CN110791528A - MicroRNA gene editing method for improving silk yield and optimizing silkworm variety - Google Patents
MicroRNA gene editing method for improving silk yield and optimizing silkworm variety Download PDFInfo
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Abstract
The invention belongs to the technical field of silkworms and silks, and discloses a method for improving the yield of silks and optimizing silkworm varieties by a genome editing technology, which comprises the following steps: and (3) carrying out sgRNA transgenic expression vector and sgRNA expression strain screening on let-7microRNA, screening a let-7 knockout strain, identifying a knockout form, and carrying out statistical analysis on the silk gland length, weight and cocoon weight after the let-7 knockout. The sgRNA expression strain is respectively hybridized with a middle silk gland and a rear silk gland Cas9 expression strain to obtain a let-7 middle silk gland specific knockout strain [ delta let-7-MSG ] and a let-7 rear silk gland specific knockout strain [ delta let-7-PSG ]; the development of silk glands and silk yield of the knockout strain silkworms are affected.
Description
Technical Field
The invention belongs to the technical field of silkworms and silks, and particularly relates to a microRNA gene knockout and screening method for improving silk yield and optimizing silkworm varieties.
Background
Conventional breeding methods commonly used include artificial mutagenesis and crossbreeding. Artificial mutation breeding refers to treating organisms by means of chemical mutagenesis or physical mutagenesis, inducing the genetic characters of the organisms to generate mutation, and screening out valuable mutants. Although artificial mutagenesis increases the frequency of genetic trait variation, induced mutation is non-directional, favorable variation is few, and the breeding process requires handling of a large number of mutant varieties or materials, which is a heavy task. And cross breeding refers to a breeding method for obtaining heterosis by crossing between different varieties in a species or between different species with relatively close relationships. The hybrid breeding can lead the dominant characters among different varieties to be continuously accumulated in hybrid varieties, but the hybridization and the screening both need to consume a large amount of time, and the obtained related characters are unstable in filial generations. Silkworm is an important economic insect and has the capability of quickly synthesizing a large amount of silk protein. The silk gland is an important organ for synthesizing and secreting silk protein of the silkworm, and the silk gland rapidly grows and synthesizes a large amount of silk protein in the period of five-instar larva of the silkworm. After the frame map and the fine map of the silkworm genome are published, people expect to obtain silkworm varieties with high silk output by controlling key genes of silk gland development and silk protein synthesis. With the development of molecular biology and genetic engineering technology, the genes are modified or regulated by means of efficient and stable transgenosis or gene editing, and the improvement of the economic traits of the silkworms at the genome level becomes possible.
Transgene interference (transgene RNAi) is a side branch of the transgene method, which does not transfer exogenous genes into organisms, but expresses a segment of double-stranded RNA (dsRNA) by the transgene method. The dsRNA is sheared in an organism to form siRNA to degrade mRNA of a specific gene, thereby exerting the post-transcriptional regulation function of the gene. Researchers often improve disease resistance or stress resistance of organisms by inhibiting susceptible genes or negative regulatory genes of the organisms to pathogens, so that the purposes of improving survival rate and yield are achieved. However, the transgene interference efficiency is affected by factors such as double-strand expression quantity and sequence specificity, the interference effect on the target gene is not expected, and the regulation function of the gene cannot be exerted.
CRISPR/Cas9 is a defense mechanism acquired by bacteria and archaea during long-term evolution to resist the invasion of viruses or foreign DNA. The exogenous DNA fragment is integrated into CRISPR, transcribed to form pre-crRNA, and then cleaved to crRNA, and the complex formed by crRNA and trans-acting crRNA (tracrRNA) can guide Cas9 protein to cleave the exogenous DNA double strand complementarily paired with crRNA. The artificially synthesized sgRNA can replace the function of a crRNA/tracrRNA compound, the sgRNA guides Cas9 to perform site-specific cleavage, and the cleaved double-stranded DNA is reconnected under the repair of a non-homologous end repair mechanism in an organism, but in the process, deletion, insertion, replacement and the like of basic groups can occur, and for a coding gene, the changes can probably cause the frame shift mutation of the gene, so that the aim of gene knockout is fulfilled. Because sgrnas have advantages of simple design, rapid synthesis, high efficiency after being combined with Cas9, and the like, CRISPR/Cas9 has been widely applied in the fields of gene function research and disease treatment.
microRNA (miRNA) is a single-stranded small RNA molecule endogenous to an organism and not encoding protein, and the length of the single-stranded small RNA molecule is about 22 nucleotides. The miRNA is shown to participate in the regulation of most of life activities and play an important post-transcriptional regulation function. let-7 mirnas are highly conserved across different species. Knocking out let-7 in silkworm silk gland cells by using a CRISPR/Cas9 technology can promote the development of silk glands, increase the expression of silk protein and improve the silk yield of silkworms, and the overexpression of let-7 in rear silk glands can inhibit the development of silk glands to obtain sericin cocoons.
In summary, the problems of the prior art are as follows:
(1) the artificial mutation is non-directional mutation, so that the variation is less, a large amount of mutant varieties or materials need to be processed in the breeding process, and the workload is large. The crossing and screening of cross breeding both need to consume a lot of time, and the obtained related characters are unstable in the filial generation.
(2) The transgene interference is influenced by factors such as double-chain expression quantity, sequence specificity and the like, the interference effect on the target gene is not expected, and the regulation and control function of the gene cannot be exerted.
(3) miRNA is used as small molecular non-coding RNA with the length of 21 nt-23 nt, and the function of the miRNA is difficult to regulate and control in a transgenic interference mode. The chemically synthesized inhibitor can effectively inhibit the function of mature miRNA, but is high in price, short in time-effect and not suitable for production application.
