CRISPR / Cas9 (clustered regularly interspaced short palindromic repeats / associated protein 9) guide sequence, primer, kit, method and application

Abstract

The invention relates to the technical field of willow leaf beetles, in particular to a CRISPR / Cas9 (clustered regularly interspaced short palindromic repeats / CRISPR associated protein 9) guide sequence, a primer, a kit, a method and application. The guide sequence is shown as SEQ ID NO: 1 to SEQ ID NO: 8. The primers are as shown in SEQ ID NO: 9 to 16. The kit comprises a guide sequence and Cas9. The method comprises the step of introducing a guide sequence and Cas9 into ova of the Laureolatiopsis palustris. The application comprises the step of constructing the salix babylonia gene mutation strain. The gene function is verified by carrying out gene editing on the salix latifolius, a basis is provided for research on nucleic acid pesticides, and the method has very important wide prospects in prevention and control of the salix latifolius and other coleoptera agricultural pests.

Description

Technical Field

[0001] The present application relates to the technical field of willow blue leaf beetles, and specifically to guide sequences, primers, kits, methods and applications of CRISPR / Cas9. Background Art

[0002] Plagiodera versicolora (Laicharting), also known as willow round leaf beetle, belongs to the family Chrysomelidae of the order Coleoptera. Plagiodera versicolora is an important pest that harms poplars, willows and other Salicaceae plants. Its distribution range is particularly wide. Both adults and larvae of Plagiodera versicolora feed on leaves. After the leaves are damaged, the net-like veins remain, causing the leaves to curl, turn brown and die, seriously affecting the growth of forests. Because Plagiodera versicolora has a short life cycle and a wide range of activities, it can have multiple generations a year. In a short period of time, the population can reach an alarming number. The outbreak period is from July to September every year, causing serious damage to Salicaceae plants in my country.

[0003] By editing the genes of the willow blue leaf beetle and studying the functions of genes related to RNA interference, the application of safe and environmentally friendly RNA pesticides in the prevention and control of the willow blue leaf beetle will be promoted, which is of great and broad significance to the development of biological pesticides. Summary of the invention

[0004] The inventors of this application have developed CRISPR / Cas9 guide sequences, primers, kits, methods and applications using CRISPR / Cas9 technology, which can knock out the transmembrane protein-related coding genes involved in dsRNA transport in the blue leaf beetle to verify the specific transport mechanism of dsRNA by the protein, and provide a research basis for the development of nucleic acid pesticides for the blue leaf beetle, which has a very important and broad prospect for the control of the blue leaf beetle and other coleoptera agricultural pests. In addition, the study of this gene can provide a basis for the research of RNA pesticides in the control of other pests.

[0005] To this end, the embodiments of the present application disclose at least the following technical solutions:

[0006] In the first aspect, the embodiment discloses a guide sequence of CRISPR / Cas9, including an RNA or DNA molecule as shown in SEQ ID NO: 1 or 2, and an RNA or DNA molecule as shown in SEQ ID NO: 3 or 4. The guide sequence as shown in SEQ ID NO: 1 to 4 guides Cas9 to cut the transmembrane protein-related coding gene SIDT1 of the willow blue leaf beetle to affect the expression of related genes. Among them, the DNA molecule as described in SEQ ID NO: 2 can be transcribed to obtain the RNA as described in SEQ ID NO: 1, and the DNA molecule as described in SEQ ID NO: 4 can be transcribed to obtain the RNA as described in SEQ ID NO: 3.

[0007] In the second aspect, the embodiment discloses a guide sequence of CRISPR / Cas9, including an RNA or DNA molecule as shown in SEQ ID NO: 5 or 6, and an RNA or DNA molecule as shown in SEQ ID NO: 7 or 8. The guide sequence as shown in SEQ ID NO: 5 to 8 guides Cas9 to cut the transmembrane protein-related coding gene SIDT2 of the willow blue leaf beetle to affect the expression of related genes. Among them, the DNA molecule as described in SEQ ID NO: 6 can be transcribed to obtain the RNA as described in SEQ ID NO: 5, and the DNA molecule as described in SEQ ID NO: 8 can be transcribed to obtain the RNA as described in SEQ ID NO: 7.

