CN112301055B - Application and method of silkworm amylase gene BmAmy1 - Google Patents
Application and method of silkworm amylase gene BmAmy1 Download PDFInfo
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- CN112301055B CN112301055B CN202011216765.0A CN202011216765A CN112301055B CN 112301055 B CN112301055 B CN 112301055B CN 202011216765 A CN202011216765 A CN 202011216765A CN 112301055 B CN112301055 B CN 112301055B
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Abstract
The invention discloses application and a method of silkworm amylase gene BmAmy1, wherein the nucleotide sequence of silkworm amylase gene BmAmy1 is shown as SEQ ID NO.3, the sequence characteristics are analyzed, the tissue and period expression characteristics are detected, BmAmy1 protein is expressed and purified through a yeast eukaryotic expression system, and the amylase activity of BmAmy1 is verified through an in vitro enzyme activity experiment; then constructing a silkworm transgenic vector, preparing a transgenic silkworm, investigating and analyzing the economic characters of the transgenic strain, and finding out that the BmAmy1 transgenic strain is superior to a control in the aspects of individual size, full cocoon weight, cocoon layer rate and the like, and the amount of residual starch in the silkworm excrement is obviously less than that of a non-transgenic control. Therefore, the BmAmy1 can be over-expressed in the domestic silkworms by a biotechnology means, so that the aim of improving economic properties such as the weight of the whole silkworm cocoon and the cocoon shell rate can be fulfilled, and a new direction is opened for cultivating more excellent domestic silkworm varieties.
Description
Technical Field
The invention relates to the technical field of biology, in particular to application of a silkworm amylase gene BmAmy1, and further relates to a specific application method.
Background
The efficiency of feed utilization by animals directly determines the economic benefits of the animal husbandry. Insects utilize their digestive systems to draw various nutrients and other substances required for their life activities from the food they obtain. Substances in food are mostly present in the form of macromolecules and other complex substances such as proteins, polysaccharides, fats and nucleic acids. These macromolecules must be broken down by catabolic reactions into smaller molecules such as amino acids and monosaccharides, which are then taken up by body cells for growth, development and reproduction. This process of decomposition is called digestion. The digestive tract of insects can be divided into foregut, midgut and hindgut. Most digestion occurs in the middle intestine, where there are abundant digestive enzymes, the nature of which is related to the nature of the insect diet. Carnivorous insects secrete mainly proteases, while phytophagous insects secrete more carbohydrases. Since the food eaten by insects contains a large amount of macromolecular polymers such as starch, protein, cellulose, hemicellulose and the like, most foods rely on the digestion of insects to obtain nutrients therein. The process of digesting these macromolecular polymers is generally divided into three stages, the early stage, the middle stage and the late stage. The primary digestion process mainly utilizes the action of polymer hydrolytic enzymes to reduce the molecular weight of food macromolecules, and the enzymes involved in the primary digestion mainly comprise alpha-amylase, trypsin, cellulase and hemicellulase. The oligomers obtained by the initial digestion are further hydrolyzed by middle-stage poly-hydrolytic enzyme or oligomer hydrolytic enzyme, and the digestive enzymes involved in the process mainly comprise alpha-amylase, aminopeptidase and the like. After the intermediate digestion dimers or smaller oligomers are obtained, such as dipeptides, maltose and cellobiose. Later digestion is with dimer hydrolases (e.g., peptidases, maltases and cellobiases) to break down dimers into monomers. The digestion process is usually carried out in the presence of digestive enzymes of the middle intestine, the staged digestion of which is associated with compartmentalization of the middle intestine (cells, inside and outside the peritrophic membrane) and the corresponding digestive enzymes. The digestive process is usually regulated by digestive enzymes and depends on its location in the insect gut. Generally, initial digestion of food is carried out within the peritrophic membrane, followed by intermediate digestion outside the peritrophic membrane, and later digestion occurs on the surface of the midgut cells, with enzymes in the microvilli or enzymes entrapped in sugar groups.
Alpha-amylase (1, 4-alpha-D-Glucan-glucanohydrolase; EC 3.2.1.1) is an enzyme class that hydrolyzes carbohydrate starch, with the systematic name 1, 4-alpha-D-Glucan hydrolase. It is widely distributed in animals (saliva, pancreas, digestive juice, etc.), plants (malt, behenic, etc.) and microorganisms. This enzyme is an endonuclease, which acts on both amylose and amylopectin as an essential factor and serves as a stabilizing factor, and which cleaves an α -1, 4-glycosidic bond in a sugar chain without distinction, resulting in high hydrolysis efficiency. The end product of the hydrolysis of amylose consists mainly of maltose and, in addition, small amounts of maltotriose and glucose, and the hydrolysis of amylopectin produces, in addition to maltose and glucose, the limiting dextrins containing alpha-1, 6-glycosidic bonds. Digestion and absorption of starch in food by animals are carried out by first decomposing starch into small linear and branched maltooligosaccharides by alpha-amylase, then hydrolyzing the starch into monosaccharides such as glucose by the action of mucosal cell alpha-glucosidase, maltose-glucoamylase (MGAM) and sucrose-isomaltase (SI), and absorbing the monosaccharides by intestinal wall cells. Alpha-amylase activity has been demonstrated in a variety of insects, including orthoptera, hemiptera, heteroptera, hymenoptera, diptera, lepidoptera, and coleoptera, among others. Most insect amylases have molecular weight of 48-60kDa and pI of 3.5-4.0, have a Michaelis constant of 0.1% in the case of soluble starch, and belong to calcium ion-dependent amylases. The most extensively studied insect amylase gene at present is the yellow mealworm amylase gene. The protein structure of this enzyme has been resolved and comprises A, B and C3 functional protein domains, of which domain a comprises catalytic amino acid residues and is the core domain. The substrate binding site is located between a long V-groove between the a and B domains, which can accommodate 6 monosaccharide units on the polysaccharide, and the amylase functions between the 3 rd and 4 th monosaccharide units of the sugar chain. The B domain binds a calcium ion that is critical to the structural integrity of the enzyme.
