WO2013056664A1 - 家蚕丝素重链基因突变序列及突变的方法和应用 - Google Patents

家蚕丝素重链基因突变序列及突变的方法和应用 Download PDF

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WO2013056664A1
WO2013056664A1 PCT/CN2012/083175 CN2012083175W WO2013056664A1 WO 2013056664 A1 WO2013056664 A1 WO 2013056664A1 CN 2012083175 W CN2012083175 W CN 2012083175W WO 2013056664 A1 WO2013056664 A1 WO 2013056664A1
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heavy chain
silk fibroin
silkworm
fibroin heavy
mutant
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PCT/CN2012/083175
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English (en)
French (fr)
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夏庆友
马三垣
徐汉福
程道军
林英
赵萍
向仲怀
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西南大学
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Priority claimed from CN2011103196373A external-priority patent/CN102358902B/zh
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Priority to JP2014536104A priority Critical patent/JP5997772B2/ja
Priority to US14/348,898 priority patent/US20150166615A1/en
Publication of WO2013056664A1 publication Critical patent/WO2013056664A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
    • A01K67/04—Silkworms
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/70—Invertebrates
    • A01K2227/706—Insects, e.g. Drosophila melanogaster, medfly
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/01—Animal expressing industrially exogenous proteins
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/02—Animal zootechnically ameliorated

Definitions

  • the invention belongs to the field of silkworm breeding and genetic engineering, and relates to a silk fibroin heavy chain based on different existing silkworm varieties. Gene mutants, methods for their preparation, mutant sequences and uses.
  • Bombyx mori has been praised for its world economy and cultural exchanges for thousands of years of breeding and domestication due to its strong ability to secrete silk and silk. It is still the industry of China, India and other countries. An important part of. However, for a long time, people have selected silkworm cultivars for the purpose of breeding, such as sputum layer, sputum rate, sedative and disease resistance, while neglecting the importance of silk strength, extensibility, dyeing performance and skin affinity. index. It is also difficult for silk to be widely used in high-end fields such as medicine and military, such as high-strength fibers such as spider silk, but only as a main reason for a single textile material.
  • Bombyx mori protein consists only of a silk fibroin heavy chain, a silk fibroin light chain and a P25 protein and a coated outermost sericin, wherein the silk fibroin heavy chain protein which determines the silk fibroin performance is composed of a highly repetitive sequence. 390kDa protein.
  • the high expression level, highly repetitive sequence and high molecular weight of silk fibroin heavy chain proteins make it difficult to study and engineer them with conventional transgenic techniques.
  • a silkworm with a body weight of about 6g eats about 25g of mulberry leaves, and can spit out 0.5g of silk composed of pure protein. This is not only a spectacular biological wonder, but also contains infinitely beautiful Value.
  • the silk gland of the silkworm has the advantages of post-translational modification processing capability of high-level biological proteins, low-scale production cost and safety for humans and animals, meets the basic requirements of a new generation of bioreactors, and has great potential for development and application.
  • Research in this field has become an important part of the expansion from the traditional silk industry to the non-filament industry. Whether in basic research or in the field of pharmaceutical and cosmetic development, extracting or expressing purified proteins plays an extremely important role, but commercial protein pure products are surprisingly high (1g most commonly used green fluorescent protein price is about 5 Millions of dollars have been plaguing many researchers. The lack of processes for large-scale expression or protein extraction severely restricts protein-related research and product development.
  • the exogenous active protein expressed by the transgenic gene in the silk gland of the silkworm can only reach 8.0% of the total weight of the silkworm cocoon. The amount of expression is too low, and the process cost of purifying a small amount of protein from the ruthenium layer is too high, and the researchers and merchants who are trying to further develop the silk gland bioreactor are all stopped.
  • Zinc finger nuclease is an artificially designed restriction enzyme consisting of a zinc finger protein domain responsible for the specific recognition of a DNA sequence and a nuclease domain responsible for cleavage of DNA. Zinc finger nucleases can cause double-strand breaks at specific sites in complex genomes.
  • NHEJ homologous end joining
  • HR homologous recombination
  • NHEJ is a special DNA double-strand break repair mechanism that binds the broken DNA ends together quickly and efficiently in the absence of DNA homologous sequences. This is an unfaithful repair mechanism and is easy to The deletion introduces a few base deletions, insertions or mutations; the design of two pairs of zinc finger nucleases in the same large DNA fragment may also result in the deletion of large fragments between the two recognition sites; In the case of exogenous DNA with the same cohesive ends at the end, NHEJ can also achieve insertion of specific small fragments.
  • HR is a common DNA fragmentation repair mechanism in the body, and its principle has been widely used in gene knockout since the 1980s.
  • zinc finger nuclease-mediated double-strand breaks can efficiently perform arbitrary operations such as site-directed replacement, repair, deletion, and insertion of target genes.
  • this technology has been widely used in gene knockout and site modification of animals and plants.
  • One of the objects of the present invention is to provide a method for mutating a silk fibroin heavy chain gene and a gene sequence obtained by the mutation method, which provides a new idea for obtaining a mutant silk fibroin heavy chain gene.
  • a second object of the present invention is to provide a novel application of two silk fibroin heavy chain mutant genes, which provides an effective way for preparing sericin and foreign proteins.
  • the silk fibroin heavy chain mutant gene is a mutant gene of the silk fibroin heavy chain gene 1325 to 1362 which is a target site recognized by zinc finger nuclease.
  • the silk fibroin heavy chain mutant gene is represented by any one of SEQ. ID NO: 2-118.
  • the silk fibroin heavy chain mutant gene is shown as any one of SEQ. ID NO: 2-14.
  • the silk fibroin heavy chain mutant gene is shown as SEQ. ID NO:11.
  • a method for mutating a silk fibroin heavy chain gene which comprises inserting a zinc finger nuclease sequence as shown in SEQ. ID NO: 119 and SEQ.
  • a recombinant vector is obtained; the recombinant vector is transcribed in vitro to obtain an mRNA encoding a zinc finger nuclease sequence, and the mRNA encoding the zinc finger nuclease sequence is applied to SEQ. ID NO:
  • the silk fibroin heavy chain gene 1325 to 1362 shown in Fig. 1 is a target site recognized by zinc finger nuclease, and the silk fibroin heavy chain mutant gene is obtained.
  • the invention utilizes the newly developed zinc finger nuclease technology to efficiently knock the silk fibroin heavy chain gene
  • a series of mutant silkworm strains including partial deletions, partial base mutations or small fragment insertions in the N-terminal non-repeat region of the silk fibroin heavy chain gene were obtained for the first time.
  • the mutants provided by the present invention have the following characteristics and advantages :) The silk gland of the silk fibroin heavy chain gene mutant provided by the present invention is seriously degraded, and the sputum layer only contains synthesis and secretion in the middle silk gland.
