CN102618528A - Deleting system for long fragments in genome based on TALEN (transcription activator-like effector Nuclease) and ssODNs (single stranded oligonucleotides) and application thereof - Google Patents

Abstract

The invention discloses a genome long fragment deletion system based on TALEN and ssODNs, which is composed of a transcriptional activator-like effector nuclease TALEN and single-stranded oligonucleotide ssODNs that cut the target sequence of the target gene; the system can accurately identify the structure as T The target sequence of (Nn)A, and long-segment deletion in the genome, also discloses the application of the genome long-segment deletion system, which has high deletion efficiency, completely changes the genetic information of the genome, obtains varieties with large genetic variation, and provides abundant resources for breeding of genetic resources.

Description

Genome Long Fragment Deletion System Based on TALEN and ssODNs and Its Application

Technical Fields

The invention relates to the field of biotechnology, in particular to a long genome fragment deletion system based on TALEN and ssODNs, and also relates to the application of the long genome fragment deletion system.

Background technique

Bombyx mori is an important economic insect and a model organism of Lepidoptera. It has been bred and domesticated for more than 5,000 years in my country. The silk industry has always been in harmony with agriculture in history, and has made great contributions to the development of the Chinese nation's economy and the promotion of culture. Silk has inherent excellent characteristics such as graceful and elegant luster, light and comfortable touch, good drapability and curve of clothing materials, moderate water absorption and affinity to human skin, etc., and has been loved by consumers for a long time. Silk is also known as the "fiber queen" for this reason. At present, the total annual output value of my country's silk industry has reached 135 billion yuan, accounting for more than 10% of the total output value of the textile industry; cocoon silk production and export volume accounted for more than 70% and 80% of the world's total, and the annual foreign exchange earnings exceeded 5 billion US dollars, which is among the best in the world. A characteristic industry with an absolute advantage in the market. There are more than 20 million farmers growing mulberry and sericulture across the country, covering more than 20 provinces and autonomous regions. Sericulture is an important part of my country's rural economy, and its annual foreign exchange earnings from exports rank among the top among agricultural products, and it is an important source of farmers' income. The silk industry is playing an increasingly important role in solving the "three rural" issues, especially in increasing farmers' income and adjusting the rural industrial structure. the

However, as an ancient traditional industry, the technology and level of sericulture and silk production have been at a plateau in the past 60 years. Major breakthroughs have not been made in the problems of silkworm varieties such as robustness, silk quality, yield and other main economic traits, and the inherent defects of silk clothing such as easy yellowing and wrinkling have not been fundamentally resolved. The renewal of variety resources is far behind the production needs, and the overall production efficiency of the silk industry is low. This is mainly because: 1) After hundreds of years of excavation, the potential of increasing production based on traditional breeding methods has been maximized. In addition to the particularity and limitations of natural conditions, it is difficult to break through the limitations of the variety resources themselves. Although there are many types and quantities of silkworm species in existence, their genetic background is single, and there is no significant difference in economic and biological characters among them. Silkworm varieties have undergone a hundred years of change, and there has been no significant improvement in economic and biological traits. 2) As a textile fiber, silk is too expensive for many consumers. In addition, silk is prone to yellowing and wrinkling, so it does not occupy a large market share among various textile fibers. In the past few decades when the silk industry has almost stagnated, the research and utilization of textile fibers such as cotton and wool have progressed well, and various man-made fibers have sprung up, which has caused a relatively large impact on the silk industry. 3) For a long time, the main purpose of human domestication and breeding of silkworms is to reel and weave silk, so the existing silkworm varieties are almost all the same multi-filament varieties, and the composition and characteristics of their cocoon silk are basically the same. Today, with the rapid development of sericulture technology, silk has been re-recognized more and more as a multifunctional biological material. the

The silkworm is an ideal model organism and an important bioreactor for studying Lepidoptera insects. The early research results of silkworm genetics and physiology have made particularly important contributions to the basic discoveries of insect pheromones, hormones, anatomy, physiology and genetics; in the silkworm genome framework map, silkworm genome fine map, silkworm gene chip and After the successive analysis of genetic variation maps, silkworm genomics and functional genomics research also plays a pivotal role in modern insect biology research. As the only organ for synthesizing and secreting silk protein, the silk gland of silkworm is the biological organ with the strongest protein synthesis ability among insects, and one silkworm can produce more than 0.5g of pure protein. Whether it is in basic research, or in the field of drug and cosmetic development, the extraction or expression of purified proteins plays an extremely important role. The cost of raising a silkworm is less than 1 dime, but it can spit out 0.5g of silk protein. If genetic engineering can be used to make the silkworm spit out 0.5g of pure protein, it will have a revolutionary role in promoting the silk industry and even the biological industry. . It is also this unique biological phenomenon and application prospects that have attracted many biologists to devote themselves to the expression regulation of silkworm silk protein and the development of silkworm bioreactors since the beginning of molecular biology. the

It is necessary to break through the limitations of traditional breeding and obtain a revolutionary breakthrough in breed selection; completely overcome the inherent defects of silk and restore the vitality of silk in the textile fiber market; develop new multi-functional silk materials to promote the single silk industry to a wider African The expansion of the silk industry, these are key issues that need to be solved urgently in modern sericulture. In the era of molecular agriculture, the solution to these problems is inseparable from the functional analysis and genetic modification of genes related to important economic traits in silkworms. To maximize the model role of silkworm in the study of Lepidoptera insects and promote the further development and application of silkworm bioreactors, important molecular tools such as transgenic, genome editing and genetic modification are also inseparable. Although the currently widely used molecular improvement tools such as transgene and RNAi have been well applied in the silkworm, the transgene and RNAi cannot completely delete the nucleic acid sequence in the genome, but at present, the endogenous genes of the silkworm can be directly edited and The transformation technology has not been reported yet, and there is no relevant report on the deletion of long-segment genome sequences. the

Contents of the invention

The purpose of the present invention is to provide a long genome fragment deletion system based on TALEN and ssODNs and its application and the application of the deletion system, which can delete the long fragment of the target gene in the genome by using the deletion system, and obtain varieties with large genetic variation. Breeding provides a wealth of genetic resources. the

In order to achieve the above purpose, the technical scheme is:

1. Genome long fragment deletion system based on TALEN and ssODNs, said deletion system is made up of transcriptional activator-like effector nuclease TALEN and single-stranded oligonucleotide ssODNs of shear target gene target sequence, said endonuclease TALEN It consists of a nuclease TALEN-L that cuts the 5' end of the target sequence and a nuclease TALEN-R that cuts the 3' end of the target sequence; the single-stranded oligonucleotide ssODNs consists of the target sequence of the target gene At least 10 consecutive nucleotides and at least 10 consecutive nucleotides at least 1 base upstream or downstream from the target sequence. the

Further, the target sequence is a nucleotide sequence of T(Nn)A, N is any base among A, G, T and C, and n is any number between 24 and 65. the

Furthermore, the target gene is the Bombyx mori gene BmBlos2 , and the target sequence is the nucleotide sequence shown in SEQ ID NO.2.