(4) Although the silk yield can be effectively improved through conventional transgenic over-expression of exogenous genes, national evaluation on the entry of transgenic species into production is relatively strict, and a long auditing period is required.
The difficulty of solving the technical problems is as follows:
transgenic RNA interference is only suitable for down-regulating the function of coding genes, and for promoting silk gland development or improving silk production yield by using a transgenic RNA interference technology, a key negative regulation gene for silk gland development or silk protein synthesis must be found, which is rarely reported. miRNA is used as a common negative regulatory factor and is involved in numerous life activity processes. By down-regulating miRNA, the negative regulation effect of miRNA is relieved, and the development of silk gland and the expression of silk protein are possibly promoted. The amount of miRNA mature bodies in cells is down-regulated, and effective methods comprise synthesis and injection of miRNA inhibitors, miRNA 'sponge' adsorption and miRNA gene knockout. Besides chemical synthesis, miRNA inhibitors can also be overexpressed by viruses, but the viruses also have great influence on the growth of silkworms and are not suitable. The miRNA sponge adsorption also needs the transgenic technology, the difficulty is similar to gene editing, but the miRNA knockout effect cannot be achieved.
The significance of solving the technical problems is as follows:
finds out the negative regulation miRNA-let-7 which is the key for the development of the silk gland of the silkworm, and realizes the knockout of the let-7 at different parts of the silk gland. The method does not modify the coding gene and does not introduce a new foreign gene. In silkworm silk gland cells, the CRISPR/Cas9 technology is used to delete the expression of let-7 non-coding RNA, thus releasing the negative regulation and control effects on silk gland development and silk protein synthesis and realizing the improvement of silk yield of silkworms. For example, after knocking out let-7 in posterior silk gland, the length of posterior silk gland is increased by nearly 100%, weight is increased by 150%, diameter of posterior silk gland is increased by 4 times, and weight of silkworm cocoon is increased by 10% after spinning. Since the silk gland can complete tissue digestion immediately after silking of the silkworm until the silk gland disappears completely, the let-7 knockout in the silk gland is not passed to offspring, and other species are not affected by gene communication among species. Therefore, the technology is safe and reliable in production and has more advantages.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a gene for improving the silk yield and optimizing the silkworm variety and a screening method.
The gene for improving the silk yield and optimizing the silkworm variety is realized in the way that the gene for improving the silk yield and optimizing the silkworm variety is a let-7 knockout strain [ let-7-2gRNA ], and the DNA sequence of the let-7-2gRNA is SEQ ID NO: 1.
the invention also aims to provide a gene screening method for improving the silk yield and optimizing silkworm varieties, which comprises the following steps:
the method comprises the following steps: constructing an sgRNA transgenic expression vector;
step two: screening an sgRNA expression strain;
step three: a let-7 knockout strain [ let-7-2gRNA ] is screened;
step four: identification of the knockout form: designing primers at the upstream and downstream of pre-let-7, amplifying a pre-let-7 sequence, and identifying the change of the silkworm silk gland cell genome DNA by a sequencing method;
step five: after let-7 knockout, silk gland phenotype was analyzed.
Further, in the first step, the specific steps of constructing the sgRNA transgenic expression vector include:
(1) inputting a let-7 secondary precursor sequence into a CCtop online prediction website, and searching gRNA spacer (SEQ ID NO: 5-8) in the sequence by using the structure of G (N20) GG; adding corresponding terminal base, and then synthesizing a primer (SEQ ID NO: 5-8) by a gene;
(2) allowing the primers to anneal to form a double strand: denaturation at 95 deg.C for 5min, gradient cooling to 25 deg.C at 95 deg.C, and decreasing by 0.1 deg.C per second for 700 cycles;
(3) preparing a system, digesting at 37 ℃ overnight, and recovering a pUC57[ U6-2gRNA ] vector framework;
(4) double chains formed by annealing sgRNA1-spacer are connected to a pUC57[ U6-2gRNA ] vector framework to construct a pUC57[ U6-sgRNA1] plasmid;
(5) preparing a system, carrying out enzyme digestion at 37 ℃ for 4h, and recovering a pUC57[ U6-sgRNA1] vector framework;
(6) double chains formed by annealing sgRNA2-spacer are connected to a pUC57[ U6-sgRNA1] vector framework to construct pUC57[ U6, let-7-2gRNA ] plasmids;
(7) preparing a system, carrying out enzyme digestion at 37 ℃ for 4h, and recovering a [ U6, let-7-2gRNA ] fragment;
(8) connecting the recovered let-7 knockout double gRNA expression fragment [ U6, let-7-2gRNA ] to a piggyBac [3xP3-EGFP ] vector to construct a let-7 knockout double gRNA transgenic expression vector: pBac [3xp3-EGFP, let-7-2gRNA ].
Further, in the second step, the specific steps of sgRNA expression line screening include:
(1) extracting pBac [3xp3-EGFP, let-7-2gRNA ] ultrapure plasmids, and mixing the plasmids with an auxiliary vector pHA3PIG ultrapure plasmid for expressing piggyBac transposase according to a molar ratio of 1: 1; injecting the mixed plasmid into D9L silkworm eggs, and then sealing the injection hole by using non-toxic instantaneous adhesive;
(2) putting the injected silkworm eggs into a biochemical incubator with the temperature of 25 ℃ and the relative humidity of 90 percent for hatching, and feeding the silkworm larvae with fresh mulberry leaves after 9-10 days to obtain G0 generation larvae;
(3) breeding G0 larvae to adults and selfing to obtain G1 generation eggs, carrying out green acceleration to the 6 th day, and carrying out transgene positive individual screening; positive individuals of embryos in the G1 generation are [ let-7-2gRNA ];
(4) g1 generation is raised to adult, the adult compound eye is screened again by fluorescence, and the screened positive individuals are used for subsequent hybridization experiments.