  • Sericin if these mutant lines are used to express foreign genes (such as spider silk, organisms) in the silk gland of the silkworm The active protein, etc.), the expression and purity of the foreign protein in the silk gland will be greatly improved, which provides a new and useful genetic material for the development of the silk gland bioreactor. ;) Since the mitochondrial layer of the mutant strain provided by the present invention has only sericin, and pure sericin has been widely used in cosmetics, the present invention provides a new source for large-scale production of sericin.
  • Figure 1 shows the structure of the silk fibroin heavy chain gene, in which the box indicates the exon, the gray solid line The regulatory region or intron sequence is shown, the number indicates the position relative to the transcription start site, the underlined portion is the nucleotide or amino acid sequence of the signal peptide sequence, and the arrow indicates the signal peptide sequence cleavage site.
  • Figure 2 shows the SNP distribution of the partial sequence of the silk fibroin heavy chain gene in forty silkworm lines, of which the upper end Boxes and solid lines indicate the location and structure of the analyzed region in the silk fibroin heavy chain gene, with the next forty-one solid lines representing 30 silkworm strains and 11
  • the nucleotide sequence of the wild silkworm strain, the diamond in the solid line indicates that the corresponding strain is mutated here relative to the reference sequence, the number indicates the position of the SNP locus relative to the transcription start site, and the number on the right side of the solid line No The number of the silkworm line.
  • Figure 3 shows the nucleotide sequence of the mutant silk fibroin heavy chain gene mutation site, where the number on the left is The number of the corresponding individual, >Ref indicates the sequence of the wild-type strain, the sequence shown in the box is the zinc finger nuclease recognition site, and the bold base is the base of the mutation, - Indicates a deletion, and the underlined base is an inserted base.
  • Figure 4 shows the anatomical view of the silk gland of the wild-type silkworm and the mutant silkworm, in which the left side is the wild type.
  • the silk gland of the fifth instar and six days, the right side is the silk gland of the mutant silkworm, five years old and six days old.
  • Figure 5 shows an observation of the mutant silkworm cocoons, in which the upper row is the silkworm cocoon of the wild type N4 and the lower row is the silkworm cocoon of the mutant silkworm.
  • Figure 6 shows the biological statistical analysis of the weight of wild-type silkworm and mutant silkworm cocoons and silkworm cocoons.
  • Figure 7 shows the wild-type silkworm, heterozygous mutant silkworm and pure-type mutant silkworm as receptors.
  • FIG. 8 shows a layered protein analysis of transgenic silkworms with wild-type silkworm, heterozygous mutant silkworm and pure-type mutant silkworm as recipients, in which Transgenic-1, Transgenic-1 and Non-transgenic represent two One transgenic line and one transgenic line, +/+, +/- and -/- respectively represent wild-type silkworm, heterozygous mutant silkworm and pure-type mutant silkworm, and the lowermost row represents the lane number, as indicated by the arrow A specific band for the exogenous green fluorescent protein fusion protein.
  • chromosomes exist in pairs, that is, two copies of the same gene. For any one mutant, both genes are normal and wild type, and one normal mutation is a heterozygous mutation. Both mutants are pure-type mutants.
  • the object of the present invention is to provide a method for efficiently realizing the targeted transformation of the silk fibroin gene, and to provide a genetic resource for the mutation of the silk fibroin heavy chain gene. That is, the silk fibroin heavy chain gene was knocked out to obtain a series of mutants of silk fibroin heavy chain gene deletion, mutation or insertion.
  • the present invention comprehensively considers the expression level of foreign proteins in the silk gland of the silk and
  • the main problems faced by silk fibroin bioreactors in the development of silk fibroin bioreactors, and the use of newly developed gene knockout technology, zinc finger nuclease technology, to screen silk fibroin genes Knock-out, and the obtained mutants were subjected to genome sequencing verification and functional verification.
  • the silk fibroin heavy chain gene mutant of the invention and the preparation method and application thereof are sequentially realized by the following steps: (1) downloading the silk fibroin heavy chain gene sequence and analyzing the zinc finger nuclease targeting site; (2) designing a specific zinc finger nuclease sequence for the site analyzed in step (1); (3) synthesizing or amplifying the nucleic acid sequence encoding the zinc finger nuclease designed in step (2) from an existing zinc finger protein library; (4) inserting the nucleic acid sequence in step (3) into a prokaryotic expression vector containing a T7 or SP6 promoter to obtain a recombinant vector; (5) performing in vitro transcription using the recombinant vector obtained in the step (4) to obtain mRNA encoding the zinc finger nuclease designed in the step (2); (6) After the silkworm eggs of the silkworm diapause cultivar are artificially hatched, they are placed in a constant temperature of 15 ° C, a relative humidity of 75%, and an absolute dark environment to promote hatching
  • the silkworm eggs of non-diapause silkworm varieties are placed at 25 °C constant temperature, relative humidity 75%, natural light environment to promote to hatch; (7) Collecting the hatched anterior silkworms one by one, and feeding them to the upper stalks with mulberry leaves or artificial feed in an environment of constant temperature, relative humidity of 75%, and natural light at 25 °C; (8) Transfer the silkworm after the captain to a constant temperature of 25 ° C, a relative humidity of 75%, and natural light for seed protection; (9) After the feathering, the male and female silk moths of the simultaneous moths were collected and mated at 25 ° C under low light conditions for 4 hours, and the female moths were placed on the sizing silkworm paper to lay eggs in a dark environment.
  • the silkworm eggs were collected once every 0.5h, and the collected silkworm eggs were protected in an environment of 25 °C; (10) After rinsing the silkworm eggs together with the silkworms with tap water, soak them in distilled water until the paste swells (about 2 minutes), then transfer the silkworm eggs to the slides sterilized with 75% alcohol with tweezers, and According to the standard of silkworm egg belly facing right, it is arranged neatly, and the arranged silkworm eggs are placed in 35-37% formaldehyde vapor for 5 min; (11) Starting at the time of collection, the mRNA obtained in step (5) was mixed by a microinjector at a molar ratio of 1:1 after 2 hours to 4 hours after collection, and then the sterilized silkworm was injected from the center of the ventral surface of the silkworm egg.
  • the modern (G0) silkworm moth is selfed or backcrossed to obtain the diapause G1 generation silkworm eggs;
  • the G1 generation silkworm eggs obtained in the step (12) are immediately subjected to pickling treatment, and then placed in an environment of constant temperature at 25 ° C, relative humidity of 75%, and natural light to promote incubation;
  • the G1 generation ant silkworm obtained in step (13) is raised to the upper stalk with mulberry leaves at a constant temperature of 25 ° C, a relative humidity of 75%, and a natural light environment, and the condition of silking and camping is observed.
  • Mutant individuals with abnormal silk or camp (15)
  • the mutant individuals obtained in step (14) are selfed or backcrossed, and the genome of the silkworm moth after planting is extracted, and the primers specific for the silk fibroin heavy chain gene are used for PCR amplification and sequencing, and further identification and confirmation are carried out.