Further, the nuclease TALEN-L is the protein encoded by the nucleotide shown in SEQ ID NO.3; the nuclease TALEN-R is the protein encoded by the nucleotide shown in SEQ ID NO.4. the

Further, the single-stranded oligonucleotide ssODNs consists of 40 consecutive nucleotides of the target sequence and 60 consecutive nucleotides 795 bases upstream of the target sequence. the

Further, the single-stranded oligonucleotide ssODNs nucleotides shown in SEQ ID NO.17. the

2. The application of the genome long fragment deletion system to modify the target gene in the target cell genome. the

Further, the target cell is a silkworm egg cell. the

Further, the target gene is the Bombyx mori oil silkworm gene BmBlos2 .

The beneficial effect of the present invention is that: the present invention utilizes TALEN and ssODNs technology, and through TALEN has the characteristic of specifically cutting nucleic acid target sequences, a protein containing a recognition target sequence is designed inside the target gene, and a nucleus is provided upstream of the recognition target sequence protein. The positioning signal NLS and the nuclease FokI enable the target protein to be located in the nucleus, and the target sequence is recognized and cleaved by the nuclease FokI; at the same time, the single-stranded oligonucleotide ssODNs has the function of repairing the nucleic acid sequence, through ssODNs and Combining upstream or downstream sequences of the target sequence, deletion and repair are completed in the genome, and finally long fragments can be deleted. The operation is simple, and any sequence can be deleted. The principle of gene homology can also be used to simultaneously delete multiple genes at once. , can obtain varieties with large genetic variation, and provide abundant genetic resources. the

Description of drawings

Figure 1 shows the schematic diagram of the structure of the silkworm gene BmBlos2 and the recognition site of the endonuclease TALEN that cuts the target sequence of the target gene, where the numbers indicate the length of the exon or intron, ATG and TAA respectively indicate the start and Stop codons, underlines indicate recognition modules of TALENs.

Fig. 2 shows the phenotype of the silkworm after the BmBlos2 gene BmBlos2 is knocked out, wherein WT represents a wild-type silkworm individual, and mutant represents a mutant.

Figure 3 shows the mutant sequence produced after injection of B3, wherein a diagram represents a partial structural schematic diagram of the silkworm gene BmBlos2 of the silkworm, the upper arrow represents the position of PCR and sequencing primers, and diagram b represents the mutant individuals from different moth circles Sequences, marks beginning with ">" represent different moth circle numbers, "△", "M" and "I" represent deletions, base substitutions and insertions respectively, and the subsequent numbers represent the bases of deletions, substitutions or insertions The base number, "X" and the following numbers represent the number of corresponding sequences, and the underlined sequence is the recognition site of TALEN.

Fig. 4 shows a schematic diagram of the structure of the Bombyx mori gene BmBlos2 and the deletion result of the target gene. Figure a shows the schematic diagram of the structure of the silkworm gene BmBlos2 , the black line indicates the design of ssODNs; Figure b shows the PCR results after co-injection of TALEN and ssODNs, the size of the target band is 556bp, and the number at the bottom indicates the mutation efficiency.

Detailed ways:

In order to make the purpose, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings. These examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. For the experimental methods not specified in the examples, the conventional conditions are generally followed, such as the conditions described in the Molecular Cloning Experiment Guide (Third Edition, J. Sambrook et al.), or the conditions suggested by the manufacturer.

The silkworm variety used in the examples is "N4", provided by the Silkworm Gene Resource Bank of Southwest University of China. the

1. Sequence Analysis of BmBlos2 Gene of Bombyx mori

The sequence of the silkworm gene BmBlos2 (number BGIBMGA002101) was downloaded from the silkDB database. Sequence analysis showed that the gene included four exons with lengths of 180bp, 131bp, 112bp and 608bp and three introns with lengths of 2465bp, 663bp and 2703bp respectively. The first exon included an untranslated region of 125bp and The structure of the 55bp coding region is shown in Figure 1. According to the general principle of gene knockout, the second and third exons of the gene are ideal target sites, and the second and third exons are used as target sequences in this embodiment.

2. Design of the third exon TALEN sequence

According to the sequence characteristics of the silkworm gene BmBlos2 of the silkworm, we selected the sequence on the third exon as the target sequence of the TALEN function, and the nucleotide sequence of the third exon of the silkworm gene BmBlos2 of the silkworm is shown in SEQ ID NO.1 , according to the TALEN principle, the nucleotide sequence with the structure T(Nn)A is the TALEN recognition target sequence, wherein N is any base in A, G, T and C, and n is any base between 24 and 65 number. According to the above principles, the target site for TALEN recognition is 5'- tcaccaagtacgcagatcta aggagcctagccgc g aacctgaacaagaccctta -3' (SEQ ID NO.2), the underlined part is the TALEN recognition module, the bold part indicates the spacer sequence, and the 5' end is underlined The nucleotide sequence is the 5' end recognition sequence, corresponding to 1-20 nucleotides shown in SEQ ID NO.2, and the 3' underlined nucleotide sequence is the 3' end recognition sequence, corresponding to SEQ ID NO.2 The 36-54 nucleotide sequence shown. The 5'-end recognition sequence and the 3'-end recognition sequence are respectively designed according to the rules of A=NI, T=NG, G=NN, C=HD to design the variable sequence of the protein that recognizes the target sequence, and couple the nuclear localization signal upstream of the protein NLS, the downstream coupling nuclease FokI, respectively, the nuclease TALEN-L composed of the nuclear localization signal NLS, the protein that recognizes the recognition sequence at the 5' end and the nuclease FokI in series, and the nuclease TALEN-L that is composed of the nuclear localization signal NLS and the recognition sequence at the 3' end The sequenced protein and the nuclease FokI are sequentially connected in series to form the nuclease TALEN-R. Design the nucleotide sequence encoding nuclease TALEN-L and nuclease TALEN-R according to the amino acid sequence of nuclease TALEN-L and nuclease TALEN-R, the nucleotide sequence of encoding nuclease TALEN-L is as SEQ ID NO. 3, wherein the 82-102 nucleotides encode the nuclear localization signal NLS, the 120-2826 nucleotides encode the protein that recognizes the 5' recognition sequence, and the 2833-3420 nucleotides encode the endonuclease FokI , the nucleotide sequence encoding the nuclease TALEN-R is shown in SEQ ID NO.4, wherein the 76th-96th nucleotides encode the nuclear localization signal NLS, and the 114th-2820th nucleotides encode the recognition 3' recognition sequence protein, the 2826-3414th nucleotides encode the nuclease FokI.