With the rapid development of molecular biology and sequencing technologies, sequences of a-amylase gene of a large number of insects were determined and functional studies were performed. In 1997, alpha-amylase protein was first purified from crude extract of coleoptera yellow meal beetle larvae by Stefan stroll et al, and the alpha-amylase contains 471 amino acid residues, has a sequence identity of 57-79% with other insect alpha-amylases, and has been analyzed for protein activity and crystal structure. The research work on silkworm amylase is the detection of physiological and biochemical activities of enzyme in intestinal fluid at present, and the research work on silkworm amylase gene sequences and protein functions is rarely reported at present. Nipaporn Ngernyuang has cloned a silkworm amylase gene in the Changlai silkworm variety Nanglai in Thailand, the ORF total length of the gene is 1503bp, 500 amino acids are coded, the similarity of the gene and amylase coding protein sequences of other insect sources is 81-54%, and the similarity of the gene and amylase coding protein sequences from mammals is more than 50%. The gene is a single copy gene, and expression characteristic analysis shows that the gene is specifically expressed in foregut. However, the functions of more silkworm amylase genes are to be researched, and a foundation is laid for cultivating more excellent silkworm varieties.
Disclosure of Invention
In view of the above, one of the objectives of the present invention is to provide an application of silkworm amylase gene bmamay 1 in breeding high-yield silkworm varieties; the second purpose of the invention is to provide the application of the protein coded by the silkworm amylase gene BmAmy1 in catalyzing starch to generate reducing sugar; the invention also aims to provide a method for cultivating the high-yield silkworm variety.
In order to achieve the purpose, the invention provides the following technical scheme:
1. the application of silkworm amylase gene BmAmy1 in breeding high-yield silkworm varieties is characterized in that the nucleotide sequence of the silkworm amylase gene BmAmy1 is shown as SEQ ID NO. 3.
Preferably, the silkworm amylase gene BmAmy1 is applied to improving the weight of the whole silkworm cocoon.
Preferably, the silkworm amylase gene BmAmy1 is applied to improving the cocoon layer rate of silkworms.
Preferably, the silkworm amylase gene BmAmy1 is applied to increasing the size of silkworm individuals.
Preferably, the silkworm amylase gene BmAmy1 is applied to reducing the residual starch content in silkworm excrement.
2. The application of the protein encoded by the silkworm amylase gene BmAmy1 in catalyzing starch to generate reducing sugar is disclosed, wherein the nucleotide sequence of the silkworm amylase gene BmAmy1 is shown as SEQ ID NO. 3.
3. A method for cultivating high-yield silkworm varieties is characterized in that silkworm amylase genes BmAmy1 are overexpressed in silkworms, and the nucleotide sequence of the silkworm amylase genes BmAmy1 is shown as SEQ ID No. 3.
Preferably, the step of overexpressing the silkworm amylase gene bmamay 1 is as follows: constructing a transgenic vector containing silkworm amylase gene BmAmy1, mixing the transgenic vector with an auxiliary vector pHA3PIG, injecting the mixture into early-stage embryos of silkworms with diapause removed, carrying out incubation until the silkworms hatch to obtain transgenic G0 generation silkworms, feeding G0 generation silkworms to adults, carrying out selfing to obtain G1 generation silkworms, and screening to obtain the transgenic silkworms.
Preferably, the construction method of the transgenic vector containing the silkworm amylase gene BmAmy1 is as follows: the nucleotide sequence of the silkworm amylase gene BmAmy1 shown in SEQ ID NO.3 is connected into a pSLfa1180fa vector containing P3P +5UI, DsRed and SV40 through BamH I and Not I to obtain a pSL1180[ P3P +5UI-BmAmy1] vector, then the obtained pSL1180[ P3P +5UI-BmAmy1] vector and piggyBac [3 xP 3-DsRed ] are subjected to single enzyme digestion by Asc I respectively, and a target fragment and a vector framework are recovered and then connected to obtain a transgenic vector containing the silkworm amylase gene BmAmy 1.
Preferably, the method for cloning the silkworm amylase gene BmAmy1 is as follows: the gene is obtained by taking sequences shown in SEQ ID NO.10 and SEQ ID NO.11 as primers and a vector containing silkworm amylase gene BmAmy1 as a template for amplification.