Further, in the third step, a let-7 knockout product [ delta let-7-MSG ] and a let-7 rear silk gland knockout product [ delta let-7-PSG ] are used. The screening method specifically comprises the following steps:
(1) hybridizing the [ let-7-2gRNA ] strain with a middle silk gland Cas9 expression strain [ Cas9-MSG ] and a rear silk gland Cas9 expression strain [ Cas9-PSG ] respectively;
(2) placing the hybridized F1 silkworm eggs into a biochemical incubator with the temperature of 25 ℃ and the relative humidity of 90% for carrying out green-forcing, and carrying out reporter gene screening when the green-forcing is carried out for 5-6 days; and obtaining a let-7 middle part silk gland knockout individual [ delta let-7-MSG ] and a let-7 rear part silk gland knockout individual [ delta let-7-PSG ] from F1 generation embryos.
Further, in the fourth step, the knockout form is identified in the treasury of the specific steps:
(1) feeding the screened knockout strains [ delta let-7-MSG ] and [ delta let-7-PSG ] larvae to five instars by using fresh mulberry leaves, and dissecting to obtain silk gland materials;
(2) extracting total DNA of silk gland cells by using a tissue DNA extraction kit;
(3) designing a primer to amplify a target site sequence, and connecting the target site sequence to a pMD-19-T simple vector for sequencing; the primer comprises: let-7 KO-F: CGTCATCAAGGATCCAGCAGT, respectively; let-7 KO-R: TTAGTGGTCTTGTGAGGAATGTT, respectively;
(4) the sequences were aligned and analyzed using BioEidt software to identify the number of base deletions.
Further, in the fifth step, after the let-7 knockout, the step of analyzing the silk gland phenotype specifically comprises:
(1) breeding the screened [ delta let-7-MSG ] and [ delta let-7-PSG ] larvae with fresh mulberry leaves until the fifth instar is from the 5 th day to the sixth day;
(2) silk glands were dissected, phenotypes observed and photographed.
Further, after the fifth step, the following steps are also required: the method comprises the following steps of carrying out statistical analysis on the silk gland length, weight and cocoon weight:
(1) breeding the screened knockout strain [ delta let-7-MSG ] and [ delta let-7-PSG ] larvae with fresh mulberry leaves until the fifth instar is from the 5 th day to the sixth day;
(2) dissecting the posterior silk gland to measure the length and weight;
(3) continuously feeding the screened knockout strains [ delta let-7-MSG ] and [ delta let-7-PSG ] larvae to spinning and cocooning; pupating the larva, weighing and comparing the weight of the silkworm cocoon after spinning is finished.
In summary, the advantages and positive effects of the invention are:
the sgRNA expression strain is respectively crossed with a middle silk gland and a rear silk gland Cas9 expression strain to obtain a strain with let-7 specifically knocked out in the middle silk gland [ delta let-7-MSG ] and a strain with let-7 specifically knocked out in the rear silk gland [ delta let-7-PSG ]. All the development of silk glands of silkworms in the knockout strain is affected, and the RNAi result is more stable and credible compared with that of different genes of silkworms.
Because of the importance of the function, the let-7 of the invention can cause the death of silkworm larvae when the silkworm knocks out or interferes with the whole body. By using a transgenic method, a line for knocking out sgRNA used by let-7 is independently established. Through hybridization with different Cas9 expression strains, the knockout of let-7 in different silk gland sections is realized, the normal growth of silkworms and the spinning and cocooning of silkworms are not inhibited, and the expansion of silk gland cells and the synthesis and secretion of silk proteins are promoted.
The technology has the significance that negative regulation miRNA-let-7 which is key to development of the silkworm silk gland is discovered for the first time, let-7 is knocked out at different parts of the silk gland, and silk gland specific knockout strains [ delta let-7-MSG ] and [ delta let-7-PSG ] are obtained. The method does not modify the coding gene and does not introduce a new foreign gene. But in the silk gland cells of the silkworms, the CRISPR/Cas9 technology is utilized to delete the expression of let-7 non-coding RNA, thereby releasing the negative regulation and control effects on silk gland development and silk protein synthesis and realizing the improvement of silk yield of the silkworms. For example, after knocking out let-7 in posterior silk gland, the length of posterior silk gland is increased by nearly 100%, weight is increased by 150%, diameter of posterior silk gland is increased by 4 times, and weight of silkworm cocoon is increased by 10% after spinning. Since the silk gland can complete tissue digestion immediately after silking of the silkworm until the silk gland disappears completely, the let-7 knockout in the silk gland is not passed to offspring, and other species are not affected by gene communication among species. Therefore, the technology is safe and reliable in production and has more advantages.
Drawings
FIG. 1 is a flow chart of a screening method for improving silk yield and optimizing genes of silkworm varieties according to an embodiment of the present invention.
Fig. 2 is a schematic diagram of the construction of a sgRNA transgenic expression vector provided in an embodiment of the present invention.
Fig. 3 is a schematic diagram of sgRNA expression line screening provided in an embodiment of the present invention.
Fig. 4 is a schematic diagram of let-7 knockout strain [ let-7-2gRNA ] screening provided in the embodiment of the present invention.
FIG. 5 is a schematic representation of the identification of a knockout form provided by embodiments of the invention.
FIG. 6 is a schematic diagram of a silk gland phenotype observation after a let-7 knockout is provided by an embodiment of the invention.
FIG. 7 is a schematic diagram of statistical analysis of silk gland length, weight and cocoon weight provided by the embodiment of the invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
In the prior art, transgene interference and transgene interference efficiency are influenced by factors such as double-strand expression quantity, sequence specificity and the like, the interference effect on a target gene cannot be expected, and the regulation and control function of the gene cannot be realized.
To solve the above problems, the following description will explain the application principle of the present invention in detail with reference to the accompanying drawings.
