CN114015690A - Construction method and application of bombyx mori shRNA expression vector - Google Patents
Construction method and application of bombyx mori shRNA expression vector Download PDFInfo
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Abstract
The invention discloses a construction method of a silkworm shRNA expression vector, which adopts a silkworm RNA polymerase U6 promoter, respectively introduces gtatcttatcatgtctggat and ggtaccaagctttaaattcg recombination sites to two ends of the promoter, linearizes a pIZT/V5-His vector through PCR amplification, and recombines and replaces an OpIE2 promoter on the vector. According to the construction method of the silkworm shRNA expression vector, the promoter OpIE2 in the skeleton vector pIZT/V5-His is recombined and replaced by the U6 promoter BmU6 in the silkworm through a recombination method, the restriction of enzyme restriction sites is avoided, and the recombination efficiency is high.
Description
Technical Field
The invention relates to the technical field of insect genes, in particular to a construction method and application of a silkworm shRNA expression vector.
Background
RNA interference (RNAi), an phenomenon of gene silencing induced by double-stranded RNA, is the inhibition of gene expression by blocking the transcription or translation of a particular gene, and when double-stranded RNA homologous to the endogenous mRNA coding region is introduced into a cell, the mRNA is degraded, resulting in silencing of gene expression.
RNAi technology is widely applied to gene function research of animals and plants at present, specific gene function analysis can be rapidly and efficiently carried out by combining a large amount of data of genome sequencing technology, dsRNA or shRNA is transferred into cells through exogenous synthesis, and the dsRNA or shRNA is cut into siRNA by utilizing intracellular RNAase to play a role in regulating and degrading target genes.
Silkworm is a model species of lepidoptera, and has a unique sex determination mode and a cell cycle regulation mode. The silkworm has about 15000 genes, most of which have unknown functions, and the problems of low transfection efficiency, low knockdown efficiency and the like generally exist when the silkworm cell line is used for gene function verification, so that the progress of the silkworm gene function research is limited, the knockdown efficiency can be greatly improved by transferring the dsRNA transmembrane channel protein SID of the nematode into the silkworm cell, but the defects of complex experimental operation, instantaneous interference effect and the like still exist.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a construction method and application of a silkworm shRNA expression vector, the OpIE2 promoter in the insect expression vector pIZT/V5-His is recombined and replaced by a BmU6 promoter, the rapid and efficient shRNA cloning expression can be realized, and the method has the characteristics of low cost and high efficiency.
(II) technical scheme
In order to achieve the purpose, the invention provides the following technical scheme: a silkworm shRNA expression vector construction method adopts a silkworm RNA polymerase U6 promoter, introduces gtatcttatcatgtctggat and ggtaccaagctttaaattcg recombination sites to two ends of the promoter respectively, and recombines and replaces an OpIE2 promoter on a vector after the pIZT/V5-His vector is linearized through PCR amplification.
Preferably, only one pair of complementary single-long-chain DNA is synthesized in vitro, KpnI and EcoRI are selected at two ends as enzyme cutting sites, a long double-chain DNA fragment containing a viscous tail end is formed by annealing after high-temperature incubation for 3 minutes, a carrier subjected to double enzyme cutting by KpnI and EcoRI and the double-chain DNA are connected by T4 DNA ligase, bacterium P after cloning and transforming escherichia coli is verified, and correct clone is selected for sequencing verification.
Preferably, two single-stranded DNAs are synthesized in vitro, and long double-stranded DNA fragments having cohesive ends are formed by in vitro annealing after diluting the fragments.
Preferably, the annealing results in the long-chain DNA fragment ending in a cohesive end and an overhanging end of 4 bases, GTAC and TTAA respectively.
Preferably, the in vitro synthesized single long chain consists of a 21nt hairpin structure and a 6nt stem-loop structure, the 6nt stem-loop structure consisting of the CTCGAG sequence.
Preferably, the DNA fragment formed after annealing can be ligated to the cohesive ends of the vector constructed in claim 1 after double cleavage.