3. Preparation of mRNA encoding TALEN-L and TALEN-R

Nucleotide sequences (SEQ ID NO.3 and SEQ ID NO.4) encoding nuclease TALEN-L and nuclease TALEN-R were artificially synthesized respectively, and then ligated with restriction endonucleases KpnI and BamHI after double digestion. The same prokaryotic expression vector pET28a was digested, then transformed into Escherichia coli, and positive clones were screened to obtain a pair of recombinant plasmids, which were named pET28a-B3L and pET28a-B3R. The resulting recombinant plasmid pairs were digested with Xho I respectively, and then transcribed in vitro with the MessageMax T7 mRNA in vitro transcription kit (purchased from Epicentre). Continue to incubate for another 15 minutes. Then the reaction solution was added to the A-plus reaction using the Epicentre A-plus tailing kit (purchased from Epicentre), and the reaction conditions were: incubate at 37°C for 30 minutes, and then use the MEGAClear kit (purchased from Ambion) to purify , the mRNA encoding nuclease TALEN-L and nuclease TALEN-R was obtained, and the obtained mRNA mixture of nuclease TALEN-L and nuclease TALEN-R was named B3, and stored at -80°C for future use.

4. Microinjection of B3

The polymorphic silkworm variety "N4" was selected and raised with mulberry leaves under the conditions of a temperature of 25°C, a relative humidity of 75%±5%, and a photoperiod of 12 hours of light and 12 hours of darkness. After eclosion, collect the male and female silkworm moths that moth at the same time, and mate for 4 hours under the conditions of temperature at 25°C and light intensity of 50-155 μmol/m2/s. The obtained silkworm eggs were directly used for microinjection. A microinjector (FemtoJet 5247 microinjector, purchased from Eppendorf) was used to inject 521 silkworm eggs among the obtained silkworm eggs with B3 at a concentration of 700ng/μL, and the injection volume of each silkworm egg was about 10nL. The injected silkworm eggs were sealed with non-toxic glue at the injection port, sterilized with 35% formaldehyde vapor for 5 minutes, and placed in a room at 25°C, with a relative humidity of 85%, and a photoperiod of 12 hours of light + 12 hours of darkness. The green hatching is accelerated in the environment, and the hatched G0 generation ant silkworms are collected and raised with artificial feed until they become moths.

5. Screening of Mutant Individuals

Among the 521 injected silkworm eggs, 126 fifth-instar larvae were obtained through artificial rearing, and 28 of them showed chimera phenotype, as shown in Figure 2. The 126 G0 generation silkworm moths obtained were self-crossed or backcrossed to obtain 41 moth circles G1 generation silkworm eggs, and the 41 moth circles were accelerated and raised separately to the third instar larvae of the G1 generation for observation. In 6 moth circles among them 277 silkworm mutants with the phenotype of oil silkworm were found.

6. Sequencing Analysis of Individuals with Inheritable Mutations

Select 51 silkworm mutants whose phenotype is oily silkworm, extract the genome, and select the sequence near the mutation site to design PCR primers for PCR amplification. PCR primers: the upstream primer is B3-F364: 5'-tccaatttgagggcaatgctac-3' (SEQ ID NO.5), the downstream primer is B3-R315: 5'-atttcaccacctcattcaactaagat-3' (SEQ ID NO.6); PCR amplification The conditions were: pre-denaturation at 94°C for 4 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 59°C for 30 s, extension at 72°C for 45 s, and extension at 72°C for 10 min. At the same time, the sequence near the mutation site was selected to design sequencing primers, and the sequencing primers are shown in SEQ ID NO.6. PCR products were detected by electrophoresis, purified, and then sequenced. The sequencing results showed that the selected 51 silkworm mutant individuals had mutations at the target sequence of the third exon, and the mutations included mutations, deletions, or insertions of small fragments. The sequencing results are shown in SEQ ID NO.7-16, and some results are shown in Shown in accompanying drawing 3.

7. Design and synthesis of ssODNs sequence

Design a single-stranded oligonucleotide (Single-stranded oligonucleotides, ssODNs) based on the target sequence of the third exon and the upstream sequence of the deletion fragment, namely the second exon. ssODNs are recognized by the 3' of the target sequence of the third exon sequence and 60 consecutive nucleotides at 792 positions upstream from the 3' recognition sequence, the specific sequence is: 5'-tgtcaccgagctgttcgagtttcgaagtcctggatccacatgatcctgtgatcagtcggt gccgcgaacctgaacaagacccttaatgaatacaacgaga -3' (SEQ ID NO.17), in which 1-60 nucleotides are deletion fragments The upstream sequence and 61-100 nucleotides are constructed according to the downstream sequence of the TALEN cleavage site, and the underlined part is the 3' end recognition site of the transcription activator-like effector nuclease TALEN.