  • the mutant (16) subsequent preservation of the mutant identified in step (15); (17) according to the amino acid sequence of the protein to be expressed in the silk gland of the silk, the coding sequence is synthesized or amplified in vitro, and constructed into a transgene vector or a homologous recombination vector targeting the silk fibroin heavy chain gene; (18) using the silkworm egg preserved in the step (16) as a receptor, and injecting the transgenic vector or the homologous recombination vector in the step (17) into the step (16) by the method described in the step (6) to the step (13)
  • the preserved silkworm embryos the hatched silkworms are bred, and the contemporary (G0) silkworm moths are selfed or backcrossed to obtain the diapause G1 silkworm eggs, and the G1 silkworm eggs are immediately acid-treated and then developed in the embryo.
  • Example 1 Sequence analysis ⁇ br/> silk fibroin heavy chain gene sequences from the NCBI database the heavy chain gene downloads silk fibroin (number AF226688), the sequence shown in Figure 1.
  • Bombyx mori heavy chain gene (+1 ⁇ +16 788, of which +1 indicates the transcription start site) includes two exons with lengths of 67 bp and 15750 bp and an intron of 971 bp in length, the first explicit
  • the sub-region includes a 25 bp untranslated region (+1 to +25) and a 42 bp coding region (+26 to +67), and the second exon contains an N-terminal non-repeat region (+1039 to +1449), and the C-terminal is non-repetitive.
  • the N-terminus of the amino acid sequence encoded by the silk fibroin heavy chain gene contains a signal peptide of 21 amino acid residues (shown underlined in Figure 1).
  • a SNP analysis on the N-terminal partial sequence (+1 ⁇ +1448) of the silk fibroin heavy chain gene. The results are shown in Figure 2. Show.
  • the N-terminal partial sequence of the silk fibroin heavy chain gene (+289 ⁇ +1448) has 10 SNP loci in 29 silkworm strains and 11 wild silkworm strains, respectively +393, +465, +555, + 556, +861, +862, +999, +1270, and +1390.
  • Example 2 Design and synthesis of the silk fibroin heavy chain gene-specific zinc finger nuclease sequence According to the sequence characteristics of the silk fibroin heavy chain gene and its distribution in different silkworm lines, the zinc finger protein is combined with the recognition of the DNA sequence.
  • CTGTTGCTCAAAGTTATGTTGCTGCTGATGCGGGAGCA as a target site for zinc finger nuclease recognition, which is located at positions +1325 to +1362 of the sequence shown in SEQ ID NO: 1, and designs and synthesizes zinc finger nuclease accordingly. Therefore, the silk fibroin heavy chain gene 1325 to 1362 is the target site for zinc finger nuclease recognition.
  • Example 3 Preparation of zinc finger nuclease mRNA
  • the nucleic acid sequence of the zinc finger nuclease (such as SEQ ID NO: 119 and SEQ ID NO: 120) was synthesized or amplified from an existing zinc finger protein library. Restriction enzymes EcoRI and XhoI (purchased from TAKARA) were used. After double digestion, the prokaryotic expression vector pET28a was digested with the same restriction enzyme, transformed into E. coli and screened for positive clones to obtain a recombinant vector.
  • the specific reaction system of the enzyme digestion was as follows: Reagent volume Plasmid 5 ⁇ l 10X H buffer 1 ⁇ l EcoRI 0.5 ⁇ l XhoI 0.5 ⁇ l Sterile water 3 ⁇ l total capacity 10 ⁇ l
  • the recombinant vector was digested with XhoI and transcribed in vitro using the MessageMax T7 mRNA in vitro transcription kit (purchased from Epicentre).
  • the reaction system was as follows: Reagent volume RNase-free water 10 ⁇ l Plasmid template (1 ⁇ g/ ⁇ l) 2 ⁇ l 10X transcription buffer 4 ⁇ l ARCA Cap/NTP Mixture 16 ⁇ l 100 mM DTT 4 ⁇ l MessageMAX T7 Enzyme Solution 4 ⁇ l total capacity 40 ⁇ l After incubating the above reaction system at 37 ° C for 30 minutes, 1 ⁇ l of DNase was added, and incubation was continued for another 15 minutes.
  • the above reaction system was subjected to a tailing reaction using an Epicentre A-plus tailing kit (purchased from Epicentre), and the reaction system was as follows: Reagent volume RNase-free water 109 ⁇ l 10X buffer 20 ⁇ l 100 mM ATP 20 ⁇ l ScriptGuard RNA Enzyme Inhibitor (40 U/ ⁇ l) 5 ⁇ l In vitro transcription reaction system 42 ⁇ l Poly (A) polymerase 4 ⁇ l total capacity 200 ⁇ l The above system was incubated at 37 ° C for 30 minutes, purified by MEGAClear kit (purchased from Ambion), and stored at -80 ° C until use.
  • MEGAClear kit purchased from Ambion
  • Example 4 Preparation of silkworm embryos for microinjection
  • the treatment method of the polygenic silkworm variety 'N4' the hatching of the silkworm eggs in the environment of constant temperature and relative humidity of 75% at 25 ° C, and the silkworm eggs produced after feeding with mulberry leaves are directly used for microinjection.
  • Treatment method of diapause silkworm variety 'Dazhi' The silkworm eggs obtained from normal rearing are placed in a constant temperature of 15 °C, a relative humidity of 75%, and a natural light environment to be hatched after routine artificial hatching treatment.
  • the silkworm eggs produced after hatching are fed at a constant temperature of 25 ° C, a relative humidity of 75%, and a natural light environment.
  • Example 5 Microinjection of zinc finger nuclease mRNA After the emergence, the male and female silk moths of the simultaneous moths were collected and mated at 25 ° C under low light conditions for 4 hours, and the female moths were placed on the sizing silkworm paper to lay eggs.
  • a total concentration of 400 ng/uL of zinc finger nuclease mRNA targeting the heavy chain gene of the silkworm was injected into several silkworm eggs by a microinjector (FemtoJet 5247 microinjector, purchased from Eppendorf).
  • the amount of silkworm eggs injected is about 10nL.
  • the silkworm eggs are sealed with non-toxic glue and disinfected for 5 minutes in 35% formaldehyde vapor. After being placed in a high humidity environment of 25 ° C and relative humidity of 85%, the hatching G0 is used.
  • Silkworms are collected from artificial diets and raised to moths.
  • Example 6 Screening of mutant individuals after microinjection
  • the polymorphic variety 'N4' and the diapause cultivar 'Dazhi' were used as the original materials, and 195 and 247 silkworm eggs were injected, and the hatched 93 and 124 G0 ants were collected and raised into artificial moths.
  • 81 and 106 G0 generation silkworm moths were obtained respectively, and 38 and 51 moths of G1 silkworm eggs were obtained by selfing or backcrossing, 38 and 51 moth circles were promoted and separately raised to the G1 generation for observation, respectively.
  • Of the 29 and 38 moth circles 250 and 105 bare or sputum thin 'silk sputum' were found.