In order to cooperate with the construction method of the bombyx mori shRNA expression vector, the following experimental steps are provided: s1, designing a gene specific recombination primer by using CE DesignV1.04 software, wherein the tail end of the primer contains gtatcttatcatgtctggat and ggtaccaagctttaaattcg recombination sites;
s2, cloning BmU6 promoter specific method is: the method comprises the following steps of using silkworm ovary tissue genome DNA as a template, amplifying a silkworm BmU6 promoter by a PCR technology, and carrying out reaction conditions: pre-denaturation at 95 ℃ for 5 min; 30s at 95 ℃, 30s at 58 ℃ and 30s at 72 ℃ (30 cycles); extension at 72 ℃ for 10min, reaction system: 2X Mix: 10 mu L of the solution; primer-F: 1 mu L of the solution; primer-R: 1 mu L of the solution; DNA template: 1 mu L of the solution; ddH 2O: make up to 20 μ L. Detecting the PCR product by agarose gel electrophoresis, and recovering and purifying the gel;
the specific method for linearization of S3 and pIZT/V5-His vector comprises the following steps: the pIZT/V5-His vector is used as a template, the pIZT/V5-His vector is linearized by PCR amplification, a forward primer pIZT-V5-F (CGAATTTAAAGCTTGGTACCGAG), a reverse primer pIZT-V5-R (ATCCAGACATGATAAGATACATTGATGA), and reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; 1min30s at 95 ℃, 2min at 58 ℃ and 2min at 72 ℃ (30 cycles); extension at 72 ℃ for 10min, reaction system: 2X Mix: 10 mu L of the solution; primer-F: 1 mu L of the solution; primer-R: 1 mu L of the solution; DNA template: 1 mu L of the solution; ddH 2O: supplementing to 20 mu L, detecting the PCR product through agarose gel electrophoresis, and recovering and purifying gel;
the specific method for recombining the S4 and pIZT/V5-His-BmU6 vectors comprises the following steps: the fragments recovered in S2 and S3 were calculated in concentration according to vector: calculating a recombination reaction system according to the ratio of the target fragment 67:20, wherein the reaction conditions are as follows: recombination at 37 ℃ for 40min, and reaction system: 5X buffer: 2 mu L of the solution; and (3) recombinase: 0.5 mu L; pIZT/V5-His linearized vector: 3.5 mu L; BmU6 mesh fragment: 4 mu L of the solution;
the specific method for verifying the S5 and pIZT/V5-His-BmU6 vectors comprises the following steps: transforming the recombinant system of S4 into 100. mu.L of Escherichia coli DH5 alpha competent cells, and carrying out reaction conditions as follows: performing ice bath for 30 min; performing water bath heat shock at 42 ℃ for 90s, immediately performing ice bath for 5min, adding 800 mu L of LB non-resistant liquid culture medium, and incubating at 37 ℃ for 1 h; uniformly coating the transformed cells with proper volume on an LB solid plate culture medium containing corresponding bleomycin, carrying out inverted culture at 37 ℃ for 18h, then selecting a single colony for colony PCR verification, and carrying out positive clone sequencing verification;
s6, the specific method for constructing the target gene shRNA vector comprises the following steps: designing shRNA target point information of a target gene through online software, synthesizing a single long chain which is composed of a 21nt hairpin structure and a 6nt annular structure and aims at a target point sequence in vitro, synthesizing a 6nt stem-loop structure by a CTCGAG sequence, and naturally cooling and renaturing the synthesized sense chain and antisense chain after in vitro heating denaturation to form a double-chain structure, wherein the reaction system comprises: sense strand (1 μ L); antisense strand (1 μ L); annealing Buffer (8 μ L); water (10 μ L). After the reaction is finished, 80 mu L of water is added, the annealing product is diluted by 5 times, and the reaction conditions are as follows: heating the water bath to 100 ℃, keeping the temperature of the reaction system for 3min, closing the water bath, naturally cooling for about 3h, and cooling to about 30 ℃ to finish annealing;
s7, the tail end of the long double-stranded DNA fragment formed by annealing is a cohesive tail end, the protruding tail end is 4 basic groups which are GTAC and TTAA respectively, the long double-stranded DNA formed by annealing is connected with a pIZT/V5-His-BmU6 vector subjected to double enzyme digestion by KpnI and EcoRI to form a shRNA interference vector of a target gene, and an enzyme digestion system is adopted: 1 μ L each of KpnI and EcoRI, 10 Xbuffer: 2 μ L, pIZT/V5-His-BmU6 vector template 1 μ L, ddH 2O: the mixture was filled to 20. mu.L and incubated at 37 ℃ for 20 min. A connection system: t4 DNA ligase: 0.5 mu L; t4 DNA Buffer: 1 mu L of the solution; KpnI and EcoRI double digested pIZT/V5-His-BmU6 vector: 1.5 mu L; annealing and diluting fragments: 1 mu L of the solution; ddH 2O: filling to 10 mu L, and connecting for 1h at 22 ℃;
s8, the specific method for verifying the target gene shRNA vector comprises the following steps: the ligation system of S7 was transformed into 100. mu.L E.coli DH 5. alpha. competent cells under the following conditions: performing ice bath for 30 min; performing water bath heat shock at 42 ℃ for 90s, immediately performing ice bath for 5min, adding 800 mu L of LB non-resistant liquid culture medium, and incubating at 37 ℃ for 1 h; the transformed cells with proper volume are evenly coated on an LB solid plate culture medium containing the corresponding bleomycin, inverted culture is carried out at 37 ℃ for 18h, then single colonies are selected for colony PCR verification, and positive clone sequencing verification is carried out.