[0008] In a third aspect, the embodiment discloses a first primer set, comprising the DNA molecules as described in SEQ ID NOs: 9-12.

[0009] In a fourth aspect, the embodiment discloses a second primer set, comprising the DNA molecules as described in SEQ ID NOs: 13-16.

[0010] In a fifth aspect, the embodiment discloses a kit comprising RNA as shown in SEQ ID NO:1, RNA as shown in SEQ ID NO:3, and Cas9 enzyme as shown in SEQ ID NO:17.

[0011] In a sixth aspect, the embodiments disclose a kit comprising RNA as shown in SEQ ID NO:5, RNA as shown in SEQ ID NO:7, and Cas9 enzyme as shown in SEQ ID NO:17.

[0012] In a seventh aspect, an embodiment discloses a method for knocking out a gene of the willow beetle, comprising the step of introducing and injecting the guide sequence and Cas9 described in the first aspect into eggs of the willow beetle.

[0013] In an eighth aspect, an embodiment discloses a method for knocking out a gene of the willow beetle, comprising the step of introducing and injecting the guide sequence and Cas9 described in the second aspect into eggs of the willow beetle.

[0014] In the ninth aspect, the embodiments disclose the use of the guide sequence described in the first or second aspect, the primer set described in the third or fourth aspect, or the kit described in the fifth or sixth aspect. The use is selected from at least one of constructing a gene-knockout willow leaf beetle strain and preparing a preparation for controlling willow leaf beetles. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The life forms of the blue leaf beetle at different stages provided in the embodiment. In the figure, 1 is the egg of the blue leaf beetle, 2 is the first-instar larva of the blue leaf beetle, 3 is the second-instar larva of the blue leaf beetle, 4 is the three-month-old larva of the blue leaf beetle, 5 is the fourth-instar larva of the blue leaf beetle, 6 is the pupa of the blue leaf beetle, and 7 is the adult of the blue leaf beetle.

[0016] Figure 2 Agarose gel images of SIDT1-sgRNA1, SIDT1-sgRNA2, sgRNA1 of SIDT2, and sgRNA2 of SIDT2 provided in the Examples.

[0017] Figure 3 A comparison of the mutation sites of the wild-type SIDT1 gene (WT) of the blue leaf beetle and its SIDT1 gene knockout mutant (Mutation1) provided in the example and an electrophoresis of the PCR amplification products. In the electrophoresis, from left to right, lane 1 is DNAMaker, lane 2 is the wild type (1026bp), and lanes 3-10 are mutants.

[0018] Figure 4 The peak diagram of the sequencing of the PCR amplification products of the wild-type SIDT1 gene (WT) of the blue leaf beetle and its SIDT1 gene knockout mutant (Mutation1) provided in the examples.

[0019] Figure 5 The example provides a comparison diagram of the mutation sites of the wild-type SIDT2 gene (WT) of the blue leaf beetle and its SIDT2 gene knockout mutant (Mutation2) and a sequencing peak diagram of the PCR amplification product.

[0020] Figure 6 The electrophoresis diagram of the PCR amplification products of the wild-type SIDT2 gene (WT) and its SIDT2 gene knockout mutant (Mutation2) of the willow leaf beetle provided in the example. In the figure, from left to right, lane 1 is DNA marker, lane 2 is wild type, and lanes 3-8 are Mutation2. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail in conjunction with the examples below. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0022] CRISPR / Cas9 guide sequence and primer set

[0023] The embodiment discloses a guide sequence of CRISPR / Cas9, including an RNA or DNA molecule as shown in SEQ ID NO: 1 or 2, and an RNA or DNA molecule as shown in SEQ ID NO: 3 or 4. The guide sequence as shown in SEQ ID NO: 1 to 4 guides Cas9 to cut the transmembrane protein-related coding gene SIDT1 of the willow blue leaf beetle to affect the expression of related genes. Among them, the DNA molecule as described in SEQ ID NO: 2 can be transcribed to obtain the RNA as described in SEQ ID NO: 1, and the DNA molecule as described in SEQ ID NO: 4 can be transcribed to obtain the RNA as described in SEQ ID NO: 3.