  • Table 1 The specific experimental statistics are shown in Table 1. Table 1.
  • the PCR primers were: Fib-HF: 5'-tgatgaggactattttgggag-3' Fib-HR: 5'-tagtgctgaaatcgctcgt-3'
  • the sequencing primer is: Fib-HF: 5'-tgatgaggactattttgggag-3'
  • the PCR reaction system is: Genomic DNA: 1.0 ⁇ l EX Taq buffer : 2.5 ⁇ l Mg2+ : 2.0 ⁇ l dNTP : 2.0 ⁇ l Ex Taq : 0.15 ⁇ l Primer Fib-HF: 0.5 ⁇ l Primer Fib-HF: 0.5 ⁇ l ddH2O : up to 25 ⁇ l
  • the PCR product is subjected to electrophoresis detection and purification, and then subjected to sequencing reaction.
  • Example 8 Phenotypic observation and anatomical observation of genetically mutated individuals Take wild-type silkworm strains and 117 mutant silkworm strains. Take 3 heads of each strain and take out the silk glands under the microscope according to the routine anatomical steps. The results are shown in Figure 4. The silk glands of the mutant individuals are significantly smaller than the wild type. Individuals, and the posterior silk glands of mutant individuals develop developmental malformations.
  • Wild type silkworm strains and 117 mutant silkworm strains were taken, and 50 silkworm pupas were taken from each line for observation.
  • the silkworm pupa layer of the mutant silkworm was thinner than the wild type.
  • 25 silkworm mites and carcasses were taken from each line, and the weight was weighed for biological statistical analysis.
  • the corpus callosum of the mutant individuals was slightly heavier than the wild type individuals, and the sputum layer weight was significantly lighter than that of the wild type individuals.
  • Protein electrophoresis analysis of silkworm pupa of wild-type silkworm strain and mutant silkworm strain showed that the mutant silkworm strain did not contain silk fibroin heavy chain protein, and only a large amount of sericin existed. See Figure 10 for lane 10.
  • Example 9 Expression of exogenous fusion green fluorescent protein in silk gland of mutant silkworm Using the plasmid containing the green fluorescent protein gene as a template, the coding sequence of the green fluorescent protein was amplified and fused with the non-repetitive region of the silk fibroin heavy chain gene to construct a transgenic vector (for the construction method and procedure, see Aichun Zhao et al. Transgenic Research, DOI 10.1007/s11248-009-9295-7). Using the preserved mutant silkworm strain (mutant sequence such as SEQ.
  • the silkworm eggs obtained under the normal feeding conditions were placed at a constant temperature of 15 ° C and a relative humidity of 75% after the conventional artificial hatching treatment. It is bred to an incubator in an absolutely dark environment, and is kept in a constant temperature of 25 ° C and a relative humidity of 75%. After the feathering, the male and female silk moths of the simultaneous moths were collected, and the male moths were mated at 25 ° C under low light conditions for 4 hours, and the female moths were placed on the sizing silkworm paper to lay eggs in a dark environment at intervals of 0.5. h Collect silkworm eggs once and protect the collected silkworm eggs in an environment of 25 °C.
  • Eppendorf microinjector was used to inject 10-15nL of total transgenic vector with a concentration of 400ng/uL into 150 silkworm eggs, seal the injection port with non-toxic glue, and pass 35%