(III) advantageous effects
Compared with the prior art, the invention provides a construction method of a bombyx mori shRNA expression vector, which has the following beneficial effects:
1. according to the construction method of the silkworm shRNA expression vector, the promoter OpIE2 in the skeleton vector pIZT/V5-His is recombined and replaced by the U6 promoter BmU6 in the silkworm through a recombination method, the restriction of enzyme restriction sites is avoided, and the recombination efficiency is high.
Drawings
FIG. 1 is a schematic diagram of the construction process of the vector pIZT/V5-His-BmU6 of the present invention;
FIG. 2 is an agarose gel electrophoresis image of the vector construction of the present invention;
FIG. 3 is a diagram showing the effect of cell transfection of a target gene using the vector of the present invention;
FIG. 4 shows the qRT-PCR detection result of the inhibition efficiency of the target gene by the vector of the present invention;
FIG. 5 shows the result of detecting H3Thr3ph inhibition of a target gene by using the vector of the present invention;
FIG. 6 shows the results of cell cycle inhibition assay of target genes using the vectors of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Please refer to fig. 1-4, the invention provides a construction method of bombyx mori shRNA expression vector, which adopts bombyx mori RNA polymerase U6 promoter, respectively introduces gtatcttatcatgtctggat and ggtaccaagctttaaattcg recombination sites to both ends of the promoter, linearizes pIZT/V5-His vector through PCR amplification, recombines and replaces OpIE2 promoter on the vector, needs to synthesize a pair of complementary single long chain DNA in vitro, selects KpnI and EcoRI as restriction sites at both ends, anneals after incubating for 3 minutes at high temperature to form a long double-chain DNA fragment containing cohesive end, connects the carrier after double restriction of KpnI and EcoRI with the double-chain DNA through T4 DNA ligase, clones transforming Escherichia coli, verifies bacterium P, selects correct clone to carry out sequencing verification, synthesizes two single-chain DNA in vitro, forms a long double-chain DNA fragment with cohesive end through in vitro annealing after diluting the fragment, anneals to form the cohesive end of the long-chain DNA fragment, the protruding ends are 4 basic groups which are GTAC and TTAA respectively, the single long chain synthesized in vitro consists of a 21nt hairpin structure and a 6nt stem-loop structure, the 6nt stem-loop structure consists of a CTCGAG sequence, and a DNA fragment formed after annealing can be connected with the cohesive end of the vector constructed in the claim 1 after double enzyme digestion.
A construction method of a silkworm shRNA expression vector comprises the following steps: s1, designing a gene specific recombination primer by using CE DesignV1.04 software, wherein the tail end of the primer contains gtatcttatcatgtctggat and ggtaccaagctttaaattcg recombination sites;
s2, cloning BmU6 promoter specific method is: the method comprises the following steps of using silkworm ovary tissue genome DNA as a template, amplifying a silkworm BmU6 promoter by a PCR technology, and carrying out reaction conditions: pre-denaturation at 95 ℃ for 5 min; 30s at 95 ℃, 30s at 58 ℃ and 30s at 72 ℃ (30 cycles); extension at 72 ℃ for 10 min. Reaction system: 2X Mix: 10 mu L of the solution; primer-F: 1 mu L of the solution; primer-R: 1 mu L of the solution; DNA template: 1 mu L of the solution; ddH 2O: make up to 20 μ L. Detecting the PCR product by agarose gel electrophoresis, and recovering and purifying the gel;
the specific method for linearization of S3 and pIZT/V5-His vector comprises the following steps: the pIZT/V5-His vector is used as a template, the pIZT/V5-His vector is linearized by PCR amplification, a forward primer pIZT-V5-F (CGAATTTAAAGCTTGGTACCGAG), a reverse primer pIZT-V5-R (ATCCAGACATGATAAGATACATTGATGA), and reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; 1min30s at 95 ℃, 2min at 58 ℃ and 2min at 72 ℃ (30 cycles); extension at 72 ℃ for 10min, reaction system: 2X Mix: 10 mu L of the solution; primer-F: 1 mu L of the solution; primer-R: 1 mu L of the solution; DNA template: 1 mu L of the solution; ddH 2O: supplementing to 20 mu L, detecting the PCR product through agarose gel electrophoresis, and recovering and purifying gel;
the specific method for recombining the S4 and pIZT/V5-His-BmU6 vectors comprises the following steps: the fragments recovered in S2 and S3 were calculated in concentration according to vector: the recombination reaction system was calculated at a ratio of 67:20 for the fragment of interest. Reaction conditions are as follows: recombination at 37 ℃ for 40min, and reaction system: 5X buffer: 2 mu L of the solution; and (3) recombinase: 0.5 mu L; pIZT/V5-His linearized vector: 3.5 mu L; BmU6 mesh fragment: 4 mu L of the solution;