8. Microcoinjection of ssODNs and B3

Silkworm eggs were prepared by using the above method for preparing silkworm eggs, and 402 silkworm eggs were taken for microinjection, specifically: B3 with a concentration of 700 ng/μL and ssODNs with a concentration of 100 mM were mixed at a volume ratio of 1:1, and then microinjected with a microscope. The microinjector (FemtoJet 5247 microinjector, Eppendorf) injected, and the injection volume of each silkworm egg was about 10nL. The injected silkworm eggs were sealed with non-toxic glue at the injection port, and sterilized with 35% formaldehyde vapor for 5 minutes, and then placed in a high-humidity environment at 25°C and 85% relative humidity for incubation, and 61 A total of 35 fifth-instar silkworms were collected from the first G0 generation of silkworms by artificial feed, and one of them was a chimera (part of the epidermis showed a transparent phenotype, and part of the epidermis was normal milky white), which was a mutant. Extract the genomes of the obtained chimeric mutants and 20 individuals with normal performance and design primers for PCR amplification. The upstream primer of the PCR primer is 5'-ttggtccagtaggtttgaagtaggt-3' (SEQ ID NO.18), and the downstream primer is such as SEQ ID NO.10 ;PCR amplification conditions were: 94°C pre-denaturation for 4 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 59°C for 30 s, extension at 72°C for 45 s; extension at 72°C for 10 min, and PCR products were analyzed by 1% agarose gel electrophoresis , the detected PCR band was 556bp. Compared with the 1351bp without mutation, the 795bp fragment was deleted in the target gene. It can be seen that a long fragment deletion occurred in the silkworm individual of the chimera phenotype, and the deletion sequence was the target gene in For the missing part of ssODNs, its proportion is 2.2%, and the results are shown in Fig. 4.

Finally, it is noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described with reference to the preferred embodiments of the present invention, those skilled in the art should understand that it can be described in the form Various changes may be made in matter and details thereof without departing from the spirit and scope of the invention as defined in the appended claims. the

<110> Southwest University

<120> Genome Long Fragment Deletion System Based on TALEN and ssODNs and Its Application

<160> 18

the

<210> 1

<211> 112

<212> DNA

<213> Silkworm ( Bombyx mori L.)

<220>

<223> Nucleotide sequence of the third exon of BmBlos2 gene of Bombyx mori

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gaacactaca tgctgcttga ggagatcaac agactcgcga tcaccaagta cgcagatcta 60

aggagcctag ccgcgaacct gaacaagacc cttaatgaat acaacgagat gt 112

the

the

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<211> 54

<212> DNA

<213> Silkworm ( Bombyx mori L.)

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<223> Exon 3 TALEN binding site

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tcaccaagta cgcagatcta aggagcctag ccgcgaacct gaacaagacc ctta 54

the

<210> 3

<211> 3426

<212> DNA

<213> Artificial sequence

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<223> Nucleotide sequence encoding TALEN-L

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atgagatctg actacaaaga ccatgacggt gattataaag atcatgacat cgattacaag 60