  • the silkworm eggs of the G1 generation of the moths were obtained.
  • the Olympus® electric macroscopic fluorescence microscope was used to screen and screen the 41 eggs of the G1 moth and obtain a positive moth circle.
  • transgenic silkworms were obtained.
  • the results of the analysis of silk gland and sputum layer of transgenic silkworm are shown in Fig. 7 and Fig. 8.
  • the fusion of silk gland and sputum layer of transgenic silkworm with heterozygous mutant silkworm and homozygous mutant silkworm as the acceptor material The content of green fluorescent protein was significantly higher than that of transgenic silkworm with wild type silkworm as the acceptor material.
  • the green fluorescent protein was fused to the silk gland and the sputum layer of the transgenic silkworm with 116 other mutant silkworm strains (mutant sequences such as SEQ. ID NO: 2 ⁇ 10 , SEQ. ID NO: 12-118).
  • the content is also significantly higher than that of wild-type silkworm as a transgenic silkworm. It can be seen that the silk fibroin heavy chain mutant gene is used in the preparation of a foreign protein.

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Abstract

提供了突变家蚕丝素重链基因的方法，具体包括将编码锌指核酸酶序列的mRNA作用于如SEQ ID NO:1所示的家蚕丝素重链基因第1325-1362位的靶位点，获得一系列家蚕丝素重链突变基因。还提供了由上述方法制备的突变序列。还提供了上述突变序列在用于制备丝胶蛋白和外源蛋白的用途。还提供了包含上述突变序列的家蚕突变体作为新型的家蚕丝腺生物反应器，该突变体的后部丝腺严重退化，其茧层中只含有中部丝腺合成和分泌的丝胶蛋白，从而可以大大提高在家蚕后部丝腺中的外源转基因的表达量和纯度，以及可以大规模生产丝胶蛋白。

Description

家蚕丝素重链基因突变序列及突变的方法和 应用 技术领域
本发明属于家蚕育种和基因工程领域，涉及基于不同现有蚕品种的家蚕丝素重链 基因突变体，及其制备方法、突变序列和用途。
背景技术
家蚕因其强大的泌丝吐丝能力而蜚声古今中外，在长达数千年的饲养和驯化中为 世界经济和文化交流做出了重要的贡献，至今也仍然是中国、印度等国家产业的重要组成 部分。但是长期以来人们选育家蚕品种都是以茧层量、茧层率、解舒和抗病性的提高作为育 种目标，而忽略了茧丝强度、延伸性、染色性能、肌肤亲和性等重要指标。这也是蚕丝难以像 蜘蛛丝等高强度纤维那样广泛的应用于医学、军事等高端领域，而仅仅只是作为单一的纺 织材料的主要原因。因此，如何快速的育成高茧丝强度、强延伸性、易染色、高肌肤亲和性等 优质的蚕丝显得尤为重要。
家蚕经过数千年的选育，虽然目前品种资源较为丰富，但就茧丝强度、延伸性、染 色性能、肌肤亲和性等特性而言，品种间几乎没有什么差异，所以通过传统育种方法改变蚕 丝的这些特性基本上不可行。随着家蚕基因组计划的全面完成，分子标记辅助育种、转基因 育种等现代农业的新型工具逐渐的加入家蚕品种分子改良的主力阵容之中。家蚕丝蛋白只 要由丝素重链、丝素轻链和 P25 蛋白及包裹的在最外层的丝胶蛋白组成，其中决定茧丝性 能的丝素重链蛋白是一个由高度重复序列构成的大约 390kDa 的蛋白。丝素重链蛋白的高 表达量、高度重复序列和高分子量使得用常规的转基因技术也难以对其进行研究和改造。
此外，一头体重约为 6g 的家蚕食下 25g 左右的桑叶，便能吐出 0.5g 由纯蛋白组成 的蚕丝，这不仅是让人叹为观止的生物学奇观，其中更是蕴藏着令人无限憧憬的应用价值。 不仅如此，家蚕丝腺还具有高等生物蛋白质的翻译后修饰加工能力、大规模生产成本低以 及对人畜安全性等优势，满足了新一代生物反应器的基本要求，有巨大的开发应用潜力。该 领域研究已成为由传统蚕丝产业向非绢丝产业拓展的重要内容。无论是在基础研究，还是 在药物、化妆品开发领域，提取或表达纯化的蛋白都扮演着极其重要的角色，但是商业化蛋 白纯品高得离奇的价格（1g 最常用的绿色荧光蛋白价格约 5 百万元）一直在困扰着众多研 究者们。大规模表达或提取蛋白的工艺的缺乏严重的制约着与蛋白相关的研究和产品的发 展。饲养一头蚕的成本不足 1 角钱，却能吐 0.5g 的丝蛋白，而如果能用基因工程手段让家 蚕吐出 0.5g 纯蛋白，这对于蚕丝产业、甚至生物产业都将产生革命性的推动作用。也正是 这一奇特的生物学现象和应用前景吸引着众多生物学家自分子生物学发端以来就长期致 力于家蚕丝蛋白的表达调控和家蚕生物反应器的开发。然而，要实现这一看似简单而美好 的梦想并非易事。尽管通过长达数十年的探索与研究，家蚕丝蛋白基因的表达调控机理还 不尽清楚，丝腺生物反应器开发的道路更是尽显曲折而收效甚微。虽然研究者们利用家蚕 丝蛋白基因的启动子先后在家蚕丝腺的不同部位成功的表达了绿色荧光蛋白、红色荧光蛋 白、胶原蛋白、人碱性成纤维细胞生长因子、猫干扰素、植酸酶、人血清白蛋白、鼠单克隆抗 体、蜘蛛牵引丝蛋白和人脑源神经营养因子等具有重大研究和应用价值的蛋白。但是目前 家蚕丝腺中转基因表达的外源活性蛋白最高只能达到蚕茧总重量的 8.0%。表达量太低，从 茧层中纯化少量蛋白的工艺成本太高，令试图进一步开发家蚕丝腺生物反应器学者和商家 们都望而止步。
不论是对于家蚕丝蛋白本身的改良，还是开发实用、高效的家蚕丝腺生物反应器， 找到一种能有效定点改造家蚕丝蛋白基因的方法或者快速育成丝蛋白突变体资源都显得 尤为重要和紧迫。然而，尽管学者们在家蚕基因打靶方面也进行了一些探索，但是目前还没 有一种可用的家蚕基因组定向改造技术。
