cacctcgatc gccgattcca agaagttcat ctcgtccttt gaccgcatca tgtacgtccc 3120
cgaggaggat ctgttcgagt ttgccctcga ctacaagaac ttcagccgca ccgacgccga 3180
ttacatcaag aagtggaagc tgtactccta cggcaaccgc atccgcatct tccgcaaccc 3240
taagaagaac aacgtctttg actgggagga ggtctgcctg acctcggcct acaaggagct 3300
cttcaacaag tacggcatca actaccagca gggagacatc cgcgccctgc tctgcgagca 3360
gtccgacaag gccttctaca gctcgtttat ggccctgatg tcgctgatgc tccagatgcg 3420
caactccatc accggccgca ccgacgtcga ttttctcatc tcgcctgtca agaactccga 3480
cggaatcttc tacgattcgc gcaactacga ggcccaggag aacgccatcc tgccaaagaa 3540
cgccgacgcc aacggcgcct acaacatcgc ccgcaaggtc ctctgggcca tcggacagtt 3600
caagaaggcc gaggacgaga agctggataa ggtcaagatc gccatcagca acaaggagtg 3660
gctggagtac gcccagacct cggtcaagca taagcgccct gccgccacca agaagcccaa 3720
gaagaagcgc aaggtcc 3737
<210> 2
<211> 1092
<212> DNA
<213> Artificial sequence
<400> 2
gtacaagtcg taatcactat taacccagac tgaccggacg tgttttgccc ttcatttgga 60
gaaataatgt cattgcgatg tgtaatttgc ctgcttgacc gactggggct gttcgaagcc 120
cgaatgtagg attgttatcc gaactctgct cgtagaggca tgttgtgaat ctgtgtcggg 180
caggacacgc ctcgaaggtt cacggcaagg gaaaccaccg acagcagtgt ctagtagcaa 240
cctgtaaagc cgcaatgcag catcactgga aaatacaaac caatggctaa aagtacataa 300
gttaatgcct aaagaagtca tataccagcg gctaataatt gtacaatcaa gtggctaaac 360
gtaccgtaat ttgccaacgg cttgtgggct aacagaagca acggcaaagc caatcttcca 420
atcgtttgtt tcttcactca gtccaatctc agctggtgat cccccaattg ggtcgcttgt 480
ttgttccggt gaagtgaaag aagacagagg taagaatgtc tgactcggag cgttttgcat 540
acaaccaagg gcagtgatgg aagacagtga aatgttgaca ttcaaggagt atttagccag 600
ggatgcttga gtgtatcgtg taaggaggtt tgtctgccga tacgacgaat actgtatagt 660
cacttctgat gaagtggtcc atattgaaat gtaagtcggc actgaacagg caaaagattg 720
agttgaaact gcctaagatc tcgggccctc gggccttcgg cctttgggtg tacatgtttg 780
tgctccgggc aaatgcaaag tgtggtagga tcgaacacac tgctgccttt accaagcagc 840
tgagggtatg tgataggcaa atgttcaggg gccactgcat ggtttcgaat agaaagagaa 900
gcttagccaa gaacaatagc cgataaagat agcctcatta aacggaatga gctagtaggc 960
aaagtcagcg aatgtgtata tataaaggtt cgaggtccgt gcctccctca tgctctcccc 1020
atctactcat caactcagat cctccaggag acttgtacac catcttttga ggcacagaaa 1080
cccaatagtc aa 1092
<210> 3
<211> 269
<212> DNA
<213> Artificial sequence
<400> 3
tctttgaaaa gataatgtat gattatgctt tcactcatat ttatacagaa acttgatgtt 60
ttctttcgag tatatacaag gtgattacat gtacgtttga agtacaactc tagattttgt 120
agtgccctct tgggctagcg gtaaaggtgc gcattttttc acaccctaca atgttctgtt 180
caaaagattt tggtcaaacg ctgtagaagt gaaagttggt gcgcatgttt cggcgttcga 240
aacttctccg cagtgaaaga taaatgatc 269
<210> 4
<211> 24
<212> DNA
<213> Artificial sequence
<400> 4
tttggccccg cctacaaggg tgtc 24
<210> 5
<211> 20
<212> DNA
<213> Artificial sequence
<400> 5
tttttttgtt ttttatgtct 20