The invention has the beneficial effects that: the invention provides a novel silkworm amylase gene BmAmy1, which is characterized in that the sequence characteristics of the silkworm amylase gene BmAmy1 are analyzed, the expression characteristics of the silkworm amylase gene in tissue and period are detected, a yeast eukaryotic expression system is used for expressing and purifying BmAmy1 protein, and the amylase activity of BmAmy1 is verified through an in vitro enzyme activity experiment. Based on the piggyBac transposon system, a silkworm transgenic vector which drives BmAmy1 to be specifically expressed in the midgut by a P3P promoter is constructed, and a transgenic positive strain is obtained by embryo microinjection. The economic characters of the transgenic line are investigated and analyzed, and the result shows that the BmAmy1 transgenic line is superior to a control in the aspects of individual size, full cocoon weight, cocoon layer rate and the like, and the amount of residual starch in silkworm excrement is obviously less than that of a non-transgenic control. Therefore, the aim of improving economic traits such as the whole cocoon weight, cocoon layer rate and the like can be realized by expressing BmAmy1 in excess in the domestic silkworm by a biotechnology means, and the BmAmy1 is shown to have important production and application potentials. In addition, the invention also opens up a new way for cultivating better silkworm varieties.
Drawings
In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 shows PCR detection of the coding region of Bombyx mori BmAmy1 gene.
FIG. 2 is the protein domain prediction of BmAmy 1.
FIG. 3 shows the alignment result of the amino acid sequences of the alpha-amylase of Bombyx mori and other species.
FIG. 4 is the silkworm alpha-amylase genetic relationship evolutionary tree clustering analysis.
FIG. 5 shows the transcriptional profiling of BmAmy1 based on qRT-PCR (A: tissue expression profile of BmAmy1 (day 3 of five years old), B: expression profile of BmAmy1 at the time of Bombyx mori midgut, He: head; Fb: adipose body; Sg: silk gland; Hc: blood cells; Ep: epidermis; Mg: midgut; Go: gonad; Mt: Mars tube; positive deviation indicates Standard Error (SEM); P <0.01(t test))
FIG. 6 is a PCR assay of pPICZ α A-BmAMY 1 recombinant vector after yeast transformation.
FIG. 7 shows the results of protein purification by BmAmy1 and western blot detection (M: protein molecular weight standard; Sup: culture supernatant; FT: flow-through).
FIG. 8 shows the enzyme activity assay of BmAmy1 (A: glucose standard curve; B: enzyme activity assay result).
FIG. 9 shows the restriction enzyme digestion verification during the construction of the transgene vector (A: pSL1180[ P3P +5UI-BmAMY1] vector double restriction enzyme digestion detection, B: piggyBac [3 XP 3-DsRed, P3P +5UI-BmAMY1] vector Asc I single restriction enzyme digestion detection M: DNA Marker, Lane 1: pSL1180[ P3P +5UI-BmAMY1] vector, Lane 2: BamH I and NotI double restriction enzyme digestion product of pSL1180[ P3P +5UI-BmAMY1] vector, Lane 5: piggyBac [3 XP 3-DsRed, P3P +5UI-BmAMY1] vector, Lane 6: piggyBac [3 XP 3-DsRed, P3P + 5-BmAMY 1] Asc vector.
FIG. 10 shows the BmAmy1 transgenic positive screen and its expression profile at transgenic silkworms (A. eyes of transgenic positive silkworms show red fluorescence under fluorescence microscope; B. expression profile of BmAmy1 gene at transgenic silkworms He: head; Fb: adipose body; Sg: silk gland; Hc: blood cells; Ep: epidermis; Mg: midgut; Mt: Ma's tube; positive deviation indicates Standard Error (SEM); P <0.001(t test)).
FIG. 11 shows the statistical analysis of the economic traits of transgenic silkworms (A. transgenic silkworms on the fourth day of five ages compared with non-transgenic individual silkworms; B. transgenic silkworm weight on the fourth day of five ages; C. transgenic silkworm cocoons compared with non-transgenic silkworm cocoons; D. transgenic silkworm cocoon layer rate statistical analysis; length of white vertical bar is1 cm; positive deviation represents Standard Error (SEM); P0.01; P0.001 (t test).
Detailed Description
The present invention is further described with reference to the following drawings and specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention.
The silkworm variety used in the embodiment of the invention is bivoltine Dazao (Dazao) and is reserved in the laboratory. Silkworm larvae are bred by using freshly picked mulberry leaves under the conditions that the ambient temperature is 25 ℃ and the relative humidity is 75% +/-5%. Dissecting and collecting materials on the 3 rd day of fifth instar of silkworm larva, respectively collecting head, fat body, silk gland, blood cell, epidermis, midgut, gonad and Mariothis tube, quickly freezing with liquid nitrogen, and storing in a refrigerator at-80 deg.C.
Example 1 identification, cloning and expression profiling of the Bombyx mori BmAmy1 Gene
1. Identification of silkworm alpha-amylase gene (BmAmy1)
The reported amino acid sequences of the silkworm alpha-amylase gene and the alpha-amylase genes of other species are downloaded at NCBI to be used as a search seed sequence, and a silkworm genome database (SilkDB) is utilized to carry out Blast comparison identification (Blast parameters are set as Blast) to obtain a candidate sequence. Then, the candidate sequences are manually screened, and finally, the structural domain is predicted on a SMART website (http:// smart.embl-heidelberg. de /), so as to obtain the nucleic acid and protein sequences of the BmAmy 1.