锌指核酸酶是一种人工设计的限制酶，由负责特异性识别 DNA 序列的锌指蛋白结 构域和负责切割 DNA 的核酸酶结构域组成。锌指核酸酶能在复杂基因组里的特定位点造成 双链断裂。从而刺激机体的 DNA 修复系统 ： 同源末端连接（NHEJ, Non-homologous End Joining）和同源重组（HR，Homologous recombination）。NHEJ 是一种特殊的 DNA 双链断 裂修复机制，其在不存在 DNA 同源序列的情况下快速、高效的强行将断裂的 DNA 末端连接 在一起，这是一种不忠实的修复机制，容易在修复处引入少数碱基的缺失、插入或突变 ； 果在同一 DNA 大片段中设计两对锌指核酸酶，也可能会造成两个识别位点之间的大片段缺 失 ； 外，在存在与断裂末端具有相同粘性末端的外源 DNA 的情况下，NHEJ 还可以实现特定 小片段的插入。HR 是机体内常见的一种 DNA 断裂修复机制，其原理自上世纪 80 年代以来已 经广泛的被应用于基因敲除中。通过人为设计各种不同类型的供体质粒，借助锌指核酸酶 介导的双链断裂，可以高效的对靶标基因进行定点置换、修复、删除、插入等任意的操作。目 前，这一技术已被广泛的应用于动植物的基因敲除和定点改造之中。
技术问题
本发明的目的之一在于提供一种家蚕丝素重链基因的突变方法和该突变方法得 到的基因序列，该方法为获得突变的家蚕丝素重链基因提供了新思路。
本发明的目的之二在于提供两种家蚕丝素重链突变基因的新应用，该应用为制备 丝胶蛋白和外源蛋白提供了有效途径。
技术解决方案
为实现上述目的，本发明的技术方案为 ：
家蚕丝素重链突变基因，所述家蚕丝素重链突变基因为家蚕丝素重链基因 1325 ～ 1362 位为锌指核酸酶识别的靶位点的突变基因。
所述家蚕丝素重链突变基因如 SEQ.ID NO:2-118 中任一种所示。
所述家蚕丝素重链突变基因如 SEQ.ID NO:2-14 中任一种所示。
所述家蚕丝素重链突变基因如 SEQ.ID NO:11 所示。
家 蚕 丝 素 重 链 基 因 的 突 变 方 法，将 含 有 如 SEQ.ID NO:119 和 SEQ.ID NO:120 所 示 锌 指 核 酸 酶 序 列 的 插 入 到 含 有 T7 ：’-taatacgactcactataggg-3’或 SP6 ： 5’-atttaggtgacactatag-3’启动子的原核表达载体中，获得重组载体 ； 重组载体进行体 外转录，获得编码锌指核酸酶序列的 mRNA，将编码锌指核酸酶序列的 mRNA 作用于如 SEQ.ID NO:1 所示的家蚕丝素重链基因 1325 ～ 1362 位为锌指核酸酶识别的靶位点，得家蚕丝素重 链突变基因。
所述的家蚕丝素重链突变基因在制备内源蛋白中的应用。
所述的家蚕丝素重链突变基因在制备丝胶蛋白中的应用。
所述的家蚕丝素重链突变基因在制备外源蛋白中的应用。
有益效果
本发明利用新近发展起来的锌指核酸酶技术，高效的对家蚕丝素重链基因进行了敲 除，首次获得了包括在丝素重链基因 N 端非重复区域部分缺失、部分碱基突变或小片段插 入的一系列突变家蚕品系。本发明提供的这些突变体具有如下的特点及优势 ：）本发明提 供的丝素重链基因突变体的后部丝腺严重退化，其茧层中只含有中部丝腺中合成与分泌的 丝胶蛋白，如果利用这些突变品系在家蚕后部丝腺中转基因表达外源蛋白（如蜘蛛丝、生物 活性蛋白等），外源蛋白在家蚕丝腺中的表达量和纯度将会大大的提高，这为家蚕丝腺生物 反应器的开发提供了一种全新的有用的遗传素材 ；）由于本发明提供的突变品系的茧层中 只有丝胶蛋白，而纯丝胶蛋白目前已经广泛的被用于化妆品，本发明为丝胶蛋白的大规模 生产提供了一种新的来源。
附图说明
图 1 显示了家蚕丝素重链基因的结构示意图，其中方框表示外显子，灰色实线表 示调控区域或内含子序列，数字表示相对于转录起始位点的位置，下划线部分为信号肽序 列的核苷酸或氨基酸序列，箭头所示为信号肽序列切割位点。
图 2 显示家蚕丝素重链基因部分序列在四十个蚕品系中的 SNP 分布，其中上端的 方框和实线表示所分析区域在丝素重链基因中的位置和结构，其下面的四十一条实线表示 30 个家蚕品系和 11 个野桑蚕品系的核苷酸序列，实线中菱形块表示相应品系在此处相对 于参照序列有变异，数字表示 SNP 位点相对于转录起始位点的位置，实线右侧的编号为不 同蚕品系的编号。
图 3 显示了突变体家蚕丝素重链基因突变位点的核苷酸序列，其中左侧的编号为 相应个体的编号，>Ref 表示野生型品系大造的序列，方框所示的序列为锌指核酸酶识别位 点，加粗的碱基为发生突变的碱基，- 表示缺失，下划线表示的碱基为插入的碱基。
图 4 显示了野生型家蚕和突变体家蚕的丝腺解剖图，其中左边为野生型品系大造 五龄六天的丝腺，右边为突变体家蚕五龄六天的丝腺。
图 5 显示了突变体家蚕蚕茧的观察图，其中上排为野生型品系 N4 的蚕茧，下排为 突变体家蚕的蚕茧。
图 6 显示了野生型家蚕和突变体家蚕蚕蛹和蚕茧重量的生物学统计分析。
图 7 显示了分别以野生型家蚕、杂合型突变体家蚕和纯和型突变体家蚕为受体的 转基因家蚕的丝腺的荧光信号，其中 +/+、+/- 和 -/- 分别表示野生型家蚕，杂合型突变体家 蚕和纯和型突变体家蚕。
图 8 显示了分别以野生型家蚕、杂合型突变体家蚕和纯和型突变体家蚕为受体的 转基因家蚕的茧层蛋白分析，其中 Transgenic-1、Transgenic-1 和 Non-transgenic 分别表 示两个转基因系和一个转基因系，+/+、+/- 和 -/- 分别表示野生型家蚕，杂合型突变体家蚕 和纯和型突变体家蚕，最下排数字表示泳道编号，箭头所示为外源绿色荧光蛋白融合蛋白 的特异条带。
在生物学中，染色体是成对存在的，即同一个基因有两份，对于任何一个突变体， 两份基因都是正常的即为野生型，一份正常一份突变即为杂合型突变体，两份都为突变即 为纯和型突变体。
本发明的最佳实施方式
本发明的实施方式
这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明 具体条件的实验方法均为本领域研究人员所熟知的方法或制造厂商所建议的条件，另外， 任何与所记载内容相似或均等的方法及材料皆可以应用于本发明中。本文中所述的实施方 法与材料仅作示范之用。
本发明的目的在于提供一种有效实现家蚕丝蛋白基因定向改造的方法，提供丝素 重链基因突变的遗传资源。即对家蚕丝素重链基因进行定向敲除，获得一系列家蚕丝素重 链基因缺失、突变或插入的突变体。
为了解决上述技术问题，本发明综合考虑外源蛋白在家蚕丝腺中的表达量和
Figure 5167
源性丝蛋白对后续蛋白纯化工艺的影响等目前家蚕丝腺生物反应器开发中所面临的主要 问题，利用新近发展起来的基因敲除技术——锌指核酸酶技术对家蚕丝素蛋白基因进行敲 除，并对获得的突变体进行基因组测序验证和功能验证。
本发明家蚕丝素重链基因突变体及其制备方法和应用，依次通过以下步骤实现 ：
(1) 下载家蚕丝素重链基因序列，分析其中锌指核酸酶靶向位点 ；
(2) 针对步骤 (1) 中分析的位点，设计特定的锌指核酸酶序列 ；
(3) 人工合成或者从已有的锌指蛋白库中扩增得到编码步骤 (2) 中设计的锌指核酸酶 的核酸序列 ；
(4) 将步骤 (3) 中的核酸序列插入到含有 T7 或 SP6 启动子的原核表达载体中，获得重 组载体 ；
（5) 利用步骤 (4) 中获得的重组载体进行体外转录，获得编码编码步骤 (2) 中设计的锌 指核酸酶的 mRNA ；
(6) 将家蚕滞育品种的蚕卵通过人工孵化处理后，置于 15℃恒温、相对湿度 75%、绝对 黑暗的环境中催青至孵化，非滞育性家蚕品种的蚕卵置于 25℃恒温、相对湿度 75%、自然光 照的环境中催青至孵化 ；
(7) 逐天收集孵化的蚁蚕，置于 25℃恒温、相对湿度 75%、自然光照的环境中用桑叶或 者人工饲料饲养至上蔟 ；
(8) 将上蔟后的家蚕转移至 25℃恒温、相对湿度 75%、自然光照的环境中进行种茧保 护；