gatgacgatg acaagatggc ccccaagaag aagaggaagg tgggcattca tggggtaccc 120

atgatgagca gaaccagact ccccagccca ccagcgccct cgccagcgtt ttcggcaggc 180

agcttttcgg acttgttacg tcagtttgac ccaagcttat tcaacactag cctttttgat 240

tccctgcctc cattcggtgc tcaccataca gaagctgcca ctggagaatg ggacgaggtt 300

caatccggac tgagggcagc agatgcacca ccacctacca tgcgtgtggc agttacggct 360

gccaggccac cgagagctaa accagcacca agaaggcgtg cagcacaacc atcagacgct 420

tctccggctg cccaggtgga tctccgcacc ttaggttact cacaacagca acaggaaaaa 480

attaagccga aagttcgttc tacagtcgcc caacaccatg aggcattggt gggacacggc 540

tttactcacg cgcatatagt tgctctgtcg caacatccgg cagcgttggg aaccgtcgct 600

gtaaagtatc aggacatgat cgctgccctc cctgaagcca cacacgaggc aattgtgggt 660

gttggaaaac agtggtcagg tgctcgcgcc ttggaagcgc tgttgacagt ggctggagag 720

ctccgtggtc cacctttaca acttgatacc ggacagctct taaagatcgc taaaaggggt 780

ggagtcacgg cagtagaagc ggtgcacgct tggagaaacg cgttaacagg agctcccctg 840

aatttgactc ctgaacaagt ggttgcaatt gcgtcgcacg acggcggtaa acaagctctt 900

gagaccgtac agaggcttct gccagtgttg tgccaagcac atggactcac gccagcacag 960

gtcgtagcta tcgccagtaa tattggaggc aaacaagcgt tagaaacagt tcagagattg 1020

ctccccgtct tatgtcaagc tcacggtctt actcctgacc aggtggttgc aatagcgtca 1080

catgatggtg gaaagcaagc tttggagacc gtacagcgct tacttccagt gctgtgccaa 1140

gcgcacggat tgacgccggc tcaagttgta gctatcgcct ctcatgatgg cggtaaacaa 1200

gccctcgaaa cagttcagcg tctgttgccc gtcctctgtc aagcacacgg cttaactcct 1260

gaacaggttg tcgcaatagc gagcaacatc ggaggcaagc aagcgctgga gaccgtacag 1320

aggctcttac cagtgctttg ccaagctcat ggtctgacgc cggaccaggt cgtagctatt 1380

gcctccaata taggtggaaa acaagccttg gagacagttc agagacttct gcccgtcttg 1440

tgtcaagcac acggactcac tcctgatcag gttgtcgcaa ttgcgtcgaa caatggcggt 1500

aaacaagcgc tcgaaaccgt acagcgcttg ctcccagtgt tatgccaagc acatggcctt 1560

acgccggaac aggtcgtagc tatagccagt aacggaggcg gtaaacaagc cctggagaca 1620

gttcagcgtt tacttcccgt cctgtgtcaa gctcacggtt tgacaccaga ccaggtggtt 1680

gcaatcgcgt caaatattgg aggcaagcaa gccttagaaa ccgtacagag gctgttgcca 1740

gtgctctgcc aagctcacgg attaactcct gagcaagtcg ttgcaatcgc ctctcatgat 1800

ggtggaaaac aagccctcga gacagttcag agactcttac ccgtcctttg tcaagctcac 1860

ggcctgactc ctcaacaggt ggttgcaatt gcgagcaata acggtggtaa acaggccctg 1920

gaaaccgtac agcgccttct gccagtcttg tgccaagctc acggtctcac tcctgagcaa 1980

gtcgtagcta tagcctccca tgacggaggc aaacaggctt tggagacagt gcagcgtttg 2040

ctccccgttt tatgtcaagc tcatggactt actcctgatc aagttgttgc aatagcgtcg 2100

aacatcggtg gaaagcaagc tctggagacc gtccagaggt tgttgccagt gctgtgccaa 2160

gcacacggat tgactcctgc acaagttgta gctattgcca gtaataacgg cggtaaacaa 2220

gccttggaaa cagttcagcg cttgctgcct gttctctgtc aagctcatgg tttaactcct 2280

gagcaagttg tcgcaattgc gtcaaacata ggtggcaagc aggccctgga gaccgtgcag 2340

cgtctcttac cagttctttg ccaagctcac ggactgacgc cgcaacaagt ggtagctatt 2400

gcctctaatg gtggaggcaa acaagccctg gaaacagtcc agagacttct gcccgtgttg 2460

tgtcaagctc acggcctcac tcctgaacag gttgtggcaa tcgcgagcca tgacggtgga 2520

aagcaggctt tggagaccgt tcagcgcttg ctcccagtct tatgccaagc tcacggattg 2580

actcctgagc aggtcgtagc tatagcatcc aacggaggtg gaaggccagc actcgagtcg 2640

atcgtggctc aattaagtag acccgaccct gcccttgcag cgctgactaa tgatcacttg 2700

gtcgcactcg cgtgcttagg cggtagaccc gcccttgatg cagtgaaaaa gggtctgcca 2760

catgctccag cactcatcaa aagaaccaat cgtcgtattc ccgagaggac atcacacagg 2820

gtggcgggat cccagctggt gaagagcgag ctggaggaga agaagtccga gctgcggcac 2880

aagctgaagt acgtgcccca cgagtacatc gagctgatcg agatcgccag gaacagcacc 2940

caggaccgca tcctggagat gaaggtgatg gagttcttca tgaaggtgta cggctacagg 3000

ggaaagcacc tgggcggaag cagaaagcct gacggcgcca tctatacagt gggcagcccc 3060

atcgattacg gcgtgatcgt ggacacaaag gcctacagcg gcggctacaa tctgcctatc 3120

ggccaggccg acgagatgca gagatacgtg aaggagaacc agaccggaa taagcacatc 3180

aaccccaacg agtggtggaa ggtgtacccct agcagcgtga ccgagttcaa gttcctgttc 3240

gtgagcggcc acttcaaggg caactacaag gcccagctga ccaggctgaa ccacaaaacc 3300

aactgcaatg gcgccgtgct gagcgtggag gagctgctga tcggcggcga gatgatcaaa 3360

gccggcaccc tgacactgga ggaggtgcgg cgcaagttca acaacggcga gatcaacttc 3420

tgataa 3426

the

<210> 4

<211> 3414

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence encoding TALEN-R

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atggactaca aagaccatga cggtgattat aaagatcatg acatcgatta caaggatgac 60