2. BmAmy1 multiple sequence alignment and phylogenetic analysis
According to the reported literature and NCBI database retrieval, protein sequences of alpha-amylase genes of a plurality of species are downloaded, Clustalx is adopted for multi-sequence comparison, and GeneDoc software is utilized for comparison to carry out graphical display on files. Phylogenetic analysis was performed using MEGA X software, the method of construction of the tree was Neighbor-Joining (Neighbor-Joining), and Bootstrap was set to 1,000.
3. RNA extraction
Taking out the tissue material from a refrigerator at minus 80 ℃ before dissection and collection, putting the tissue material into a mortar fully pre-cooled by liquid nitrogen, and carefully grinding for 3-4 times until the material is in a fine powder state. An appropriate amount of tissue powder was put into a 1.5mL centrifuge tube, and then 1mL of TRIzol solution was added thereto, and the mixture was shaken and mixed, followed by extraction of RNA according to the manual of TRIzol reagent. And adding 40-50 mu L DEPC water into the white flocculent RNA precipitate obtained by the last step of centrifugation, after the RNA precipitate is completely dissolved, flicking and uniformly mixing, measuring and recording the concentration and purity of the RNA, and storing the mixture in a refrigerator at the temperature of-80 ℃ for later use. The cDNA was prepared using a kit from Beijing Quanji corporation. The amplification conditions were: 30min at 42 ℃; 85 ℃, 5s (inactivation of TransScript RT/RI and gDNA Remover). The 20. mu.L cDNA was diluted 5-fold, dispensed and stored at-20 ℃ for further use.
4. Cloning of the coding region of the BmAmy1 Gene
According to the full-length sequence of the gene BmAmy1 identified by informatics, a gene-specific Primer BmAmy1-F was designed by using Primer Premier 6.0 software: 5'-atgcctcagttgcgattg-3' (SEQ ID NO.1) and BmAmy 1-R: 5'-tcatatcttctgcttgatctgg-3' (SEQ ID NO. 2). The cDNA of the middle intestine of the silkworm obtained by the reverse transcription is taken as a template, and the full-length primers BmAmy1-F and BmAmy1-R of BmAmy1 are used for PCR amplification. The PCR amplification procedure was: pre-denaturation at 95 ℃ for 3 min; then denaturation at 95 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 1-2 kb/min, and circulation from denaturation to extension for 32 times; finally extending for 5-10 min at 72 ℃; the product was stored at 12 ℃. After the PCR amplification is finished, 5. mu.L of nucleic acid Loading Buffer (containing fluorescent dye) is added into the reaction product and mixed uniformly, 5. mu.L of PCR product is taken firstly and subjected to electrophoresis detection by 1% agarose gel under the electrophoresis condition of 160V for 25min, and the result is shown in figure 1. The result shows that the electrophoresis detection result shows that the band is single and consistent in size and prediction, is about 1,500bp, and the band is recovered and is connected to a pEASY-T5 vector through TA clone and is named as T5-BmAMY 1. Sequencing the recombinant vector to obtain the sequence information of the BmAmy1, and the result is shown in SEQ ID NO. 3. The bioinformatics analysis result shows that the gene coding region of BmAmy1 has the length of 1518bp, contains 9 introns, is positioned on chromosome 8, and predicts that the protein size is 57.82KDa and the isoelectric point of the protein is 6.97.
The domain of BmAmy1 protein was predicted online using SMART website (http:// SMART. embl-heidelberg. de /), and the prediction results show that the protein encoded by BmAmy1 gene has a typical alpha-amylase domain and all contain a C-terminal domain, which is considered to be related to the activity of amylase, and at the same time, the amino terminals of the amino acid sequence of BmAmy1 respectively contain a Signal peptide (Signal peptide) region with the length of 18 amino acids, indicating that it may be a secreted protein.
The amino acid sequence of the BmAmy1 gene was aligned with the amino acid sequences of α -amylases of Diatraea saccharalis, Spodoptera frugiperda, Heliothis armigera, Anopheles merus, Drosophila melanogaster, Tribolium castaneum, cattle (Bos Taurus) and humans (Homo sapiens) and the results are shown in FIG. 3. The comparison result shows that: the predicted amino acid sequence of bmamay 1 has 54% sequence similarity to previously reported bombyx mori alpha-amylase (bmamay 2); black boxes indicate 7 motifs conserved in all animal alpha-amylases; the blue mask region represents the catalytically active site conserved in all species of alpha-amylase, 2 aspartic acid residues (D212 and D314) and 1 glutamic acid (E249) residue, respectively; red arrows indicate three histidine residues, His123, His216 and His313, respectively, which are involved in substrate recognition and conserved; the green mask represents the four predicted calcium binding sites, asparagine (N119), arginine (R173), aspartic acid (D182), and histidine (H216), respectively; the gray masked area represents 12 cysteine residues, 8 of which are conserved in all species and form 4 disulfide bonds, different numbers of which may be associated with differences in enzyme activity; in addition, three amino acid residues related to chloride ion binding, all marked with red masks, arginine (R207), asparagine (N309) and arginine (R346) in that order, were found, and the last amino acid residue R346, which is a conserved site of the mammalian and other insect α -amylase sequences, plays a significant role in chloride ion binding.