The gene for increasing the yield of silk and optimizing the variety of the silkworm provided by the embodiment of the invention is a let-7 knockout strain [ let-7-2gRNA ], and the DNA sequence of the let-7-2gRNA is SEQ ID NO: 1.
as shown in fig. 1, the screening method for improving silk yield and optimizing the genes of the silkworm varieties provided by the embodiment of the present invention specifically includes the following steps:
s101: and constructing an sgRNA transgenic expression vector.
S102: sgRNA expression line screening: the sgRNA transgene expression plasmid is injected into newly laid silkworm eggs by a microinjection method, successfully hatched larvae are fed by fresh mulberry leaves to obtain G0 generation adults, and the G0 generation adults are mated to obtain G1 generation silkworm eggs. And carrying out green fluorescence screening when the silkworm eggs are induced to be green for 5-6 days.
S103: let-7 knockout strain [ let-7-2gRNA ] is screened.
S104: identification of the knockout form: primers are designed on the upstream and downstream of pre-let-7, the sequence is amplified, and the change of the silkworm silk gland cell genome DNA is identified by a sequencing method.
S105: after let-7 knockout, silk gland phenotype was observed.
S106: statistical analysis was performed on silk gland length, weight and cocoon weight.
In step S101, the construction of the sgRNA transgene expression vector provided in the embodiment of the present invention specifically includes the steps of:
(1) and (3) inputting a let-7 secondary precursor sequence into a CCtop online prediction website, and searching a gRNA spacer in the sequence by using the structure of G (N20) GG to avoid sequences with high off-target rate. After addition of the corresponding terminal base, the primers were synthesized.
(2) The primers were allowed to anneal to form double strands under the following conditions: denaturation at 95 ℃ for 5min, gradient cooling at 95 ℃ to 25 ℃ and 0.1 ℃ per second reduction for a total of 700 cycles.
(3) The following system was prepared, and pUC57[ U6-2gRNA ] vector backbone was recovered after digestion overnight at 37 ℃.
(4) Double chains formed by annealing sgRNA1-spacer were ligated to pUC57[ U6-2gRNA ] vector backbone to construct pUC57[ U6-sgRNA1] plasmid.
(5) The following system was prepared, and after digestion at 37 ℃ for 4 hours, the pUC57[ U6-sgRNA1] vector backbone was recovered.
(6) Double chains formed by annealing sgRNA2-spacer are connected to a pUC57[ U6-sgRNA1] vector framework to construct pUC57[ U6, let-7-2gRNA ] plasmid.
(7) The following system is prepared, enzyme digestion is carried out for 4h at 37 ℃, and then [ U6, let-7-2gRNA ] fragments are recovered.
(8) Connecting the recovered let-7 knockout double gRNA expression fragment [ U6, let-7-2gRNA ] to a piggyBac [3xP3-EGFP ] vector to construct a let-7 knockout double gRNA transgenic expression vector: pBac [3xp3-EGFP, let-7-2gRNA ].
In step S102, the sgRNA expression strain screening provided by the embodiment of the present invention specifically includes:
(1) extracting pBac [3xp3-EGFP, let-7-2gRNA ] ultrapure plasmid, and mixing with auxiliary vector pHA3PIG ultrapure plasmid which is stored in a laboratory and expresses piggyBac transposase according to the molar ratio of 1: 1. The mixed plasmid is injected into D9L silkworm eggs within one hour after egg laying by a microinjection instrument, and then an injection hole is sealed by non-toxic instant adhesive.
(2) And (3) putting the injected silkworm eggs into a biochemical incubator with the temperature of 25 ℃ and the relative humidity of 90% for hatching, and after 9-10 days, feeding the silkworm larvae with fresh mulberry leaves, wherein the silkworm larvae are G0 generation larvae.
(3) Breeding the G0 generation larvae to adults and selfing to obtain G1 generation eggs, carrying out green acceleration to the 6 th day, and screening transgenic positive individuals by using a macroscopic electric fluorescence microscope. The green-emitting individuals of the embryonic eyes in the G1 generation are positive individuals and are named as [ let-7-2gRNA ].
(4) G1 generation is raised to adult, the adult compound eye is screened again by green fluorescence, and the screened positive individuals are used for subsequent hybridization experiments.
In step S103, the let-7 knockout strain [ let-7-2gRNA ] provided in the embodiment of the present invention is screened, specifically including the steps of:
(1) the [ let-7-2gRNA ] strain was crossed with the middle silk gland Cas9 expression strain [ Cas9-MSG ] and the rear silk gland Cas9 expression strain [ Cas9-PSG ], respectively.
(2) And (3) placing the hybridized F1 silkworm eggs into a biochemical incubator with the temperature of 25 ℃ and the relative humidity of 90% for carrying out incubation, and screening the reporter genes by using a macroscopic electric fluorescence microscope when the incubation lasts for 5-6 days. The F1 generation embryo eyes emitting green light and red light simultaneously are let-7 middle part silk gland knockout individual [ delta let-7-MSG ] and let-7 rear part silk gland knockout individual [ delta let-7-PSG ].
In step S104, the identification of the knockout form provided by the embodiment of the present invention specifically includes:
(1) feeding the screened [ delta let-7-MSG ] and [ delta let-7-PSG ] larvae with fresh mulberry leaves to five instars, and dissecting to obtain silk gland materials.
(2) And extracting total DNA of the silk gland cells by using a tissue DNA extraction kit.
(3) Primers were designed to amplify the target site sequence and ligated into the pMD-19-T simple vector for sequencing. Let-7 KO-F:
CGTCATCAAGGATCCAGCAGT SEQ ID NO：3
>let-7KO-RTTAGTGGTCTTGTGAGGAATGTT SEQ ID NO：4。
(4) the sequences were aligned and analyzed using BioEidt software to identify the number of base deletions.