the specific method for verifying the S5 and pIZT/V5-His-BmU6 vectors comprises the following steps: transforming the recombinant system of S4 into 100. mu.L of Escherichia coli DH5 alpha competent cells, and carrying out reaction conditions as follows: performing ice bath for 30 min; performing water bath heat shock at 42 ℃ for 90s, immediately performing ice bath for 5min, adding 800 mu L of LB non-resistant liquid culture medium, and incubating at 37 ℃ for 1 h; uniformly coating the transformed cells with proper volume on an LB solid plate culture medium containing corresponding bleomycin, carrying out inverted culture at 37 ℃ for 18h, then selecting a single colony for colony PCR verification, and carrying out positive clone sequencing verification;
s6, the specific method for constructing the target gene shRNA vector comprises the following steps: the shRNA target point information of a target gene is designed through online software, a single long chain which is aimed at a target point sequence and consists of a 21nt hairpin structure and a 6nt annular structure is synthesized in vitro, and a 6nt stem-loop structure consists of a CTCGAG sequence. Through synthesizing sense strand and antisense strand, the double-stranded structure is formed through natural cooling and renaturation after the in vitro heating denaturation of the synthesized sense strand and antisense strand, and the reaction system is as follows: sense strand (1 μ L); antisense strand (1 μ L); annealing Buffer (8 μ L); water (10 μ L), 80 μ L of water was added after the reaction was completed, the annealed product was diluted 5-fold, and the reaction conditions: heating the water bath to 100 ℃, keeping the temperature of the reaction system for 3min, closing the water bath, naturally cooling for about 3h, and cooling to about 30 ℃ to finish annealing;
s7, the tail end of the long double-stranded DNA fragment formed by annealing is a cohesive tail end, the protruding tail end is 4 basic groups which are GTAC and TTAA respectively, the long double-stranded DNA formed by annealing is connected with a pIZT/V5-His-BmU6 vector subjected to double enzyme digestion by KpnI and EcoRI to form a shRNA interference vector of a target gene, and an enzyme digestion system is adopted: 1 μ L each of KpnI and EcoRI, 10 Xbuffer: 2 μ L, pIZT/V5-His-BmU6 vector template 1 μ L, ddH 2O: make up to 20. mu.L, incubate at 37 ℃ for 20min, link system: t4 DNA ligase: 0.5 mu L; t4 DNA Buffer: 1 mu L of the solution; KpnI and EcoRI double digested pIZT/V5-His-BmU6 vector: 1.5 mu L; annealing and diluting fragments: 1 mu L of the solution; ddH 2O: filling to 10 mu L, and connecting for 1h at 22 ℃;
s8, the specific method for verifying the target gene shRNA vector comprises the following steps: the ligation system of S7 was transformed into 100. mu.L E.coli DH 5. alpha. competent cells under the following conditions: performing ice bath for 30 min; performing water bath heat shock at 42 ℃ for 90s, immediately performing ice bath for 5min, adding 800 mu L of LB non-resistant liquid culture medium, and incubating at 37 ℃ for 1 h; the transformed cells with proper volume are evenly coated on an LB solid plate culture medium containing the corresponding bleomycin, inverted culture is carried out at 37 ℃ for 18h, then single colonies are selected for colony PCR verification, and positive clone sequencing verification is carried out.
pIZT/V5-His-BmU6 vector construction
The pIZT/V5-His-BmU6 vector is constructed by taking pIZT/V5-His as a starting vector, and the specific construction method comprises the following steps: the OpIE2 promoter on the pIZT/V5-His vector is replaced by a domestic silkworm BmU6 promoter, and the specific method comprises the following steps: the silkworm ovary tissue genome DNA is adopted as a template, and a silkworm BmU6 promoter is amplified by PCR (forward primer BmU 6-F:gtatctt atcatgtctggatAGGTTATGTAGTACACATTGTTGTAAATCA the recombination sites are underlined; reverse primer BmU 6-R:g gtaccaagctttaaattcgACTTGTAGAGCACGATATTTTGTATATATACC underlined is a recombination site), 2% agarose gel electrophoresis was performed after the PCR reaction was completed, and the result showed that the amplified band length was 467bp (fig. 2A), which is consistent with the expected band size, and the vector was linearized by PCR amplification using pzt/V5-His as a template (forward primer pzt-V5-F: CGAATTTAAAGCTTGGTACCGAG, respectively; the reverse primer pIZT-V5-R: ATCCAGACATGAT AAGATACATTGATGA), performing agarose gel electrophoresis with the concentration of 1% after the PCR reaction is finished, and displaying that the length of an amplified band is 3336bp (figure 2B) which is consistent with the expected band size, performing recombination reaction after the PCR product is purified and recovered, and transforming, identifying and sequencing the recombination reaction product to finally obtain pIZT/V5-His-BmU6 vector (a vector map is shown in figure 1, and the BmU6 promoter sequence is shown in a sequence list 1).