[0024] The nucleotide sequence of the SIDT1 gene is as follows:

[0025] ATGACGTTCCTGATATACATCTAAAATGGTGTTGAAGTATCTCATCACGTTAACACAGAACTAGCC

[0026] TAGGCAGGGAATTAGGCAATCCAGCGATCTGTCAAATCGAAAAAAACAATGTGGCGGCTTGGGGCAC

[0027] ACATTTGTGATTTGAGTGTGAAAACTGTTACATTTAGTGATCTCTAGTGCGTCTTTTATGTGTTTCTTCAA

[0028] TTGTGGTTGGTTATTAAGTCTTTTCCTGAAAAAAATGCCTGTCACGTGCATTGCAGAATACAACTAACA

[0029] CAAGGTATCGCATTCTGTGCCCTAATCCGCCACATTGAATGTCACGTGGTTTGGATTGCCTAATGTTGA

[0030] ATAATTTTAATAAGTAATCACAGTACGTGAGCAGTCCTAGACACGCTAACGTAAGAAATATGACGTTCC

[0031] TGATATACATCTAAAATGGTGTTGAAGTATCCCATCACGTTAGCACAGAACTCGCCTAGGCAGTGAATG

[0032] GTAAATAATTTTAATAATCTCAACTTCCAATGCTGTTTTATGTAGTTTAACTGTATGTCCTGTGCCGTTCG

[0033] CAAGTTATCGTTCTGGTTTCGTGAGATTTCGGTTAGCGTACGTTCCCTCTTTTAGCACCCGTAAGATAG

[0034] CGTGAGTAACGTACACCCGTAGACCCACAAAATACGAAAACGTGAAAACTTGTGCAATGCGCATGCC

[0035] CAGATGTCAGAATGAATCCTAACGTTACCGTTCGTGGAACAACTTTAAGTTTCAACCTGTGCTTCAAC

[0036] TCTCTAATGTCATTGTGGTTTGATTTGAGTTTCAGATATAAATAAACGGAATTGAATTTAAATATTGATA

[0037] GGAATTAGGAAATATGTATTCCAAGAATTATTTCTTCTTATGGAGTTTAGTTATTATAATATATTTGAAAC

[0038] ATAACTCTGCACTAGAACTAGAATATAATAAAAATTACACATTTATGATAAACAATACAATAGATTACGT

[0039] TTTGGGATTTACAGACAAAGTAAGGATAGATAAAATTTTTTGGTGTAGTTTTAATTTTATCCTAATTCAG

[0040] ATAAATGATACTAGAAATGTACCACCAAGAATATGGATTGAAAGTAATGCTACTACTTTTGCACCCTTG

[0041] ATGATAAGTGCACGTCAGACACAAGAAATGCTTTCTTGGCAGCTGCCTTTAGAACTGGAAAGAGTTTC

[0042] CGGAGAAATATTATACAACTTCACATCTAGAACTCTATGCCATGACATATTGAAACATTACAAACTTTC

[0043] GGGTAAGGATTTTATTTAACTGAAGAAAGAAAAATAGATGGTTCAAGTGATATTATTTGGTGAAATACT

[0044] CAATGAAGTTTGATATATCATTTTTGTTGTTGATTATGCTTGTTCTAAAAACATTAGTGTAGAATATGTCC

[0045] AAATAAATCGAGAAGGTTGTATTAATATTTCAAAATATATAAACAAATGAATAAATATACCTCTATTGATT

[0046] TATGCAAAATAAACAAGAAAATACTCAATACTACAAAAAATTTATAGGTCATATTGAGATTCCCTCTTA

[0047] CCTTCTATTTTCTGAAGCTTAGTTATTAGTAAAGTTAGGGTCTTGTTTCTACACACTACATCTGTACAGA