(9) 待羽化后收集同时化蛾的雌雄蚕蛾，在 25℃、弱光的条件下交配 4h 后拆对，并将雌 蛾投放于上浆的蚕连纸上，在黑暗的环境中产卵，每间隔 0.5h 收集一次蚕卵，并将收集的 蚕卵置于 25℃的环境中保护 ；
(10) 将蚕卵连同蚕连纸片用自来水冲洗以后，于蒸馏水中浸泡至浆糊膨润（约经 2min），而后用镊子将蚕卵转移至用 75% 酒精消毒的载玻片上，并按照蚕卵腹面向右的标准 将其排列整齐，排列好的蚕卵置于 35-37% 的甲醛蒸气中消毒 5min ；
（11）以收集时间开始记时，于收集后 2h-4h 用显微注射仪将步骤 (5) 中获得的 mRNA 按摩尔比 1:1 的比例混合后从蚕卵腹面中央注入已经消毒的蚕卵中，并用无毒的胶水封闭卵 壳上注射留下的小孔 ；
(12) 将注射完成的蚕卵在 35-37% 的甲醛蒸气中消毒 5min 后，置于 25℃、相对湿度 85% 以上的湿度条件下，进行催青直到孵化，饲养孵化出来的蚁蚕，将当代（G0）的蚕蛾进行自交 或回交，获得滞育性的 G1 代蚕卵 ；
(13) 将步骤 (12) 中获得的 G1 代蚕卵经即时浸酸处理后置于 25℃恒温、相对湿度 75%、 自然光照的环境中催青至孵化 ；
(14) 将步骤 (13) 中获得的 G1 代蚁蚕在 25℃恒温、相对湿度 75%、自然光照的环境中用 桑叶饲养至上蔟，观察其吐丝和营茧的情况，筛选获得吐丝或营茧异常的突变个体 ；
(15) 将步骤 (14) 中获得的突变个体自交或回交制种，并提取制种后蚕蛾的基因组，用 丝素重链基因特异的引物进行 PCR 扩增、测序，进一步鉴定和确认突变体 ；
(16) 将步骤 (15) 中鉴定的突变体进行继代保存 ；
(17) 根据在家蚕丝腺中拟表达蛋白的氨基酸序列，体外合成或者扩增得到其编码序 列，并将其构建成转基因载体或者靶向家蚕丝素重链基因的同源重组载体 ；
(18) 以步骤 (16) 中保存的蚕卵作为受体，通过步骤 (6) 至步骤 (13) 所述的方法将步 骤 (17) 中转基因载体或同源重组载体注射到步骤 (16) 保存的家蚕胚胎中，饲养孵化出来 的蚁蚕，将当代（G0）的蚕蛾进行自交或回交，获得滞育性的 G1 代蚕卵，G1 代蚕卵经即时浸 酸处理后于胚胎发育第六天在荧光显微镜下扫描获得转基因个体 ； 后将获得的转基因个 体正常饲养、继代，获得相应的转基因家蚕 ；
(19) 对步骤 (18) 中获得的转基因家蚕的丝腺和茧层进行观察和分析，提取和纯化其 中的目的蛋白。
实施例 1 ： 蚕丝素重链基因的序列分析
从 NCBI 数据库下载家蚕丝素重链基因的序列（编号为 AF226688），其序列结构如图 1 所 示。家蚕丝素重链基因（+1 ～ +16 788，其中 +1 表示转录起始位点）包括两个长分别为 67bp 和 15750bp 的外显子以及一个长 971bp 的内含子，第一外显子包括 25bp 的非翻译区（+1 ～ +25）和 42bp 的编码区（+26 ～ +67），第二外显子包含 N 端非重复区域（+1039 ～ +1449）、C 端非重复区域（+16396 ～ +16788）和高度重复区域（+1450 ～ +16395，图 1 灰色方框所示）。 家蚕丝素重链基因编码的氨基酸序列的 N 端含有一个长 21 氨基酸残基的信号肽（图 1 下划 线所示）。
为了选择在各个蚕品系中均能发挥作用的锌指核酸酶靶位点，我们对家蚕丝素重 链基因的 N 端部分序列（+1 ～ +1448）做了 SNP 分析，结果如图 2 所示。家蚕丝素重链基因 的 N 端部分序列（+289 ～ +1448）在 29 个家蚕品系和 11 个野桑蚕品系中存在 10 个 SNP 位 点，分别为 +393、+465、+555、+556、+861、+862、+999、+1270 和 +1390。
实施例 2 ： 家蚕丝素重链基因特异锌指核酸酶序列的设计与合成 根据家蚕丝素重链基因的序列特征及其在不同蚕品系中 SNP 分布，结合锌指蛋白识别 DNA 序列的特性，我们选择 CTGTTGCTCAAAGTTATGTTGCTGCTGATGCGGGAGCA 作为锌指核酸酶识 别的靶位点，该靶位点位于如 SEQ ID NO:1 所示的序列的 +1325 ～ +1362 位，并据此设计和 合成锌指核酸酶。
所以，家蚕丝素重链基因 1325 ～ 1362 位为锌指核酸酶识别的靶位点。
实施例 3 ： 锌指核酸酶 mRNA 的制备
人工合成或者从已有的锌指蛋白库中扩增得到锌指核酸酶的核酸序列 ( 如 SEQ ID NO:119 及 SEQ ID NO:120) 用限制性内切酶 EcoRI 和 XhoI（购自 TAKARA）双酶切后与经过 同样酶切的原核表达载体 pET28a 进行连接反应，转化大肠杆菌并筛选阳性克隆，获得重组 载体，酶切的具体反应体系如下 ：
试剂 体积
质粒 5 μl
10X H buffer 1 μl
EcoRI 0.5 μl
XhoI 0.5 μl
无菌水 3 μl
总体积 10 μl

将重组载体 用XhoⅠ酶切消化后用MessageMax T7 mRNA体外转录试剂盒（购自Epicentre）进行体外转录，其反应体系如下：
试剂 体积
无RNA酶水 10 μl
质粒模板 (1μg/μl) 2 μl
10X 转录缓冲液 4 μl
ARCA Cap/NTP 混合物 16 μl
100 mM DTT 4 μl
MessageMAX T7 酶液 4 μl
总体积 40 μl

将上述反应体系在37℃孵育30分钟后加1μlDNA酶,再继续孵育15分钟。
将上述反应体系用Epicentre A-plus加尾试剂盒（购自Epicentre）进行加尾反应，其反应体系如下：
试剂 体积
无RNA酶水 109 μl
10X 缓冲液 20 μl
100 mM ATP 20 μl
ScriptGuard RNA 酶抑制剂 (40 U/μl) 5 μl
体外转录反应体系 42 μl
Poly （A）聚合酶 4 μl
总体积 200 μl

将上述体系在37℃孵育30分钟后用MEGAClear试剂盒（购自Ambion）进行纯化后-80℃保存备用。
实施例4： 显微注射用家蚕胚胎的制备
多化性家蚕品种'N4'的处理方法：在25℃恒温、相对湿度75%的环境中催青孵化、用桑叶饲养之后所产的蚕卵直接用于显微注射。
滞育性家蚕品种'大造'的处理方法：将正常饲养所得的蚕卵在常规的人工孵化处理后置于15℃恒温、相对湿度75%、自然光照的环境中催青至孵化。孵化后的蚕在25℃恒温、相对湿度75%、自然光照的环境饲养之后产的蚕卵用于注射。待羽化后收集同时化蛾的雌雄蚕蛾，在25℃、弱光的条件下交配4h后拆对，并将雌蛾投放于上浆的蚕连纸上产卵。
实施例5： 锌指核酸酶mRNA的显微注射
羽化后收集同时化蛾的雌雄蚕蛾，在25℃、弱光的条件下交配4h后拆对，并将雌蛾投放于上浆的蚕连纸上产卵。并于产卵后立即将总浓度400ng/uL靶向家蚕重链基因的锌指核酸酶mRNA用显微注射仪（FemtoJet 5247 显微注射仪，购自Eppendorf）注射到若干蚕卵之中，每粒蚕卵注射量大约10nL。注射后的蚕卵用无毒胶水封住注射口，并经35%的甲醛蒸气中消毒5min以后，置于25℃、相对湿度85%的高湿度环境中催青孵化，将孵化的G0代蚁蚕用人工饲料收集饲养至化蛾。
实施例6： 显微注射后突变个体的筛选
分别用多化性品种'N4'和滞育性品种'大造'作为原始材料，共注射蚕卵195和247粒，将孵化的93和124头G0代蚁蚕用人工饲料收集饲养至化蛾。分别获得的81和106个G0代蚕蛾，通过自交或回交获得38和51蛾圈G1代蚕卵，将38和51个蛾圈催青并单独饲养至G1代上蔟观察，分别在其中的29和38个蛾圈中发现250和105头裸蛹或者茧层稀薄的'丝胶茧'。具体实验统计结果见表1.