The pIZT/V5-His-BmU6 vector is used for inhibiting the expression of the Haspin gene (gene number: XM _012694372.1) in silkworm cells, and the specific construction method comprises the following steps:
(1) shRNA interference sequence design for inhibiting Haspin gene expression
Inputting the silkworm Haspin gene sequence (shown in a sequence list 2) into shRNA online design software (http:// retrieval. umn. edu/sRecords /) to obtain 8 matched RNA interference sequences with the length of 21bp, and designing three small interference RNAs aiming at the silkworm Haspin gene by combining the verified human Haspin gene shRNA sequence, wherein the sequences are as follows: Bomb-haspin-shRNA-1: GCAATCAAACGGATGCTATAC;
Bomb-haspin-shRNA-2:GCTTTGGATCTACATGCTATA；
Bomb-haspin-shRNA-3:GCAATGATTGGAAGAACTTTG
according to shRNA design scheme, adding a KpnI cohesive end, a 21bp chain, a 6bp stem-loop sequence, a 21bp reverse complementary chain, a transcription termination region TTTT and an EcoRI cohesive end respectively, wherein the sequences of a sense chain and an antisense chain corresponding to the three sequences are as follows:
| BmU6-BmH-shRNA1-F | gtaccGTAATTAAATGGATGTTATATctcgaggTATAgCATCCgTTTgATTgCTTTTTT |
| BmU6-BmH-shRNA1-R | aattAAAAAAGcAATcAAAcGGATGcTATAcctcgagATATAACATCCATTTAATTACg |
| BmU6-BmH-shRNA2-F | gtaccGTTTTGGATTTATATGTTATActcgagTATAgCATgTAgATCCAAAgCTTTTTT |
| BmU6-BmH-shRNA2-R | aattAAAAAAGcTTTGGATcTAcATGcTATActcgagTATAACATATAAATCCAAAACg |
| BmU6-BmH-shRNA3-F | gtaccGTAATGATTGGAAGAACTTTGctcgagCAAAGTTCTTCCAATCATTgCTTTTTT |
| BmU6-BmH-shRNA3-R | aattAAAAAAGcAATGATTGGAAGAACTTTGctcgagCAAAGTTCTTCCAATCATTACg |
the synthesized sequences were dissolved in sterile water without DNA/RNase to 10. mu.M/. mu.L
(2) shRNA interference vector construction for inhibiting silkworm Haspin gene expression
Annealing each pair of sense strand and antisense strand synthesized in step (1) to form double-stranded DNA. 20 μ L of reaction system: sense strand (1 μ L); antisense strand (1 μ L); annealing Buffer (8 μ L); water (10 μ L), reaction conditions: heating the water bath to 100 ℃, keeping the temperature of the reaction system for 3min, closing the water bath, naturally cooling for about 3h, cooling to about 30 ℃ to finish annealing, adding 80 mu L of water after the reaction is finished, and diluting the annealing product by 5 times for later use.
Carrying out KpnI and EcoRI double digestion on pIZT/V5-His-BmU6 vector, connecting the vector with annealed and diluted double-stranded DNA, transforming Escherichia coli DH5 alpha competent cells by a connecting product, screening on a bleomycin resistant plate, verifying positive clones by colony PCR, carrying out sequencing verification, and verifying the correct clone to be named as pIZT/V5-BmU6-BmH-shRNA 1/2/3.
(3) pIZT/V5-BmU6-BmH-shRNA1/2/3 interference vector transfection silkworm BmN cell
The pIZT/V5-BmU6-BmH-shRNA1/2/3 vector constructed in the step (2) is used for transfecting bombyx mori BmN cells by using FUGENE (Promega), the transfection method is operated according to the instructions of the FUGENE, and the transfection efficiency is observed under a fluorescence microscope after 48 hours (figure 3).
(4) Silkworm Haspin gene expression level identification
And (3) after transfection for 48h, respectively collecting cells, extracting total RNA, and carrying out quantitative PCR verification, wherein the primer of the quantitative PCR is BmHaspin-RT-F: GGCGAAGGTGTGTACGGGGAAGT, respectively; BmHaspin-RT-R: AGCGTCAGTAT CATTCCCTTCGTCAA, the internal reference primer is BmA 3-F: ATGTGCGACGAAGAAGTTGC, respectively; BmA 3-R: GTCTCCTACGTACGAGTCCT are provided.