[0048] TGATAAAGACGTTTTATAGGGCTTACACCAGAAAAATATTTTCAATTGTCCAGGAATTCCCAAAAAAAG

[0049] TGTAATGTGAGTTTTTTCAAAAGCAATACATATTTGTTTGTTCCATTCAACAAAAAAAAAAAAATTCTGC

[0050] ATAACTACTAGAAAAAAATTGACAGCAAAGGTCAAAGTACCATATTTAATGACATTCATATTAGATACAG

[0051] CCTGAGAAGATTATTGAGCAAGTTTTGCAAAATTTAGTGGTAACCTTTTTCAATTTTAAAGCACAATAT

[0052] CTTTCCGTTTTGAAAAACTGAAGTCACTGTATATTTCAATGTCATTCAATATGCTTCATGACAGTCTATT

[0053] TAACAAATTGATTTATATATTTTCAATACTACTAGTGCAAAAACAGTTAACCATCAGAATTTCAATTCCA

[0054] TGAGACCTTAGAATTGTTAATAATTCCAAATTAAACTCCTATTTTTTGCATGATTTACAAAATATTTGTGT

[0055] ATAATGAAAATGTTATTTGAATTATGAACAATCTTCAGTTATTTTTTGTTTTTCTCTTGAAAATAAGAG

[0056] TAGCAACACATTTCACTATTTACTTTTAATAAATAATAAAATTATATCATGGACCATTAGTTATCACTAGT

[0057] TTGTATATTATCTCAATTTGATAATTCAAAGGACAGAGGACAGCAGAATTGTTTTATGATTTTTATTATAT

[0058] AGGCCATTAAAATCTGCAATCTTCAGTTATTGCTTTGTAGTTATCTGTCTAATGGGTATGAATGTTTTTC

[0059] AAAATCTATTATATTGCTATTCTCTGCTGACACCATGAAAATAAAATATTCTTATATTTCAGGAAAAATTG

[0060] AGATTAAACATGATAAGCCTATAGTATCTGTAGCAACTGCCTCTAATGAAACAATCCAATTTACCATAAA

[0061] TGTTCATGATCAGAAACATTTTCATATTGAGCACTCAAAAGAATATAAAACTGCCATTTCTCCAGGAGA

[0062] GCCAAGGTAATCAAATGGTATTTAATGAAATTAAATGTTGTTAAACATCAATAGACATGATTCTTGAGT

[0063] AATTGTTTTGATGTGAAGAAAACCGATATCCAAATGATATATTGAATTATATCTCGCATGTATTAGAGAT

[0064] CAGACCATCATATCGTCGGTCCTAAGGCAAAACCACGAATGTGAACTGATTGGGTAATTATACACATTT

[0065] ATTCTAATTCACTTGAAACAACATTATTGATGAACGAAGATGGTCGTGGGTTTGATTAAGTATGCAACC

[0066] TGATTTTCGGAAGCCAATAGAAACTTCAGACCTTGTTTTTTCAAAATGACGAATCTGCACATTCGTGG

[0067] TTTTGCTTTAGGACCGACGATATGAAATCAGATATTAATTTTGTAATGTTGTGAACTGTTGCTAATACAA

[0068] TTAGTGCCCGAATGGTCTCAGCAAATTATATCAATGGTGAACTACATATTCATTTTCTTCATCACAGTTG

[0069] AACAAAATGTTATAATTGTTTATTTTCCAATTTGAATAGAAAGTATTATGTGATTGAATCGAGATTTTATG

[0070] ATAAAACATCGACTTTATATATCTATTTCACACAGAAACAGTCTTGTGAATAACCCCAGAATTTATTTTC

[0071] ATATTCAACATGAGTTGTGACAGCTTGTTAGAACATGCACACTTGCCGTTTTTAATGATTGTTGCATAAT