表1. 显微注射和突变筛选结果
家蚕品种 注射蚕卵数 孵化数（率） G0 代蛾 G1 蛾圈数 突变蛾圈数（率） 突变个体数
N4 195 93(48%) 81 38 29(76%) 250
大造 247 124(50%) 106 51 38(75%) 105

实施例7： 可遗传突变个体的测序分析
从事实例6中选取117个表现型均为裸蛹或'丝胶茧'的突变个体， 提取基因组，选取家蚕丝素重链基因突变位点附近序列设计PCR引物和测序引物，PCR引物为：
Fib-H-F: 5'-tgatgaggactattttgggag-3'
Fib-H-R: 5'-tagtgctgaaatcgctcgt-3'
测序引物为：Fib-H-F: 5'-tgatgaggactattttgggag-3'
PCR 反应体系为：
基因组DNA： 1.0 μl
EX Taq buffer ： 2.5 μl
Mg2+ ： 2.0 μl
dNTP ： 2.0 μl
Ex Taq ： 0.15μl
引物Fib-H-F： 0.5μl
引物Fib-H-F： 0.5μl
ddH2O ： up to 25 μ l
PCR 产物经电泳检测、纯化后进行测序反应。测序结果表明所选取的117个突变个体在锌指核酸酶靶位点均发生了突变，具体序列如SEQ ID NO:2-118，这些突变包括变异、缺失和小片段的插入,部分结果如图3所示。
实施例8： 可遗传突变个体的表型观察与解剖学观察
取野生型家蚕品系大造和 117 个突变体家蚕品系，每个品系取3头按常规的解剖步骤取出其丝腺于显微镜下观察，结果如图4所示，突变个体的丝腺明显小于野生型个体，而且突变个体的后部丝腺出现发育畸形的现象。
取野生型家蚕品系大造和117个突变体家蚕品系，每个品系取50个蚕茧进行观察，结果如图5所示，突变体家蚕的蚕茧茧层较野生型个体要薄。每个品系取25头蚕茧和蛹体，称取其重量进行生物学统计分析，结果如图6所示，突变个体的蛹体较野生型个体稍重，其茧层重量明显比野生型个体轻。对野生型家蚕品系和突变体家蚕品系的蚕茧进行蛋白电泳分析，结果表明突变体家蚕品系中不含有丝素重链蛋白，只有大量的丝胶蛋白存在，详见图8的泳道10。
可见， 所述的 家蚕丝素重链突变基因在制备内源蛋白中的应用。
实施例9： 外源性融合绿色荧光蛋白在突变体家蚕丝腺中的表达
以含有绿色荧光蛋白基因的质粒为模板，扩增得到绿色荧光蛋白的编码序列，并将其与家蚕丝素重链基因的非重复区域融合，构建成转基因载体（构建方法和流程参见Aichun Zhao等，Transgenic Research， DOI 10.1007/s11248-009-9295-7）。利用保存的突变体家蚕品系（突变序列如 SEQ.ID NO:11 ）作为受体材料，将正常饲养条件下所得的蚕卵在常规的人工孵化处理后置于15℃恒温、相对湿度75%、绝对黑暗的环境中催青至孵化，在25℃恒温、相对湿度75%的环境中进行饲养和种茧保护。待羽化后收集同时化蛾的雌雄蚕蛾，在25℃、弱光的条件下交配4h后拆对，并将雌蛾投放于上浆的蚕连纸上，在黑暗的环境中产卵，每间隔0.5h收集一次蚕卵，并将收集的蚕卵置于25℃的环境中保护。于产卵后3h用Eppendorf显微注射仪，将10-15nL、总浓度400ng/uL的构建好的转基因载体注射进入150粒蚕卵中，用无毒胶水封住注射口，并经35%的甲醛蒸气中消毒5min以后，置于25℃、相对湿度85%的高湿度环境中催青孵化，将孵化的69头G0代蚁蚕用桑叶收集饲养至化蛾，通过自交或回交共获得11蛾圈G1代蚕卵，用 Olympus®电动宏观荧光显微镜观察筛选了41蛾圈G1代蚕卵并获得1个阳性蛾圈，共获得11头转基因家蚕。对转基因家蚕的丝腺和茧层观察分析结果如图7和图8所示，以杂合型突变体家蚕和纯合型突变体家蚕为受体材料的转基因蚕的丝腺和茧层中融合绿色荧光蛋白的含量要显著高于以野生型家蚕为受体材料的转基因蚕。同样，以其他116个突变体家蚕品系（突变序列如 SEQ.ID NO:2 ～ 10 ， SEQ.ID NO:12 ～ 118 ）为受体材料的转基因蚕的丝腺和茧层中融合绿色荧光蛋白的含量也显著高于以野生型家蚕为受体材料的转基因蚕。
可见， 所述的 家蚕丝素重链突变基因在制备外源蛋白中的应用。
最后说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管通过参照本发明的优选实施例已经对本发明进行了描述，但本领域的普通技术人员应当理解，可以在形式上和细节上对其作出各种各样的改变，而不偏离所附权利要求书所限定的本发明的精神和范围。
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Claims (1)

1. 家蚕丝素重链突变基因 , 其特征在于 ： 述家蚕丝素重链突变基因为家蚕丝素重链 基因 1325 ～ 1362 位为锌指核酸酶识别的靶位点的突变基因。
2. 根据权利要求 2 所述的家蚕丝素重链突变基因，其特征在于，所述家蚕丝素重链突 变基因如 SEQ.ID NO:2-118 中任一种所示。
3. 根据权利要求 2 所述的家蚕丝素重链突变基因，其特征在于，所述家蚕丝素重链突 变基因如 SEQ.ID NO:2-14 中任一种所示。
4. 根据权利要求 2 所述的家蚕丝素重链突变基因，其特征在于，所述家蚕丝素重链突 变基因如 SEQ.ID NO:11 所示。
5. 家蚕丝素重链基因的突变方法，其特征在于，将含有如 SEQ.ID NO:119 和 SEQ.ID NO:120 所示锌指核酸酶序列的插入到含有 T7 ：’-taatacgactcactataggg-3’或 SP6 ： 5’-atttaggtgacactatag-3’启动子的原核表达载体中，获得重组载体 ； 重组载体进行体 外转录，获得编码锌指核酸酶序列的 mRNA，将编码锌指核酸酶序列的 mRNA 作用于如 SEQ.ID NO:1 所示的家蚕丝素重链基因 1325 ～ 1362 位为锌指核酸酶识别的靶位点，得家蚕丝素重 链突变基因。
6. 权利要求 1 所述的家蚕丝素重链突变基因在制备内源蛋白中的应用。
7. 根据权利要求 5 所述的应用，其特征在于，所述内源蛋白为丝胶蛋白。
8. 权利要求 1 所述的家蚕丝素重链突变基因在制备外源蛋白中的应用。
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