(5) Analysis of H3Thr3ph expression in knockdown cell lines
The pIZT/V5-BmU6-BmH-shRNA1 with the highest knockdown efficiency is selected for the next cytological verification. Knowing that the Haspin gene can catalyze phosphorylation modification of histone H3Thr3 site, H3Thr3ph is recognized by BIR domain on Survivin subunit so as to position a chromosome passenger complex at metaphase centromere to control the smooth proceeding of mitotic cycle, we detected the change of H3Thr3ph in mN Bcell line transfected with pZT/V5-BmU 6-BmH-shRNA1 plasmid (FIG. 5). The specific method comprises the following steps: 48H transfected BmN cells were fixed with 4% paraformaldehyde at room temperature for 15min, rinsed 3 times with 1 XPBS, permeabilized with 0.25% Triton X-100 for 10min, rinsed 3 times with 1 XPBS, blocked with 3% Bovine Serum Albumin (BSA) at room temperature for 1H, rinsed 3 times with 1 XPBS, incubated with histone H3Thr3 phosphorylated antibody (1:500 dilution, v/v) overnight at 4 ℃, and incubated with Alexa Fluor 594 labeled Goat Anti-Rabbit IgG (Goat Anti-Rabbit IgG, Jackson ImmunoResearch) (1: 2000, v/v) for 2H at 37 ℃. Finally, 4', 6-diamidino-2-phenylindole (DAPI,0.01mg/mL + 90% glycerol) is mounted in a mounting plate, and a ZEISS laser confocal microscope is used for observing a fluorescence signal.
(6) Analysis of cell cycle phenotypic changes in knockdown cell lines
To further explore the effect of the alteration of H3Thr3ph expression by Haspin gene knock-down on cell cycle, we combined immunofluorescence with cell slide and seen cells transfected with pIZT/V5-BmU6-BmH-shRNA1 plasmid 48H in more abnormal states as shown in FIG. 6.
BmU6 DNA sequence information:
>BmU6-2
AGGTTATGTAGTACACATTGTTGTAAATCACTGAATTGTTTTAGATGATTTTAACAATTAGTACTTATTAATATTAAATAAGTACATACCTTGAGAATTTAAAAATCGTCAACTATAAGCCATACGAATTTAAGCTTGGTACTTGGCTTATAGATAAGGACAGAATAAGAATTGTTAACGTGTAAGACAAGGTCAGATAGTCATAGTGATTTTGTCAAAGTAATAACAGATGGCGCTGTACAAACCATAACTGTTTTCATTTGTTTTTATGGATTTTATTACAAATTCTAAAGGTTTTATTGTTATTATTTAATTTCGTTTTAATTATATTATATATCTTTAATAGAATATGTTAAGAGTTTTTGCTCTTTTTGAATAATCTTTGTAAAGTCGAGTGTTGTTGTAAATCACGCTTTCAATAGTTTAGTTTTTTTAGGTATATATACAAAATATCGTGCTCTACAAGT
silkworm Haspin gene sequence information:
Bombyx mori putative serine/threonine-protein kinase haspin homolog(LOC101745149),mRNA。XM_012694372.1
ATGCATCTTACCCTGCAATACACAGGATTTTTGAGCGACGATTGTGATGATACGATTGTCGGGTTATCAAAACTCTCGCTCGATGATGTGGAACCGGAGATCACCGTTCTAGGTATACATGATACTTCGAATCGTGTTGCTACAGCTCGAGATTACGTGTTACGTCGATGCAATCAAACGGATGCTATACTATTTGATGAATGCTATCCAGACGCACTTTTGAAAAACTGTCACAAGATTGGCGAAGGTGTGTACGGGGAAGTATTTCTTTGGCGTGCTCGAGATGGCAGAGCTCGAGTAATGAAAATAGTTCCAATTGCTGGTCATACCAAGGTCAATGGAGAAGACCAAAAGGACTATCACGAGATTATCTCGGAAATTGTGATTGCTATGGAATTGAGCGCCCTGCGCGCTCCCATAGCCGATATCGAACGACATTTTGACGAAGGGAATGATACTGACGCTTTGGATCTACATGCTATAGGCAATGCAACGGATGTTTTTAATCAGGTTCTAGCAGTAAGGTGTGTGTACGGCAGCTACCCATCACGTTTGCTAGATCTCTGGGACTTGTACGACGAATGTAAAGGTTCCGAGAACGACAATCCGGCCATCTTGCCCGTCGATCAACAATACATCGTACTAGAGCTGGCCAACGCCGGACAAGATTTGGAGAGCTACCAGTTCAACAACGCTGAACAAGCACACGCGCTGTTCCTCCAGGTGGCATTCGGTCTGGCCGTGGGCGAGGAGGCCTACCAGTTCGAGCATCGCGATCTGCACTGGGGAAACGTTCTCATCGCGCCCACCGAACAAAAGTTTGCCACGTTCGTGCTCCGCGGGCGGCGGCACTGCACGCCCCGCTGCGGCGTCGCGGCCACCATCATCGACTACTCGCTGTCGCGCGTGTCGCTGCCGGCGAGCGCGCCCGCACGCTGCGCCGCGCTCTACAACGACCTGGCGGACGACGACGGACTCTTCGACGCCGTCGGAGACTATCAGTTTGAGGTCTACCGCCTCATGAAAAGCAAGTTGGGCAATGATTGGAAGAACTTTGAACCATATACAAACATACTGTGGCTACATTACACTGTTGATAAAATGATAACGGCCCTACGCTACAAAAGAACCAATACAAAGATACACAAGCATTACATAGACAAATTGAAGGGCATCAAGAATAGAATTCTCGATTACAAGAGTGCAACCGATTTTGTTTTAACGGACAACGAATATTAA。