gatgacaaga tggcccccaa gaagaagagg aaggtgggca tccacggggt acccatgatg 120

tctcgcactc gtctccctag ccctccagca ccgtcaccag cattctcggc aggtccttc 180

tcggacttat tacgccagtt tgacccgagc ttattcaata ctagcctttt tgattccctg 240

cctccattcg gtgctcacca tacagaagct gccactggcg aatgggacga ggttcaatcc 300

ggtctgaggg cagcagatgc accacacct accatgagag tggctgttac ggctgccagg 360

ccaccgagag ctaaaccagc accaagaagg cgtgcagcac agccatcaga cgcttctccg 420

gctgcccaag tggatctcag gaccttaggt tactcacaac agcaacagga aaaaatcaaa 480

ccaaaagtta gatctacagt cgcccagcac catgaggcat tggtgggcca cggttttact 540

cacgcgcata tcgttgctct gtcgcagcat ccggcagcgt tgggaaccgt cgctgtaaag 600

tatcaagaca tgattgctgc cctccccgaa gccaacacg aggcaatagt gggagttggc 660

aaacaatggt caggagctag ggccttggaa gcgctgttga cagtggctgg agagctcaga 720

ggacccccctt tacaacttga taccggccag ctcttaaaga ttgctaaacg cggtggagtc 780

acggcagtag aagcggtgca cgcttggcgt aacgcgttaa caggagctcc cctgaatttg 840

actcctgaac aggtggttgc aatagcgtcg cacgacggcg gtaaacaagc ccttgagacc 900

gtacagcgcc ttctgccagt gttgtgccaa gcacatggtc tcacgccgca acaggtcgta 960

gctatcgcca gtaacattgg aggcaaacaa gcgttagaaa cagttcagcg tttgctcccc 1020

gtcttatgtc aagctcacgg acttactcct caacaggtgg ttgcaatcgc gtcaaacaat 1080

ggtggaaagc aagccttgga gaccgtacag aggttacttc cagtgctgtg ccaagcacat 1140

ggcttgacgc cggaacaggt cgtagctatt gcctctaata agggcggtaa acaagcgctc 1200

gagacagttc agagactgtt gcccgtcctc tgtcaagcac acggtttaac accagagcaa 1260

gtggttgcaa tagcgagcaa caatggaggc aaacaagccc tggagaccgt acaggcactc 1320

ttaccagtgc tttgccaagc ccatggactt actcctgagc aagttgtagc tatcgcctcc 1380

aacggtggag gcaagcaagc gttggagaca gttcaggctc ttctgcccgt cttgtgtcaa 1440

gctcacggcc tcacaccaga acaggtggtt gcaattgcgt cgcatgatgg tggaaaacaa 1500

gccctcgaga ccgtacaggc attgctccca gtgttatgcc aagctcacgg tcttactcct 1560

gaacaggtcg tagctatagc cagtaacggc ggtggaaagc aagctctgga gacagttcag 1620

cgcttacttc ccgtcctgtg tcaagcccat ggactgacac ccgagcaagt tgttgcaatc 1680

gcgtcaaatg gcggtggaaa acaagcattg gagaccgtac agcgtctgtt gccagtgctc 1740

tgccaggctc acggcttaac tcctcagcaa gttgttgcta ttgcctctaa caatggcggt 1800

aaacaagccc tcgaaacagt tcagaggctc ttacccgtcc tttgtcaagc tcatggtctc 1860

acacccgagc aggttgtcgc aatagcgagc aacggaggcg gtaaacaagc gctggagacc 1920

gtacaggctc ttctgccagt cttgtgccaa gctcacggac tcactccaga gcaagtcgta 1980

gctatcgcct ccaatggagg cggtaaacaa gccttggaga cagtgcagag attgctcccc 2040

gttttatgtc aagctcacgg ccttacaccc gagcaggttg tggctattgc gtcgcatgac 2100

ggtggcaaac aggccctgga gaccgtccag cgcttgttgc cagtgctgtg ccaagctcat 2160

ggtctgactc cagagcaggt tgttgctata gccagtaaca tcggaggaaa gcaggccttg 2220

gagacagttc agcgtttgct gcctgttctc tgtcaagctc acggattaac acccgagcag 2280

gttgttgcaa tagcgtcaaa taacggtggt aaacaggctt tggagaccgt gcagagactc 2340

ttaccagttc tttgccaagc tcatggcctg actcctcagc aagtggttgc tatcgcctct 2400

aacaaaggag gccgccccgc cctggaaaca gtccaacgtc ttctgcctgt gttgtgtcag 2460

gctcacggtc tcactccaga acaagtggtt gcaattgcga gcaatggagg tggcaaacaa 2520

gctttggaga ccgttcagcg cttgctcccg gtcttatgcc aggctcacgg acttacgccc 2580

cagcaagtgg tcgctatcgc atccaacggt ggaggaaggc ctgcactcga atcgattgtg 2640

gcacaattaa gtcgtcccga ccctgccctt gcagcgctga ctaatgatca cttggtcgca 2700

ctcgcgtgct taggtggaag acctgccctt gatgcagtga aaaagggtct gccacatgct 2760

cccgcactca tcaagagaac taatagacgc atacccgaga gaaccagcca ccgtgttgct 2820

ggatcccagc tggtgaagag cgagctggag gagaagaagt ccgagctgcg gcacaagctg 2880

aagtacgtgc cccacgagta catcgagctg atcgagatcg ccaggaacag cacccaggac 2940

cgcatcctgg agatgaaggt gatggagttc ttcatgaagg tgtacggcta caggggaaag 3000

cacctgggcg gaagcagaaa gcctgacggc gccatctata cagtgggcag ccccatcgat 3060

tacggcgtga tcgtggacac aaaggcctac agcggcggct acaatctgcc tatcggccag 3120

gccgacgaga tggagagata cgtggaggag aaccagaccc ggaataagca cctcaacccc 3180

aacgagtggt ggaaggtgta ccctagcagc gtgaccgagt tcaagttcct gttcgtgagc 3240

ggccacttca agggcaacta caaggcccag ctgaccaggc tgaaccacat caccaactgc 3300

aatggcgccg tgctgagcgt ggaggagctg ctgatcggcg gcgagatgat caaagccggc 3360

accctgacac tggaggaggt gcggcgcaag ttcaacaacg gcgagatcaa cttc 3414

the

<210> 5

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> The third exon upstream detection primer B3-F364

<400> 5

tccaatttga gggcaatgct ac

the

<210> 6

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> The third exon downstream detection primer B3-R315

<400> 6

atttcaccac ctcattcaac taagat 26

the

<210> 7

<211> 674

<212> DNA

<213> Silkworm ( Bombyx mori L.)

<220>

<223> Sequencing results of silkworm mutant Bb-1-01

<400> 7

tccaatttga gggcaatgct acaatattag aactgcacta ttaaaaataa tcgatttaat 60

gaatgaaaga ttaataaatc tacagtttat ttcgaaataa atagtatcag atctttgttt 120

tgtataaatt tttttatgaa tatctataac cagtttagag aaaatctgta agaataataa 180

atccttaagc aaattgttgc aagggactga ttgttgtatc acaccaaggt acttaagacc 240

cagagaatta ccagaaacaa gacccatatg gttatttcaa gtaaatattt gatacaggaa 300

cactacatgc tgcttgagga gatcaacaga ctcgcgatca ccaagtacgc agatctaagg 360

aaccgcgaac ctgaacaaga cccttaatga atacaacgag atgtgtgagt ttttaactgg 420

aaccatcaat tttttaatga atgtattgcc agcaaatgtg tcataataca aagcaacttg 480

tgtttcagtt atcaaataat ttgataaaca ctgtggctag cttatgtgtc atttgttttt 540

gtgaaggcat agaattgaaa taatccctca tatcaactat tgctataatc tctaaattgc 600

attctgtcaa cgcctggcct attagaattt aggcctaact aacacaagat cttagttgaa 660

tgaggtggtg aaat 674

the

<210> 8

<211> 673

<212> DNA

<213> Silkworm ( Bombyx mori L.)

<220>

<223> Sequencing results of silkworm mutant Bb-1-05

<400> 8

tccaatttga gggcaatgct acaatattag aactgcacta ttaaaaataa tcgatttaat 60

gaatgaaaga ttaataaatc tacagtttat ttcgaaataa atagtatcag atctttgttt 120

tgtataaatt tttttatgaa tatctataac cagtttagag aaaatctgta agaataataa 180

atccttaagc aaattgttgc aagggactga ttgttgtatc acaccaaggt acttaagacc 240

cagagaatta ccagaaacaa gacccatatg gttatttcaa gtaaatattt gatacaggaa 300

cactacatgc tgcttgagga gatcaacaga ctcgcgatca ccaagtacgc agatctaagg 360

agcgcgaacc tgaacaagac ccttaatgaa tacaacgaga tgtgtgagtt tttaactgga 420

accatcaatt ttttaatgaa tgtattgcca gcaaatgtgt cataatacaa agcaacttgt 480

gtttcagtta tcaaataatt tgataaacac tgtggctagc ttatgtgtca tttgtttttg 540

tgaaggcata gaattgaaat aatccctcat atcaactatt gctataatct ctaaattgca 600

ttctgtcaac gcctggccta ttagaattta ggcctaacta acacaagatc ttagttgaat 660

gaggtggtga aat 673

the

the

<210> 9

<211> 680

<212> DNA

<213> Silkworm ( Bombyx mori L.)

<220>

<223> Sequencing results of silkworm mutant Bb-1-07

<400> 9

tccaatttga gggcaatgct acaatattag aactgcacta ttaaaaataa tcgatttaat 60

gaatgaaaga ttaataaatc tacagtttat ttcgaaataa atagtatcag atctttgttt 120

tgtataaatt tttttatgaa tatctataac cagtttagag aaaatctgta agaataataa 180

atccttaagc aaattgttgc aagggactga ttgttgtatc acaccaaggt acttaagacc 240

cagagaatta ccagaaacaa gacccatatg gttatttcaa gtaaatattt gatacaggaa 300

cactacatgc tgcttgagga gatcaacaga ctcgcgatca ccaagtacgc agatctaagg 360

agcagaagcc gcgaacctga acaagaccct taatgaatac aacgagatgt gtgagttttt 420

aactggaacc atcaattttt taatgaatgt attgccagca aatgtgtcat aatacaaagc 480

aacttgtgtt tcagttatca aataatttga taaacactgt ggctagctta tgtgtcattt 540

gtttttgtga aggcatagaa ttgaaataat ccctcatatc aactattgct ataatctcta 600

aattgcattc tgtcaacgcc tggccttatta gaatttaggc ctaactaaca caagatctta 660

gttgaatgag gtggtgaaat 680

the

the

<210> 10

<211> 678

<212> DNA

<213> Silkworm ( Bombyx mori L.)