In order to analyze the genetic evolution relationship between bombyx mori bmamay genes and other species, bmamay 1 genes are used as query sequences, Blast programs of databases such as NCBI, Ensemble and the like are used for on-line retrieval and comparison to obtain the amino acid sequences of alpha-amylase of other insects and other organisms, MEGA-X software is used for carrying out phylogenetic analysis on the sequences, a phylogenetic tree (figure 4) is constructed by using a Neighbor-Joining method, and the results show that: the evolutionary tree is largely divided into 6 major classes of lepidoptera, coleoptera, diptera 1, diptera 2, non-insect arthropods and other invertebrates and the phylum vertebrata, with BmAmy1 clustering into a subclass of lepidopteran amylase gene cluster.
Example 2 expression profiling of BmAmy1 Gene
The expression characteristic analysis of the BmAmy1 is carried out by adopting a fluorescent quantitative PCR method, wherein the fluorescent quantitative PCR primer of the BmAmy1 is BmAmy 1-qRT-F: 5'-ccatcatccgtcctgctctat-3' (SEQ ID NO.4) and BmAmy 1-qRT-R: 5'-ggcaagttgtgattcaagtcct-3' (SEQ ID NO. 5). The fluorescent quantitative PCR primer of the internal reference gene is sw 22934-F: 5'-ttcgtactggctcttctcgt-3' (SEQ ID NO.6) and sw 22934-R: 5'-caaagttgatagcaattccct-3' (SEQ ID NO. 7). The qPCR experiment used SYBR Premix Ex Taq II kit from Takara and the fluorescent quantitative PCR instrument was Jena qTOWER, Germany3touch quantitative PCR instrument. The PCR procedure was as follows: pre-denaturation at 95 ℃ for 30s, followed by denaturation at 95 ℃ for 3s and annealing at 60 ℃ for 30s, with 40 cycles. Where each sample was run for 3 experiments and Ct values were collected for data analysis. Through 2-ΔΔCtThe relative expression level of the target gene was calculated, and the results are shown in FIG. 5. The results showed that bmamay 1 was expressed in domestic silkworms with a high intestinal tissue specificity and little expression in other tissues (fig. 5, a); in addition, expression characteristics of the bmamay 1 in the middle intestine of silkworms in the period of time were also examined, and using the middle intestine tissue cDNA from the beginning of the fifth instar of silkworms (L5D0) to the 7 th day of the fifth instar (L5D7) as a template, it was revealed that the bmamay 1 gene exhibited high expression in the 3 rd day of the fifth instar (L5D3) to the 5 th day of the fifth instar (L5D5) (fig. 5, B), which is the full-term of the fifth instar of silkworms in three days.
Example 3 eukaryotic expression, purification and Activity detection of the Bombyx mori BmAmy1 Gene
The eukaryotic expression is selected from Pichia pastoris X-33 strain, wherein the X-33 Pichia pastoris contains AOX1 gene, and the generated transformant is Mut+The genotype of the plant. The expression vector is pPICZ alpha A vector containing bleomycin (Zeocin) resistance.
(1) Construction and transformation of yeast eukaryotic expression vector
According to the sequence characteristics of BmAmy1 and the structural characteristics of a pPICZ alpha A vector, gene specific primers EcoRI-BmAmy1-F with EcoR I and Not I enzyme cutting sites are designed: 5' -cggaattcaatcatcataaaaggacgaacc-3’(SEQ ID No.8) and NotI-BmAmy 1-R: 5' -atttgcggccgctatcttctgcttgatctggag-3' (SEQ ID NO. 9). A gene fragment with EcoR I and Not I enzyme cutting sites is amplified by using TransTaq HiFi DNA polymerase PCR by taking the previously constructed T5-BmAmy1 vector as a template. And (3) cutting and recovering the gel, performing TA cloning again on the gene fragment with the enzyme cutting site, performing ligation transformation, selecting monoclonal sequencing verification, performing amplification culture on positive clones, extracting plasmids and storing for later use.
(2) Construction of BmAmy1-pPICZ alpha A recombinant vector
Carrying out double enzyme digestion on the product with the EcoR I enzyme digestion sites and the Not I enzyme digestion sites and the pPICZ alpha A vector, and respectively recovering the target fragment and the pPICZ alpha A vector skeleton. And after enzyme digestion is finished, sucking 1 mu L of original plasmid and 5 mu L of enzyme digestion product for electrophoresis detection, judging whether the enzyme digestion is correct or not according to strip size comparison, after the correctness is confirmed, respectively carrying out gel cutting recovery on the target gene and the pPICZ alpha A vector skeleton, and connecting to obtain the pPICZ alpha A-BmAMY 1 recombinant vector.