although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Sequence listing
<110> south Yang college of learning
<120> construction method and application of bombyx mori shRNA expression vector
<130> ZD11111
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 467
<212> DNA
<213> Bombyx mori
<400> 1
aggttatgta gtacacattg ttgtaaatca ctgaattgtt ttagatgatt ttaacaatta 60
gtacttatta atattaaata agtacatacc ttgagaattt aaaaatcgtc aactataagc 120
catacgaatt taagcttggt acttggctta tagataagga cagaataaga attgttaacg 180
tgtaagacaa ggtcagatag tcatagtgat tttgtcaaag taataacaga tggcgctgta 240
caaaccataa ctgttttcat ttgtttttat ggattttatt acaaattcta aaggttttat 300
tgttattatt taatttcgtt ttaattatat tatatatctt taatagaata tgttaagagt 360
ttttgctctt tttgaataat ctttgtaaag tcgagtgttg ttgtaaatca cgctttcaat 420
agtttagttt ttttaggtat atatacaaaa tatcgtgctc tacaagt 467
Claims (7)
1. A construction method of a silkworm shRNA expression vector is characterized by comprising the following steps: a bombyx mori RNA polymerase U6 promoter is adopted, gtatcttatcatgtctggat recombination sites and ggtaccaagctttaaattcg recombination sites are respectively introduced into two ends of the promoter, and an OpIE2 promoter on a replacement vector is recombined after pIZT/V5-His vector is linearized through PCR amplification.
2. The method for constructing bombyx mori shRNA expression vector according to claim 1, which is characterized in that: only one pair of complementary single-long-chain DNA needs to be synthesized in vitro, KpnI and EcoRI are selected at two ends as enzyme cutting sites, a long double-chain DNA fragment containing a viscous tail end is formed by annealing after high-temperature incubation for 3 minutes, a carrier subjected to double enzyme cutting by KpnI and EcoRI and the double-chain DNA are connected through T4 DNA ligase, a bacterium P is verified after cloning and transforming escherichia coli, and correct clone is selected for sequencing verification.
3. The method for constructing bombyx mori shRNA expression vector according to claim 1, which is characterized in that: two single-stranded DNAs are synthesized in vitro, and long double-stranded DNA fragments with cohesive ends are formed by in vitro annealing after diluting the fragments.
4. The method for constructing bombyx mori shRNA expression vector according to claim 1, which is characterized in that: annealing to form long-chain DNA fragment with sticky end and 4 bases as protruding end, GTAC and TTAA.
5. The method for constructing bombyx mori shRNA expression vector according to claim 1, which is characterized in that: the single long chain synthesized in vitro consists of a 21nt hairpin structure and a 6nt stem-loop structure, and the 6nt stem-loop structure consists of a CTCGAG sequence.
6. The method for constructing bombyx mori shRNA expression vector according to claim 1, which is characterized in that: the DNA fragment formed after annealing can be ligated to the cohesive ends of the vector constructed in claim 1 after double digestion.