In step S105, observing the silk gland phenotype after the let-7 provided by the embodiment of the present invention is knocked out, specifically including the steps of:
(1) and breeding the screened [ delta let-7-MSG ] and [ delta let-7-PSG ] larvae with fresh mulberry leaves until the fifth-instar larvae grow from the 5 th day to the sixth day.
(2) Silk glands were dissected for phenotype and photographed with a camera.
In step S106, the statistical analysis of the silk gland length, weight and cocoon weight provided by the embodiment of the present invention specifically includes the steps of:
(1) and breeding the screened [ delta let-7-MSG ] and [ delta let-7-PSG ] larvae with fresh mulberry leaves until the fifth-instar larvae grow from the 5 th day to the sixth day.
(2) The posterior silk gland was dissected and its length and weight were measured.
(3) Continuously feeding the screened [ delta let-7-MSG ] and [ delta let-7-PSG ] larvae until the larvae spin and cocoon. Pupating the larva, weighing and comparing the weight of the silkworm cocoon after spinning is finished.
The application of the principles of the present invention will be further illustrated with reference to specific embodiments.
Example 1.
As shown in fig. 2, a schematic diagram of a sgRNA transgene expression vector construction provided in the embodiments of the present invention.
In the figure: A. a suitable sgRNA targeting site was found on let-7 secondary precursor (pre-let-7), consistent with the G (N20) GG structure.
B. The sgRNA expression vector simultaneously expresses two sgRNAs by using a U6 promoter, a promoter 3XP3 specifically expressed in eyes and nerves is selected as a screening marker to start green fluorescent EGFP, and a lepidoptera insect transposon piggyBac is used as a transgenic basic skeleton of the transgenic vector.
The method comprises the following steps:
(1) let-7 secondary precursor sequences are input into a CCtop online prediction website (https:// crispr. cos. uni-heidelberg. de /), and gRNA spacers in the sequences are searched by the structure of G (N20) GG, so that the sequences with high miss ratio are avoided. After adding corresponding terminal base, sending a primer for synthesizing the Huahua big gene:
(2) the primers were allowed to anneal to form double strands under the following conditions: denaturation at 95 ℃ for 5min, gradient cooling at 95 ℃ to 25 ℃ and 0.1 ℃ per second reduction for a total of 700 cycles.
(3) Preparing the following system, digesting at 37 ℃ overnight, and recovering a pUC57[ U6-2gRNA ] (the vector is a double gRNA expression vector) vector framework:
(4) double chains formed by annealing sgRNA1-spacer were ligated to pUC57[ U6-2gRNA ] vector backbone to construct pUC57[ U6-sgRNA1] plasmid.
(5) The following system was prepared, and after digestion at 37 ℃ for 4h, the pUC57[ U6-sgRNA1] vector backbone was recovered:
(6) double chains formed by annealing sgRNA2-spacer are connected to a pUC57[ U6-sgRNA1] vector framework to construct pUC57[ U6, let-7-2gRNA ] plasmid.
(7) Preparing the following system, carrying out enzyme digestion at 37 ℃ for 4h, and recovering [ U6, let-7-2gRNA ] fragments:
(8) connecting the recovered let-7 knockout double gRNA expression fragment [ U6, let-7-2gRNA ] to a piggyBac [3xP3-EGFP ] vector to construct a let-7 knockout double gRNA transgenic expression vector: pBac [3xp3-EGFP, let-7-2gRNA ].
As shown in fig. 3, the sgRNA expression line screening provided in the present embodiment is schematically illustrated.
The sgRNA transgene expression plasmid is injected into newly laid silkworm eggs by a microinjection method, successfully hatched larvae are fed by fresh mulberry leaves to obtain G0 generation adults, and the G0 generation adults are mated to obtain G1 generation silkworm eggs. And carrying out green fluorescence screening when the silkworm eggs are induced to be green for 5-6 days.
A # represents positive eggs screened at G1 generation and emitting green light to eyes. B.b # indicates positive adults at passage G1.
The method comprises the following steps:
(1) extracting pBac [3xp3-EGFP, let-7-2gRNA ] ultrapure plasmid, and mixing with auxiliary vector pHA3PIG ultrapure plasmid which is stored in a laboratory and expresses piggyBac transposase according to the molar ratio of 1: 1. The mixed plasmid is injected into D9L silkworm eggs within one hour after egg laying by a microinjection instrument, and then an injection hole is sealed by non-toxic instant adhesive.
(2) And (3) putting the injected silkworm eggs into a biochemical incubator with the temperature of 25 ℃ and the relative humidity of 90% for hatching, and after 9-10 days, feeding the silkworm larvae with fresh mulberry leaves, wherein the silkworm larvae are G0 generation larvae.
(3) Breeding the G0 generation larvae to adults and selfing to obtain G1 generation eggs, carrying out green acceleration to the 6 th day, and screening transgenic positive individuals by using a macroscopic electric fluorescence microscope. Because EGFP fluorescent protein is used as a reporter gene, green light emitting individuals of embryo eyes in the G1 generation are positive individuals and are named as [ let-7-2gRNA ].
(4) G1 generation is raised to adult, the adult compound eye is screened again by green fluorescence, and the screened positive individuals are used for subsequent hybridization experiments.
As shown in FIG. 4, a schematic diagram of screening of specific knockout strains [ Δ let-7-MSG ] and [ Δ let-7-PSG ] of let-7microRNA provided by the embodiment of the invention in silkworm silk glands is shown.
In the figure: A. [ let-7-2gRNA ] individuals. B. [ Cas9-PSG ] individual. C. The individuals expressing green light and red light simultaneously are hybrid individuals, namely let-7 rear silk gland knockout individuals [ delta let-7-PSG ].