(3) BmAmy1-pPICZ alpha A recombinant vector linearization treatment and pichia pastoris transformation
10 mu g of ultrapure plasmid is extracted for linearization, Sac I enzyme cutting sites are selected, enzyme cutting is carried out for about 2 hours at 37 ℃, and then 5 mu L of product is taken for electrophoresis detection to determine whether the enzyme cutting is complete. After the completion of the enzyme digestion, the enzyme digestion product was recovered in a clean manner, and the detailed procedures refer to the use instruction of easy pure PCR Purification Kit of Beijing Quanji corporation. Finally, the linearized plasmid was concentrated to a volume of 10 μ L with a freeze dryer and then transformed into pichia pastoris competent yeast cells. The results of PCR measurements of pPICZ α A-BmAMY 1 recombinant vector after yeast transformation are shown in FIG. 6.
(4) Large-scale inducible expression and purification of BmAmy1 protein
Expanding and culturing a Pichia pastoris recombinant containing a BmAmy1 gene, and performing shaking culture at a rotation speed of 250rpm overnight for 16-18 h until the OD is reached600After centrifugation at 1,500 Xg for 5min, the supernatant was discarded, BMMY was added to resuspend yeast cells and diluted to OD600After 1.0, 100% methanol was added every 24h to a final concentration of 0.5%, and protein expression was induced at 28 ℃ at 250 rpm. After 96h of induction culture, collecting bacterial liquid, and centrifuging at 8,000rpm and 4 ℃ for 1Collecting supernatant for 5min, collecting 50 μ L supernatant, preparing protein sample, adjusting pH of the remaining supernatant to 7.0 with 1M Tris, filtering with 0.45 μ M water system filter membrane, and placing on ice for next protein purification.
As the expressed recombinant protein has His label, the protein is purified by a nickel column affinity chromatography method, the nickel column filler is Ni Sepharose excel of GE company, and the main operation steps are as follows (taking purified 1L supernatant as an example):
1) approximately 5mL of the filler was added to the culture supernatant and incubated at 80rpm for more than 4 hours at 4 deg.C (overnight incubation was also possible).
2) Carefully adding the mixed solution of the filler and the culture medium supernatant into a small chromatographic column, collecting the flow-through liquid (FT) without controlling the flow rate, storing at 4 ℃, and preparing a flow-through liquid protein sample.
3) Eluting with 0mM, 20mM, 50mM, 100mM, 200mM, 300mM, 500mM and 1M imidazole concentration buffer solution, wherein the elution speed of 0mM and 20mM buffer solution can be slightly faster, the elution speed of 50-500 mM buffer solution is controlled, the elution speed is kept for 2-3 s/drop, and finally all proteins in the filler are removed by 1M imidazole.
4) Protein samples were prepared by sampling all the above gradient-eluted samples, and SDS-PAGE was performed on the culture Supernatant (Supernatant) and the Flow-through (Flow through) protein samples, followed by simultaneous Western blotting (Myc mouse antibody), and the results of protein purification were confirmed as shown in FIG. 7. The results showed that the protein of interest expressed by secretion was highly pure and that the bmamay 1 protein was successfully detected in the culture supernatant, indicating that the recombinant protein was expressed by secretion as expected. Finally, the purified protein is subjected to ultrafiltration desalination and concentration, then is subjected to liquid nitrogen quick freezing, and is stored in a refrigerator at the temperature of minus 80 ℃ for the next activity detection experiment.
(5) Enzyme Activity detection of BmAmy1
The purified recombinant BmAmy1 protein is measured for enzyme activity by using a3, 5-dinitrosalicylic acid colorimetric method, the basic principle is that amylase decomposes starch to generate reducing sugar, the reducing sugar can react with the 3, 5-dinitrosalicylic acid to generate a brownish red substance, an absorption peak is arranged at 540nm, the increase rate of an absorbance value (namely the generation rate of the reducing sugar) at 540nm is measured within a certain range, and then the enzyme activity of the amylase is calculated, and the specific operation details refer to the specification of a Solebao alpha-amylase activity detection kit. Firstly, a standard curve (fig. 8, a) is drawn by using a gradient diluent of a glucose standard solution and measuring a light absorption value after reaction for subsequent enzyme activity calculation. The result of the enzyme activity determination is shown in a figure 8B, an alpha-amylase standard (derived from bacillus) is added in the experiment, and the result shows that the BmAmy1 shows obvious alpha-amylase activity, and the average activity of the BmAmy1 is 0.52mg/min/mg protein. Definition of enzyme activity unit: the catalytic production of 1mg reducing sugar per mg (mg) of protein per minute was 1 enzyme activity unit at 40 ℃, pH 7.0.