7. The method for constructing the bombyx mori shRNA expression vector according to claim 1, which comprises the following steps: s1, designing a gene specific recombination primer by using CE DesignV1.04 software, wherein the tail end of the primer contains gtatcttatcatgtctggat and ggtaccaagctttaaattcg recombination sites;
s2, cloning BmU6 promoter specific method is: the method comprises the following steps of using silkworm ovary tissue genome DNA as a template, amplifying a silkworm BmU6 promoter by a PCR technology, and carrying out reaction conditions: pre-denaturation at 95 ℃ for 5 min; 30s at 95 ℃, 30s at 58 ℃ and 30s at 72 ℃ (30 cycles); extension at 72 ℃ for 10 min. Reaction system: 2X Mix: 10 mu L of the solution; primer-F: 1 mu L of the solution; primer-R: 1 mu L of the solution; DNA template: 1 mu L of the solution; ddH 2O: make up to 20 μ L. Detecting the PCR product by agarose gel electrophoresis, and recovering and purifying the gel;
the specific method for linearization of S3 and pIZT/V5-His vector comprises the following steps: the pIZT/V5-His vector is used as a template, the pIZT/V5-His vector is linearized by PCR amplification, a forward primer pIZT-V5-F (CGAATTTAAAGCTTGGTACCGAG), a reverse primer pIZT-V5-R (ATCCAGACATGATAAGATACATTGATGA), and reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; 1min30s at 95 ℃, 2min at 58 ℃ and 2min at 72 ℃ (30 cycles); extension at 72 ℃ for 10 min. Reaction system: 2X Mix: 10 mu L of the solution; primer-F: 1 mu L of the solution; primer-R: 1 mu L of the solution; DNA template: 1 mu L of the solution; ddH 2O: supplementing to 20 mu L, detecting the PCR product through agarose gel electrophoresis, and recovering and purifying gel;
the specific method for recombining the S4 and pIZT/V5-His-BmU6 vectors comprises the following steps: the fragments recovered in S2 and S3 were calculated in concentration according to vector: calculating a recombination reaction system according to the ratio of the target fragment 67:20, wherein the reaction conditions are as follows: recombination at 37 ℃ for 40min, and reaction system: 5X buffer: 2 mu L of the solution; and (3) recombinase: 0.5 mu L; pIZT/V5-His linearized vector: 3.5 mu L; BmU6 mesh fragment: 4 mu L of the solution;
the specific method for verifying the S5 and pIZT/V5-His-BmU6 vectors comprises the following steps: transforming the recombinant system of S4 into 100. mu.L of Escherichia coli DH5 alpha competent cells, and carrying out reaction conditions as follows: performing ice bath for 30 min; performing water bath heat shock at 42 ℃ for 90s, immediately performing ice bath for 5min, adding 800 mu L of LB non-resistant liquid culture medium, and incubating at 37 ℃ for 1 h; uniformly coating the transformed cells with proper volume on an LB solid plate culture medium containing corresponding bleomycin, carrying out inverted culture at 37 ℃ for 18h, then selecting a single colony for colony PCR verification, and carrying out positive clone sequencing verification;
s6, the specific method for constructing the target gene shRNA vector comprises the following steps: designing shRNA target point information of a target gene through online software, synthesizing a single long chain which is composed of a 21nt hairpin structure and a 6nt annular structure and aims at a target point sequence in vitro, synthesizing a 6nt stem-loop structure by a CTCGAG sequence, and naturally cooling and renaturing the synthesized sense chain and antisense chain after in vitro heating denaturation to form a double-chain structure, wherein the reaction system comprises: sense strand (1 μ L); antisense strand (1 μ L); annealing Buffer (8 μ L); water (10 μ L). After the reaction is finished, 80 mu L of water is added, the annealing product is diluted by 5 times, and the reaction conditions are as follows: heating the water bath to 100 ℃, keeping the temperature of the reaction system for 3min, closing the water bath, naturally cooling for about 3h, and cooling to about 30 ℃ to finish annealing;
s7, the tail end of the long double-stranded DNA fragment formed by annealing is a cohesive tail end, the protruding tail end is 4 basic groups which are GTAC and TTAA respectively, the long double-stranded DNA formed by annealing is connected with a pIZT/V5-His-BmU6 vector subjected to double enzyme digestion by KpnI and EcoRI to form a shRNA interference vector of a target gene, and an enzyme digestion system is adopted: 1 μ L each of KpnI and EcoRI, 10 Xbuffer: 2 μ L, pIZT/V5-His-BmU6 vector template 1 μ L, ddH 2O: make up to 20. mu.L, incubate at 37 ℃ for 20min, link system: t4 DNA ligase: 0.5 mu L; t4 DNA Buffer: 1 mu L of the solution; KpnI and EcoRI double digested pIZT/V5-His-BmU6 vector: 1.5 mu L; annealing and diluting fragments: 1 mu L of the solution; ddH 2O: filling to 10 mu L, and connecting for 1h at 22 ℃;
s8, the specific method for verifying the target gene shRNA vector comprises the following steps: the ligation system of S7 was transformed into 100. mu.L E.coli DH 5. alpha. competent cells under the following conditions: performing ice bath for 30 min; performing water bath heat shock at 42 ℃ for 90s, immediately performing ice bath for 5min, adding 800 mu L of LB non-resistant liquid culture medium, and incubating at 37 ℃ for 1 h; the transformed cells with proper volume are evenly coated on an LB solid plate culture medium containing the corresponding bleomycin, inverted culture is carried out at 37 ℃ for 18h, then single colonies are selected for colony PCR verification, and positive clone sequencing verification is carried out.
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