The method comprises the following steps:
(1) the [ let-7-2gRNA ] strain was crossed with the middle silk gland Cas9 expression strain [ Cas9-MSG ] and the rear silk gland Cas9 expression strain [ Cas9-PSG ], respectively.
(2) And (3) placing the hybridized F1 silkworm eggs into a biochemical incubator with the temperature of 25 ℃ and the relative humidity of 90% for carrying out incubation, and screening the reporter genes by using a macroscopic electric fluorescence microscope when the incubation lasts for 5-6 days. The F1 generation embryo eyes emitting green light and red light simultaneously are let-7 middle part silk gland knockout individual [ delta let-7-MSG ] and let-7 rear part silk gland knockout individual [ delta let-7-PSG ].
As shown in FIG. 5, a schematic representation of the identification of a knockout form provided by embodiments of the invention.
Primers are designed on the upstream and downstream of pre-let-7, the sequence is amplified, and the change of the silkworm silk gland cell genome DNA is identified by a sequencing method. Compared with a normal sequence, the two different sgRNA target points are subjected to different forms of base deletion, and the deletion is different from 2 to 83 bases.
The method comprises the following steps:
(1) feeding the screened [ delta let-7-MSG ] and [ delta let-7-PSG ] larvae with fresh mulberry leaves to five instars, and dissecting to obtain silk gland materials.
(2) And extracting total DNA of the silk gland cells by using a tissue DNA extraction kit.
(3) Primers were designed to amplify the target site sequence and ligated into the pMD-19-T simple vector for sequencing. Let-7 KO-F: CGTCATCAAGGATCCAGCAGT > let-7 KO-R: TTAGTGGTCTTGTGAGGAATGTT are provided.
(4) The sequences were aligned and analyzed using BioEidt software to identify the number of base deletions.
As shown in fig. 6, a schematic diagram of silk gland phenotype observation after let-7 knockout is provided in the present invention.
In the figure: A. after the [ let-7-2gRNA ] individuals and the [ Cas9-MSG ] individuals are hybridized, four different light-emitting forms appear, and comparison of the central silk glands of the four different light-emitting forms shows that: [ Delta let-7-MSG ] the posterior part of the middle silk gland of the individual is obviously enlarged.
B. After the [ let-7-2gRNA ] individuals and the [ Cas9-PSG ] individuals are hybridized, four different light-emitting forms appear, and the comparison of the rear silk glands of the four different light-emitting forms shows that: [ Delta let-7-PSG ] individual posterior silk gland is obviously lengthened and thickened.
The method comprises the following steps:
(1) and breeding the screened [ delta let-7-MSG ] and [ delta let-7-PSG ] larvae with fresh mulberry leaves until the fifth instar is 5-6 days.
(2) Silk glands were dissected for phenotype and photographed with a camera.
As shown in fig. 7, a schematic diagram of statistical analysis of silk gland length, weight and cocoon weight provided by the embodiment of the present invention is provided.
A. [ Delta let-7-PSG ] the posterior silk gland length of the individual increased by 60%.
B. [ Delta let-7-PSG ] the posterior silk gland weight of the individual increased 150%.
C. [ Delta let-7-PSG ] individual cocoon weight gain 12%.
D. [ Delta let-7-MSG ] individual cocoon weight reduction 26%.
The method comprises the following steps:
(1) and breeding the screened [ delta let-7-MSG ] and [ delta let-7-PSG ] larvae with fresh mulberry leaves until the fifth instar is 5-6 days.
(2) The posterior silk gland was dissected and its length and weight were measured.
(3) Continuously feeding the screened [ delta let-7-MSG ] and [ delta let-7-PSG ] larvae until the larvae spin and cocoon. Pupating the larva, weighing and comparing the weight of the silkworm cocoon after spinning is finished.
Through hybridizing with different Cas9 expression strains, knockout of let-7 in different tissues is realized, and normal growth and spinning and cocooning of silkworms are not influenced.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Sequence listing
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Claims (8)
1. The non-coding RNA knockout strain for increasing the silk yield and optimizing the silkworm variety is characterized in that the non-editing RNA for increasing the silk yield and optimizing the silkworm variety is let-7microRNA, the knockout strains in the middle silk gland and the rear silk gland of the silkworm are [ delta let-7-MSG ] and [ delta let-7-PSG ] respectively, and the DNA sequence of the let-7-2gRNA for knockout is SEQ ID NO. 1.
2. The sgRNA vector, let-7sgRNA expression strain screening method and knockout strain screening method for increasing silk yield and optimizing silkworm varieties according to claim 1, wherein the sgRNA vector and knockout strain screening method for increasing silk yield and optimizing silkworm varieties comprises:
the method comprises the following steps: constructing an sgRNA transgenic expression vector of let-7;
step two: let-7sgRNA expression strain screening;
step three: screening the let-7 knockout strains delta let-7-PSG and delta let-7-MSG;
step four: identification of the knockout form: designing primers on the upstream and downstream of pre-let-7, amplifying a pre-let-7 sequence, and identifying sequence change of let-7 sites on the DNA of the silk gland cell genome of the silkworm through sequencing;
step five: after let-7 knockout, the aberrant phenotype of the silk gland was analyzed.