Example 4 construction of transgenic vector of Bombyx mori BmAmy1 Gene, microinjection of embryo, screening of Positive transgene, and shape investigation
The silkworm variety subjected to transgenic microinjection is a diversified variety D9L, and is reserved in the laboratory. The transgenic operation is specifically as follows:
(1) construction of pSL1180[ P3P +5UI-BmAmy1] recombinant vector
Using the constructed T5-BmAmy1 vector as a template, PCR-amplifying the desired fragment with gene-specific primers BamH I and Not I, respectively, and cloning the desired fragment into pSL1180 vector containing P3P +5UI (pSL1180[ P3P +5UI-DsRed ], P3P +5UI and DsRed linked to pSLfa 11852 vector containing SV40 termination signal upstream of SV40 (pSLfa 1180fa vector containing SV40 termination signal, Chinese patent publication No. CN102296071A, P3P +5UI see Liang Jiang, etc. the 5-gen intron of the midgut-specific BmA84 gene, source UIs and expression of expression vector BmAMM. P) to obtain specific primers BmAMY1 and expression vector 855. BmAMY1 sequence [ P5 + 5M ] as follows:
BamHI-BmAmy1-F：5’-cgggatccatgcctcagttgcgattg-3’(SEQ ID NO.10)；
NotI-BmAmy1-R：5’-atttgcggccgctcatatcttctgcttgatctgg-3’(SEQ ID NO.11)。
the vector was subjected to double digestion with BamH I and Not I, and the electrophoresis revealed a band of about 1,500bp (FIG. 9, A) corresponding in size to the expected size of the BmAmy1 fragment. The vector is sent to Huada gene for sequencing verification, and the sequencing result shows that the fragment has no mutation, which indicates that the pSL1180[ P3P +5UI-BmAmy1] vector is successfully constructed. And then carrying out single enzyme digestion on the obtained pSL1180[ P3P +5UI-BmAmy1] vector and piggyBac [3 xP 3-DsRed ] by using Asc I, recovering a target fragment and a vector skeleton, and then connecting to obtain the piggyBac [3 xP 3-DsRed, P3P +5UI-BmAmy1] vector. Asc I single enzyme digestion is carried out on the gene, electrophoresis detection shows that a band (figure 9, B) appears at about 3,800bp, the size of the band is consistent with that of an expected single enzyme digestion fragment [ P3P +5UI-BmAmy1], and the construction success of a transgene vector piggyBac [3 xP 3-DsRed, P3P +5UI-BmAmy1] is proved, and the gene can be used for the next step of transgene microinjection.
(2) Acquisition, insertion site analysis and expression abundance analysis of BmAmy1 transgenic overexpression silkworms
And (3) uniformly mixing the transgenic vector and the auxiliary vector pHA3PIG, injecting the mixture into the silkworm eggs, feeding the silkworm eggs to the generation G1, and screening the silkworm eggs by using a fluorescence microscope when the silkworm eggs grow for 5-6 days in the generation G1. Since DsRed or EGFP driven by an eye-and nerve-specific promoter was used as a marker gene, the eyes of transgenic silkworms fluoresce red or green under a fluorescent microscope (fig. 10, a). In this way we screened for transgenic silkworms. We named the transgenic silkworm strain overexpressing bmamay 1 gene as P3P: amy 1.
Extraction of P3P: the genome of Amy1 transgenic silkworm imago is cut by Hae III enzyme and then cyclized by T4 DNA ligase, then the cyclized genome is taken as a template, PCR amplification is carried out by respectively using a piggyBac vector left arm primer pBacL-F/R and a right arm primer (pBacR-F/R), a fragment larger than 500bp is recovered, and TA cloning and sequencing are carried out. And (3) carrying out sequence alignment on the sequencing results of the left arm and the right arm in a SilkBase database, wherein the alignment and positioning results are shown in Table 1. The insertion site for P3P: Amy1 is on chromosome 22 and is in the intergenic region. The transgenic silkworms are bred to G2 generations, and the tissues of the transgenic silkworms and wild type controls thereof are dissected on the 3 rd day of the fifth age, and total RNA is extracted respectively. After the total RNA is inverted into cDNA, qRT-PCR detection is carried out by using a specific primer of the BmAmy1 gene. The results showed that bmamay 1 significantly up-regulated expression in the midgut of the corresponding transgenic silkworms, indicating successful specific overexpression (fig. 10, B).
TABLE 1 analysis of transgene insertion sites
(3) Relevant economic character investigation statistical analysis of transgenic silkworm
According to the feeding standard described in the method, the transgenic silkworms and the non-transgenic control silkworms were carefully fed under the identical conditions, and data statistics were performed on the 4 th day of the five-year-old according to the male and female analyses. The results show that the individual size of P3P: Amy transgenic silkworms was significantly larger than non-transgenic silkworms, both male and female (fig. 11, a). The weight of 60 heads of P3P Amy1 transgenic silkworms and non-transgenic control silkworms was weighed respectively, and the results showed that the average body weight ratio of the male and female transgenic silkworms was 11.9% and 6.8% of that of the non-transgenic silkworms (FIG. 11, B). The statistics of the total cocoon weight and the cocoon layer rate in the later period also obtain the results of the same trend, namely the cocoon of the transgenic line is obviously larger than the non-transgenic cocoon (figure 11, C), and the cocoon layer rates of the female transgenic silkworm and the male transgenic silkworm are correspondingly increased by 11.2 percent and 5.7 percent compared with the control (figure 11, D).