[0072] TTACCTTCATAGAAACTACCATTATATTGCATATATATTATTATAGAAGTTATCTATATAGAACGGGGTGA

[0073] CCGCAAAATATCGATGTCGGATTTTTTTTCATCTGGACCTTCATTTTCCAGATATTGAGGTAACTGAAAT

[0074] TTGAGCTTGATTAAAAGACGCTAACCCGTGCCATTCGTCGCTCCAAAGCTGACATATGAAGCCATAAC

[0075] TCCACGTTCAATATTTAAAAATAAAGCAAATCTTTCACCAGAATTATATTGTATACATAATTGTTGTTATA

[0076] TTAAGTCGAACCGAGTTAAAACAGCCTTTAACCCCAAAAACGTGGCCTAGTACCAATAGGAACAACT

[0077] TCGTAGATTTTGTTTTTGAGTGGTAAATTTTCCTTTTTTTGGTATATTTGAGTTTACAGTAGAAATATAAT

[0078] AAAAAACAAGTTCCAAAGTATAAAAATTTGGCATATAATACTTACAAAACAGACATAGAAAAGATACA

[0079] AACATAGTACCTACATAAGTTAATAATTTTACATAGGTATTTAATCAATTCTAGAACATTTGTTGGAGGA

[0080] ATTTTGAAACATCAGGACATAATTCGGTATTTGCTTACTACTCGCAGGTCATTGCTTTTGATATTCATC

[0081] TCTCAACAGAAACGACTCGTTAAACTCGGTTATTTATATATTACGCTCAGTCATATCATTAACTACTTTT

[0082] AATATTGTGACAATTTTCAATAATCTGACATTTAAGTTTGACAGTCCATCTGTCAAAATTTTTCGAAAA

[0083] AATGGAATATCTCAAACAAATCACGATAGAGAAGCGATTCAAAATGCAACAGACCCAAAATTCTAC

[0084] GTAAAAGTTTGAAGTTGATATCTCTATCAATTCTCCCATAAACCATAAATGAATCAGTGAGGTGAATCAC

[0085] CCTGTATATATTATTTTATTTTTATATCACTTGTTTTTCATTATATTGCTAGTGTAAACTTTGAAAAAGCA

[0086] AAATTTATCACTTTACAAAATTCTAAGTTTGCTCCTAGTGGTTGGAGGCTGTTGTAACTTGGTATGACT

[0087] TGAAACAACAATAATTTTGTATAGATTGTTGATTTTGAAGAAGAACATTATCTCATTTTGAGATATTCAG

[0088] AGTGTTTGTGTTTCTATTATTTACCGTTCAGGAGTTATGGCTCCATATGTCAGTCTTTCAGGCAAATATT

[0089] GGGATTTTTTTCAAACTTCGAGCAATACGGGTTAACGTCGTTTAATCGAGTTCGAATTTCTCAATATTT

[0090] GGGAAATAAAATCGTTTTGGGGGTCCAGACGAACAAGATTCCGACGTTTAATTTCCTCCATGAAATCC

[0091] TGTCACCGTAATATAGAAGTTATATGTCATACAAAGCATGCAAAGTAAGGGAGAATACAGGGTGTTTC

[0092] ACAGTGGATGGAATTAGAAGTATCTGAAAAACTGAAAGACAGATCATTTCGAAAATTCGTGGGCGT

[0093] GGATAATCGAACTTGATCTATCCGTCTAAAATATTTTTATCAATACAGTGTTGCCGCTTGTATCAGAAAG

[0094] TAGCAGCTACATTGTTATTTCAAATAGAACACCTCGTATATTATATCATTTTTGGAATCCTCATAAGATTT

[0095] CTCACAACTTTTCCCACATAATTCCCTATACCTATCTGTGACGATTTTCGAGATAAAAATTATTTTGTCCA