<220>

<223> Sequencing results of silkworm mutant Be-4-02

<400> 10

tccaatttga gggcaatgct acaatattag aactgcacta ttaaaaataa tcgatttaat 60

gaatgaaaga ttaataaatc tacagtttat ttcgaaataa atagtatcag atctttgttt 120

tgtataaatt tttttatgaa tatctataac cagtttagag aaaatctgta agaataataa 180

atccttaagc aaattgttgc aagggactga ttgttgtatc acaccaaggt acttaagacc 240

cagagaatta ccagaaacaa gacccatatg gttatttcaa gtaaatattt gatacaggaa 300

cactacatgc tgcttgagga gatcaacaga ctcgcgatca ccaagtacgc agatgaaagg 360

agctagccgc gaacctgaac aagaccctta atgaatacaa cgagatgtgt gagtttttaa 420

ctggaaccat caatttttta atgaatgtat tgccagcaaa tgtgtcataa tacaaagcaa 480

cttgtgtttc agttatcaaa taatttgata aacactgtgg ctagctttg tgtcatttgt 540

ttttgtgaag gcatagaatt gaaataatcc ctcatatcaa ctattgctat aatctctaaa 600

ttgcattctg tcaacgcctg gcctattaga atttaggcct aactaacaca agatcttagt 660

tgaatgaggt ggtgaaat 678

the

<210> 11

<211> 673

<212> DNA

<213> Silkworm ( Bombyx mori L.)

<220>

<223> Sequencing results of silkworm mutant Be-4-04

<400> 11

tccaatttga gggcaatgct acaatattag aactgcacta ttaaaaataa tcgatttaat 60

gaatgaaaga ttaataaatc tacagtttat ttcgaaataa atagtatcag atctttgttt 120

tgtataaatt tttttatgaa tatctataac cagtttagag aaaatctgta agaataataa 180

atccttaagc aaattgttgc aagggactga ttgttgtatc acaccaaggt acttaagacc 240

cagagaatta ccagaaacaa gacccatatg gttatttcaa gtaaatattt gatacaggaa 300

cactacatgc tgcttgagga gatcaacaga ctcgcgatca ccaagtacgc agatctaagg 360

agccgcgaac ctgttaagac ccttattgaa tacaacgaga tgtgtgagtt tttaactgga 420

accatcaatt ttttaatgaa tgtattgcca gcaaatgtgt cataatacaa agcaacttgt 480

gtttcagtta tcaaataatt tgataaacac tgtggctagc ttatgtgtca tttgtttttg 540

tgaaggcata gaattgaaat aatccctcat atcaactatt gctataatct ctaaattgca 600

ttctgtcaac gcctggccta ttagaattta ggcctaacta acacaagatc ttagttgaat 660

gaggtggtga aat 673

the

the

<210> 12

<211> 669

<212> DNA

<213> Silkworm ( Bombyx mori L.)

<220>

<223> Sequencing results of silkworm mutant Be-4-06

<400> 12

tccaatttga gggcaatgct acaatattag aactgcacta ttaaaaataa tcgatttaat 60

gaatgaaaga ttaataaatc tacagtttat ttcgaaataa atagtatcag atctttgttt 120

tgtataaatt tttttatgaa tatctataac cagtttagag aaaatctgta agaataataa 180

atccttaagc aaattgttgc aagggactga ttgttgtatc acaccaaggt acttaagacc 240

cagagaatta ccagaaacaa gacccatatg gttatttcaa gtaaatattt gatacaggaa 300

cactacatgc tgcttgagga gatcaacaga ctcgcgatca ccaagtacgc agatctaagg 360

agcgcctgaa caagaccctt aatgaataca acgagatgtg tgagttttta actggaacca 420

tcaatttttt aatgaatgta ttgccagcaa atgtgtcata atacaaagca acttgtgttt 480

cagttatcaa ataatttgat aaacactgtg gctagcttat gtgtcatttg tttttgtgaa 540

ggcatagaat tgaaataatc cctcatatca actattgcta taatctctaa attgcattct 600

gtcaacgcct ggcctattag aatttaggcc taactaacac aagatcttag ttgaatgagg 660

tggtgaaat 669

the

the

<210> 13

<211> 669

<212> DNA

<213> Silkworm ( Bombyx mori L.)

<220>

<223> Sequencing results of silkworm mutant Be-4-09

<400> 13

tccaatttga gggcaatgct acaatattag aactgcacta ttaaaaataa tcgatttaat 60

gaatgaaaga ttaataaatc tacagtttat ttcgaaataa atagtatcag atctttgttt 120

tgtataaatt tttttatgaa tatctataac cagtttagag aaaatctgta agaataataa 180

atccttaagc aaattgttgc aagggactga ttgttgtatc acaccaaggt acttaagacc 240

cagagaatta ccagaaacaa gacccatatg gttatttcaa gtaaatattt gatacaggaa 300

cactacatgc tgcttgagga gatcaacaga ctcgcgatca ccaagtacgc agatctaagg 360

agcacctgaa caagaccctt aatgaataca acgagatgtg tgagttttta actggaacca 420

tcaatttttt aatgaatgta ttgccagcaa atgtgtcata atacaaagca acttgtgttt 480

cagttatcaa ataatttgat aaacactgtg gctagcttat gtgtcatttg tttttgtgaa 540

ggcatagaat tgaaataatc cctcatatca actattgcta taatctctaa attgcattct 600

gtcaacgcct ggcctattag aatttaggcc taactaacac aagatcttag ttgaatgagg 660

tggtgaaat 669

the

the

<210> 14

<211> 678

<212> DNA

<213> Silkworm ( Bombyx mori L.)