3. The method for screening the knockout line of the silkworm for improving the yield of silk and optimizing the variety of silkworms according to claim 2, wherein in the first step, the specific steps of constructing the sgRNA transgenic expression vector comprise:
(1) inputting a let-7 secondary precursor sequence SEQ ID NO 2 into a CCtop online prediction website, and finding a gRNA spacer SEQ ID NO 5-8 in a sequence by using the structure of G (N20) GG; adding corresponding basic groups and let-7sgRNA primers SEQ ID NO of 5-8 into the 5' -end of the gRNA spacer;
(2) allowing the primers to anneal to form a double strand: denaturation at 95 deg.C for 5min, gradient cooling to 25 deg.C at 95 deg.C, and decreasing by 0.1 deg.C per second for 700 cycles;
(3) preparing a system, digesting at 37 ℃ overnight, and recovering a pUC57[ U6-2gRNA ] vector framework sequence SEQ ID NO 9;
(4) double chains formed by annealing sgRNA1-spacer are connected to a pUC57[ U6-2gRNA ] vector framework to construct a pUC57[ U6-sgRNA1] plasmid;
(5) preparing a system, carrying out enzyme digestion at 37 ℃ for 4h, and recovering a pUC57[ U6-sgRNA1] vector framework;
(6) double chains formed by annealing sgRNA2-spacer are connected to a pUC57[ U6-sgRNA1] vector framework to construct pUC57[ U6, let-7-2gRNA ] plasmids;
(7) preparing a system, carrying out enzyme digestion at 37 ℃ for 4h, and recovering a [ U6, let-7-2gRNA ] fragment;
(8) connecting the recovered let-7 knockout double gRNA expression fragment [ U6, let-7-2gRNA ] to a piggyBac [3xP3-EGFP ] vector to construct a let-7 knockout double gRNA transgenic expression vector: pBac [3xp3-EGFP, let-7-2gRNA ].
4. The method for screening genes for improving silk yield and optimizing silkworm varieties according to claim 1, wherein in the second step, the specific steps of screening sgRNA expression strains comprise:
(1) extracting pBac [3xp3-EGFP, let-7-2gRNA ] ultrapure plasmids and auxiliary vector pHA3PIG ultrapure plasmids for expressing piggyBac transposase according to a molar ratio of 1:1, mixing; injecting the mixed plasmid into D9L silkworm eggs, and then sealing the injection hole by using non-toxic instantaneous adhesive;
(2) putting the injected silkworm eggs into a biochemical incubator with the temperature of 25 ℃ and the relative humidity of 90 percent for hatching, and feeding the silkworm larvae with fresh mulberry leaves after 9-10 days to obtain G0 generation larvae;
(3) breeding G0 larvae to adults and selfing to obtain G1 generation eggs, carrying out green acceleration to the 6 th day, and carrying out transgene positive individual screening; positive individuals of embryos in the G1 generation are [ let-7-2gRNA ];
(4) g1 generation is raised to adult, the adult compound eye is screened again by fluorescence, and the screened positive individuals are used for subsequent hybridization experiments.
5. The gene screening method for improving silk yield and optimizing silkworm varieties according to claim 1, wherein in the third step, the screening of the let-7 knockout strains [ Δ let-7-MSG ] and [ Δ let-7-PSG ] comprises the following specific steps:
(1) hybridizing the [ let-7-2gRNA ] strain with a middle silk gland Cas9 expression strain [ Cas9-MSG ] and a rear silk gland Cas9 expression strain [ Cas9-PSG ] respectively;
(2) placing the F1 silkworm eggs obtained by hybridization into a biochemical incubator with the temperature of 25 ℃ and the relative humidity of 90% for carrying out green-forcing, and carrying out reporter gene screening when the green-forcing is carried out for 5-6 days; and obtaining a let-7 middle part silk gland knockout individual [ delta let-7-MSG ] and a let-7 rear part silk gland knockout individual [ delta let-7-PSG ] from F1 generation embryos.
6. The method for screening genes for improving silk yield and optimizing silkworm varieties according to claim 1, wherein in the fourth step, the identification of the knockout form comprises the following specific steps:
(1) feeding the screened [ delta let-7-MSG ] and [ delta let-7-PSG ] larvae to five instars by using fresh mulberry leaves, and dissecting to obtain silk gland materials;
(2) extracting total DNA of silk gland cells by using a tissue DNA extraction kit;
(3) designing a primer to amplify a target site sequence, and connecting the target site sequence to a pMD-19-T simple vector for sequencing; the primer comprises: let-7 KO-F: CGTCATCAAGGATCCAGCAGT, respectively;
>let-7KO-R：TTAGTGGTCTTGTGAGGAATGTT；
(4) the sequences were aligned and analyzed using BioEidt software to identify the number of base deletions.
7. The method for screening genes for improving silk yield and optimizing silkworm varieties according to claim 1, wherein in the fifth step, after the let-7 knockout, the specific steps of analyzing the silk gland phenotype comprise:
(1) breeding the screened [ delta let-7-MSG ] and [ delta let-7-PSG ] larvae with fresh mulberry leaves to the 6 th day of five instars;
(2) silk glands were dissected, phenotypes observed and photographed.