The research shows that economic traits such as the weight of the whole cocoons and the cocoon layer rate can be improved by over-expressing BmAmy1 in the silkworms, so that BmAmy1 has important production and application potentials. In addition, the invention opens up a new way for cultivating more excellent silkworm varieties.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Sequence listing
<110> university of southwest
Application and method of <120> silkworm amylase gene BmAmy1
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 1
atgcctcagt tgcgattg 18
<210> 2
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 2
tcatatcttc tgcttgatct gg 22
<210> 3
<211> 1518
<212> DNA
<213> silkworm (Bombyx mori)
<400> 3
atgcctcagt tgcgattgct tctcgtgtgt ttaatcttgg gtttcgtgct atgcaatcat 60
cataaaagga cgaaccaaga tctcaaccgg tcgacaattg tgcatctctt cgaatggaaa 120
tggaaggata ttgctgatga atgcgagaga ttccttgcgc cgaaaggctt cggtggagtt 180
caagtatctc caccctcgga gaatgttatc attcgtttat cagacggtac acgaccttgg 240
tatgaacgtt accaagtaat gtcctacaag ttgatcacca gatcaggaaa tcaagatgaa 300
tttttgaaca tgacccgaag atgtaatgat gttgggataa gaatttacgc tgacgtggta 360
attaatcata tgacgagtga caatagggaa tctgttggta cagggggtag tacagccaac 420
tataaagagt acagctaccc cgctgtgccg tacacgagag accacttcca tcatccgtcc 480
tgctctatta ataattataa tgatgctgca caggtaaggg cctgcgattt ggttggattg 540
aaggacttga atcacaactt gccgtatgtt cgtgacaaaa tcgttgaata cctaaacaag 600
cttattgcat taggagttgc cggattcaga gtggatgcag caaaacatat gtggccacac 660
gacctcaaag agatgtacaa tagacttaat aatctcaatg tagattttgg cttcctacct 720
aatacaaaac cgtacattta ccaagaagta atttatagag ggaatgagcc tatccaacat 780
actgaataca caccctttgg tgacgtcaca gaatttcggg tcggatacga gttaaaacca 840
gtttttgagg gtaaaaatcc attgaaatgg ttgaagtcct ggggcgagaa ttggaactta 900
tcacccagtg accgcgttgt tgtattcatt gacaatcacg acacgcagag gtcgaatgaa 960
gttttgacgt acaaaaatgc caagtcttat aaggcagcta tagcgtttac actggcacac 1020
ccatatggcg aacctcgcgt gatgagcggt tatttcttcg acagaaccga ccaaggtcca 1080
ccaagcgacg gtaacgagga aattttatct ccaatcataa atgatgacga cacctgcggc 1140
aatggctggg tttgcgagca tcgttggcga cagatctatc aaatggtcgc atttagaaat 1200
gccgtccgag atactagagt tgaaaattgg tgggacaacg gtcttaatca gatagctttc 1260
agtcgaggaa ataaaggctt tatcgctatt aacgccgagg gtcgagattt ggatgttatt 1320
ttacagactg gtcttccatc tggtacttat tgcgatgtta taagcgggaa aattcaaggc 1380
caagaatgtt ccggaagaaa aataacagtt ggcagtgatg gtcgtgcgca tatttatgta 1440
tccaaagacg gggaggacat gcatcttgcc acacatgttg ggccggagtc attactccag 1500
atcaagcaga agatatga 1518
<210> 4
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ccatcatccg tcctgctcta t 21
<210> 5
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ggcaagttgt gattcaagtc ct 22
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
<210> 7
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
caaagttgat agcaattccc t 21
<210> 8
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
cggaattcaa tcatcataaa aggacgaacc 30
<210> 9
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
atttgcggcc gctatcttct gcttgatctg gag 33
<210> 10
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
cgggatccat gcctcagttg cgattg 26
<210> 11
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
atttgcggcc gctcatatct tctgcttgat ctgg 34
Claims (7)
1. The application of silkworm amylase gene BmAmy1 in cultivating high-yield silkworm varieties is characterized in that: the nucleotide sequence of the silkworm amylase gene BmAmy1 is shown in SEQ ID NO. 3.
2. Use according to claim 1, characterized in that: the silkworm amylase gene BmAMY1 is applied to improving the weight of the whole silkworm cocoon.
3. Use according to claim 1, characterized in that: the silkworm amylase gene BmAmy1 is applied to improving the silkworm cocoon layer rate.
4. Use according to claim 1, characterized in that: the silkworm amylase gene BmAmy1 is applied to improving the size of silkworm individuals.
5. Use according to claim 1, characterized in that: the silkworm amylase gene BmAmy1 is applied to reducing the residual starch content in silkworm excrement.
6. A method for cultivating high-yield silkworm varieties is characterized in that: through overexpression of a silkworm amylase gene BmAmy1 in silkworms, the nucleotide sequence of the silkworm amylase gene BmAmy1 is shown as SEQ ID NO. 3.
7. The method of claim 6, wherein: the steps of the overexpression of the silkworm amylase gene BmAmy1 are as follows: constructing a transgenic vector containing silkworm amylase gene BmAmy1, mixing the transgenic vector with an auxiliary vector pHA3PIG, injecting the mixture into early-stage embryos of silkworms with diapause removed, carrying out incubation until the silkworms hatch to obtain transgenic G0 generation silkworms, feeding G0 generation silkworms to adults, carrying out selfing to obtain G1 generation silkworms, and screening to obtain the transgenic silkworms.
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