<220>

<223> Sequencing results of silkworm mutant Be-4-12

<400> 14

tccaatttga gggcaatgct acaatattag aactgcacta ttaaaaataa tcgatttaat 60

gaatgaaaga ttaataaatc tacagtttat ttcgaaataa atagtatcag atctttgttt 120

tgtataaatt tttttatgaa tatctataac cagtttagag aaaatctgta agaataataa 180

atccttaagc aaattgttgc aagggactga ttgttgtatc acaccaaggt acttaagacc 240

cagagaatta ccagaaacaa gacccatatg gttatttcaa gtaaatattt gatacaggaa 300

cactacatgc tgcttgagga gatcaacaga ctcgcgatca ccaagtacgc agatctaagg 360

agctagccgc gaacctgaac aagaccctta atgaatacaa cgagatgtgt gagtttttaa 420

ctggaaccat caatttttta atgaatgtat tgccagcaaa tgtgtcataa tacaaagcaa 480

cttgtgtttc agttatcaaa taatttgata aacactgtgg ctagctttg tgtcatttgt 540

ttttgtgaag gcatagaatt gaaataatcc ctcatatcaa ctattgctat aatctctaaa 600

ttgcattctg tcaacgcctg gcctattaga atttaggcct aactaacaca agatcttagt 660

tgaatgaggt ggtgaaat 678

the

the

<210> 15

<211> 667

<212> DNA

<213> Silkworm ( Bombyx mori L.)

<220>

<223> Sequencing results of silkworm mutant Be-4-17

<400> 15

tccaatttga gggcaatgct acaatattag aactgcacta ttaaaaataa tcgatttaat 60

gaatgaaaga ttaataaatc tacagtttat ttcgaaataa atagtatcag atctttgttt 120

tgtataaatt tttttatgaa tatctataac cagtttagag aaaatctgta agaataataa 180

atccttaagc aaattgttgc aagggactga ttgttgtatc acaccaaggt acttaagacc 240

cagagaatta ccagaaacaa gacccatatg gttatttcaa gtaaatattt gatacaggaa 300

cactacatgc tgcttgagga gatcaacaga ctcgcgatca ccaagtacgc agatctaagg 360

aacctgaaca agacccttaa tgaatacaac gagatgtgtg agtttttaac tggaaccatc 420

aattttttaa tgaatgtatt gccagcaaat gtgtcataat acaaagcaac ttgtgtttca 480

gttatcaaat aatttgataa acactgtggc tagcttatgt gtcatttgtt tttgtgaagg 540

catagaattg aaataatccc tcatatcaac tattgctata atctctaaat tgcattctgt 600

caacgcctgg cctattagaa tttaggccta actaacacaa gatcttagtt gaatgaggtg 660

gtgaaat 667

the

the

<210> 16

<211> 667

<212> DNA

<213> Silkworm ( Bombyx mori L.)

<220>

<223> Sequencing results of silkworm mutant Bc-21-11

<400> 16

tccaatttga gggcaatgct acaatattag aactgcacta ttaaaaataa tcgatttaat 61

gaatgaaaga ttaataaatc tacagtttat ttcgaaataa atagtatcag atctttgttt 121

tgtataaatt tttttatgaa tatctataac cagtttagag aaaatctgta agaataataa 181

atccttaagc aaattgttgc aagggactga ttgttgtatc acaccaaggt acttaagacc 241

cagagaatta ccagaaacaa gacccatatg gttatttcaa gtaaatattt gatacaggaa 301

cactacatgc tgcttgagga gatcaacaga ctcgcgatca ccaagtacgc agatctaagg 361

agccgcgaac ctgaacaaga cccttaatga atacaacgag atgtgtgagt ttttaactgg 421

aaccatcaat tttttaatga atgtattgcc agcaaatgtg tcataataca aagcaacttg 481

tgtttcagtt atcaaataat ttgataaaca ctgtggctag cttatgtgtc atttgttttt 541

gtgaaggcat agaattgaaa taatccctca tatcaactat tgctataatc tctaaattgc 601

attctgtcaa cgcctggcct attagaattt aggcctaact aacacaagat cttagttgaa 661

tgaggtggtg aaat 674

the

the

<210> 17

<211> 100

<212> DNA

<213> Artificial sequence

<220>

<223> ssODNs nucleotide sequence

<400> 17

tgtcaccgag ctgttcgagt ttcgaagtcc tggatccaca tgatcctgtg atcagtcggt 60

gccgcgaacc tgaacaagac ccttaatgaa tacaacgaga 100

the

<210> 18

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> mutant amplification upstream primer

<400> 18

ttggtccagt aggtttgaag taggt 25

the

Claims (9)

1. delete system based on the genome long segment of TALEN and ssODNs; It is characterized in that: said deletion system is made up of the transcriptional activation increment effector nucleicacidase TALEN and the single stranded oligonucleotide ssODNs that shear the goal gene target sequence, and the nucleicacidase TALEN-R that said restriction endonuclease TALEN is held by the nucleicacidase TALEN-L that shears said target sequence 5 ' end and the said target sequence 3 ' of shearing forms; Said single stranded oligonucleotide ssODNs is made up of at least 10 continuous nucleotides of 10 continuous nucleotides in the said target sequence and the distance said target sequence upper reaches or 1 base in downstream at least at least.

2. genome long segment according to claim 1 is deleted system, and it is characterized in that: said target sequence is the nucleotide sequence of T (Nn) A, and N is A, G, and the arbitrary base among T and the C, n is the arbitrary numeral between 24 to 65.

3. genome long segment according to claim 1 and 2 is deleted system, and it is characterized in that: said goal gene is a silkworm oily silkworm gene BmBlos2, said target sequence is nucleotide sequence shown in SEQ ID NO.2.

4. genome long segment according to claim 1 is deleted system, it is characterized in that: the protein of said nucleicacidase TALEN-L nucleotide coding shown in SEQ ID NO.3; The protein of said nucleicacidase TALEN-R nucleotide coding shown in SEQ ID NO.4.

5. genome long segment according to claim 1 is deleted system, it is characterized in that: said single stranded oligonucleotide ssODNs is made up of continuous 60 Nucleotide of 795 bases of continuous 40 Nucleotide and the distance said target sequence upper reaches of said target sequence.

6. genome long segment according to claim 1 is deleted system, and it is characterized in that: said single stranded oligonucleotide ssODNs is Nucleotide shown in SEQ ID NO.17.

7. each said genome long segment of claim 1-6 is deleted system transforms goal gene in the target cell genome application.

8. application according to claim 7 is characterized in that: said target cell is the silkworm egg cell.

9. according to claim 7 or 8 described application, it is characterized in that: said goal gene is a silkworm oily silkworm gene BmBlos

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