8. The method for screening genes for improving silk yield and optimizing silkworm varieties according to claim 1, wherein after the fifth step, the following steps are carried out: the method comprises the following steps of carrying out statistical analysis on the silk gland length, weight and cocoon weight:
(1) breeding the screened [ delta let-7-MSG ] and [ delta let-7-PSG ] larvae with fresh mulberry leaves to the 6 th day of five instars;
(2) dissecting the posterior silk gland to measure the length and weight;
(3) continuously feeding the screened [ delta let-7-MSG ] and [ delta let-7-PSG ] larvae until the larvae spin and cocoon; pupating the larva, weighing and comparing the weight of the silkworm cocoon after spinning is finished.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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| CN114480509A (en) * | 2022-03-04 | 2022-05-13 | 西南大学 | Transgenic method for preparing pure sericin cocoon by inducing complete degradation of silkworm silk secretion organs and silkworm variety |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005054494A2 (en) * | 2003-11-26 | 2005-06-16 | University Of Massachusetts | Sequence-specific inhibition of small rna function |
| CN101139597A (en) * | 2007-08-07 | 2008-03-12 | 西南大学 | Cultivated silkworm metamorphosis development switch gene bmo-let-7 |
| CN106222203A (en) * | 2016-08-10 | 2016-12-14 | 云南纳博生物科技有限公司 | CRISPR/Cas technology is utilized to obtain bombyx mori silk fibroin heavy chain gene mutant and mutation method and application |
| CN106636208A (en) * | 2016-11-10 | 2017-05-10 | 西南大学 | Method of utilizing bombyx mori nuclear polyhedrosis virus vector system to excessively express microRNA |
| CN107760715A (en) * | 2017-10-17 | 2018-03-06 | 云南瑞火生物科技有限公司 | A kind of precisely efficient transgene carrier and its construction method and application |
| US20190111179A1 (en) * | 2016-11-21 | 2019-04-18 | Nantong University | Construction of microrna gene-mediated novel tissue engineered nerve and applications thereof in repairing nerve defect |
-
2019
- 2019-08-15 CN CN201910754929.6A patent/CN110791528B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005054494A2 (en) * | 2003-11-26 | 2005-06-16 | University Of Massachusetts | Sequence-specific inhibition of small rna function |
| CN101139597A (en) * | 2007-08-07 | 2008-03-12 | 西南大学 | Cultivated silkworm metamorphosis development switch gene bmo-let-7 |
| CN106222203A (en) * | 2016-08-10 | 2016-12-14 | 云南纳博生物科技有限公司 | CRISPR/Cas technology is utilized to obtain bombyx mori silk fibroin heavy chain gene mutant and mutation method and application |
| CN106636208A (en) * | 2016-11-10 | 2017-05-10 | 西南大学 | Method of utilizing bombyx mori nuclear polyhedrosis virus vector system to excessively express microRNA |
| US20190111179A1 (en) * | 2016-11-21 | 2019-04-18 | Nantong University | Construction of microrna gene-mediated novel tissue engineered nerve and applications thereof in repairing nerve defect |
| CN107760715A (en) * | 2017-10-17 | 2018-03-06 | 云南瑞火生物科技有限公司 | A kind of precisely efficient transgene carrier and its construction method and application |
Non-Patent Citations (16)
| Title |
|---|
| SHIPING LIU等: "Characterization and expression patterns of let-7 microRNA in the silkworm (Bombyx mori)", 《BMC DEVELOPMENTAL BIOLOGY》 * |
| SHIPING LIU等: "Characterization and expression patterns of let-7 microRNA in the silkworm (Bombyx mori)", 《BMC DEVELOPMENTAL BIOLOGY》, 25 July 2007 (2007-07-25), pages 88, XP021027775, DOI: 10.1186/1471-213X-7-88 * |
| WEI WANG等: "Let-7 microRNA is a critical regulator in controlling the growth and function of silk gland in the silkworm", 《RNA BIOLOGY》 * |
| WEI WANG等: "Let-7 microRNA is a critical regulator in controlling the growth and function of silk gland in the silkworm", 《RNA BIOLOGY》, 4 February 2020 (2020-02-04), pages 703 - 717 * |
| YUANYUAN LIU等: "Tissue-specific genome editing of laminA/C in the posterior silk glands of Bombyx mori", 《JOURNAL OF GENETICS AND GENOMICS》 * |
| YUANYUAN LIU等: "Tissue-specific genome editing of laminA/C in the posterior silk glands of Bombyx mori", 《JOURNAL OF GENETICS AND GENOMICS》, 14 September 2017 (2017-09-14), pages 451 - 459 * |
| 何婷 等: "利用Bac-to-Bac杆状病毒系统超量表达家蚕let-7簇microRNAs", 《中国农业科学》 * |
| 何婷 等: "利用Bac-to-Bac杆状病毒系统超量表达家蚕let-7簇microRNAs", 《中国农业科学》, 31 January 2016 (2016-01-31), pages 581 - 592 * |
| 凌琳: "家蚕发育相关microRNA let-7和miR-2的功能研究", 《中国博士论文全文数据库》 * |
| 凌琳: "家蚕发育相关microRNA let-7和miR-2的功能研究", 《中国博士论文全文数据库》, 18 January 2019 (2019-01-18) * |
| 周挺: "家蚕let-7 miRNA的靶基因克隆和激素诱导", 《中国优秀硕士学位论文全文数据库（电子期刊）基础科学辑》 * |
| 周挺: "家蚕let-7 miRNA的靶基因克隆和激素诱导", 《中国优秀硕士学位论文全文数据库（电子期刊）基础科学辑》, 15 December 2011 (2011-12-15), pages 006 - 91 * |
| 王伟: "let-7 microRNA调控家蚕丝腺发育的分子机制", 《中国博士学位论文全文数据库（电子期刊）农业科技辑》 * |
| 王伟: "let-7 microRNA调控家蚕丝腺发育的分子机制", 《中国博士学位论文全文数据库（电子期刊）农业科技辑》, 15 May 2020 (2020-05-15), pages 051 - 2 * |
| 陈燕飞: "家蚕tRNA-isopentenyltransferase（BmIPT）基因的克隆及其功能研究", 《中国优秀硕士学位论文全文数据库（电子期刊）基础科学辑》 * |
| 陈燕飞: "家蚕tRNA-isopentenyltransferase（BmIPT）基因的克隆及其功能研究", 《中国优秀硕士学位论文全文数据库（电子期刊）基础科学辑》, 15 January 2019 (2019-01-15), pages 55 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114480509A (en) * | 2022-03-04 | 2022-05-13 | 西南大学 | Transgenic method for preparing pure sericin cocoon by inducing complete degradation of silkworm silk secretion organs and silkworm variety |
| CN114480509B (en) * | 2022-03-04 | 2023-09-05 | 西南大学 | Transgenic method for preparing pure sericin cocoons by inducing complete degeneration of silk-secreting organs of silkworms and silkworm varieties thereof |
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