[0096] ATTTTTGTGCAACCTGACCATTGAGAAATATCAATTTTTTCCCAATGCAAACTTAAAAATATTGCA

[0097] ATTTGGGTTATAGCTGCATAAGACCATCAAGTATCCTGTCGATGAACTGGATCTCAGTTTCAGCATACA

[0098] GGGAGGCTCAGAAGTGAGTGCCTTTCGGCGACTCATGAAAATGACTGAAACTCCGTTTTTGCTTGAA

[0099] TCATCATGAAAAACATGTGTATTAATGAATCTAGGAAGATTTCAAAAACATTTCAAAATTTCTCTTCAAT

[0100] CTAATGAATCAGCATACTTTTTAATTTAATTAATTTAAGTATTTAGGATGTATGATTGATGAATGTTTGGA

[0101] GATGAATTTCTCATTGTAAATACATCTGTAACAAAATTTCAAAAAAATTATATTTTTCACAAGAATTTCTG

[0102] CACAACTTTCTCTATCTAGCAGAACAACTGTATATAAAGCAATAATCCAGCCGCATTTCGATTACTGTTC

[0103] TTCTATTTTATATATTTTACAAAAAAATGAAATCCAAATATTACAAAAATTGCAGAACAGAGGAATGCG

[0104] AGTAATTTTACATTGCAACAGATACACCCCAATAAAAACAATGCTCGCAGCATTGAAATGGCAATCAAT

[0105] AGATCAAAGATTGTATTATATGGCCATGCTATTTGTCTTTAAAATCACAAAGAATATGTTTCCATTTACTT

[0106] CAACCAATTTGTTACATACAACAATGAACTCCATAACTATAATACACGGTTCCGTACATAATTTTCATATC

[0107] AATAGGACAAAGCTGAAGAGTTCAATGAAGTCTATATTTTTCAAAGGATTGAATGAATATAATCATTTA

[0108] CCAGAGGAAATAAAACCATGTAATACGATATATTGCTTCAAAACGAAATTGTTATCCTACATTCTATCAA

[0109] GAGACGGCTAACACTGGTATTGGTTGTTTTGCGGCGCTCTCAGCATTTGGCTGTGGCTGTTGTACTCAT

[0110] GTTTTTGTTTATATTTTTATTACTAACTCATGTCATTTTATTATTTGTCTTTTAATTGGTCACATTTTATATT

[0111] GCGTCTTATGTTTGATTCTTGATGTTACAATTAGGTTTAGTAACTGATTTTTATCAAATTGTATTTTGTAA

[0112] ATTTTATATGAAATTACGTATGTATATTAGGGTATCCTATTTGAAATAAACCTTTCTTTCTTCTTCTTTATC

[0113] AAACTCTCTGCATGTTCTACTGATATTCTTGTTACATTGACCATAAATGATGAATAAATCCATCAAAACC

[0114] ACATTGAAATAATTATTTTCTGTCATAGTACTTTCACACCCATGAAACTTGAATTCAGGATGACAATGA

[0115] AGGAATAACATGTTAGTTCTCTGTCAGAATGAAATTCATAGCCTACTATCTAAAGCACTATTCAATATTT

[0116] ATTTTATTGAAGACGAATTCATAAGTGATCATAACTAAAAAACTGTACGAGATAGGTATAGGGAAAGTA

[0117] TACTCAATTAAATTGGTGTTTCATTAAAAAAAACGGAGTTTCAGTTATTTTCATGAGTCCCCGAAAGGC

[0118] ACACTTCTGAGCCTCCCTGTATGCTGAAACTTAGATCTAGTTCATCGACAGAATACTTGATGGTCTTAT

[0119] GCAGCTATAACCCAAATTGCAATATTTTGTAACTTTGCATTGGGAAAAAATTTATATTTTTCAATGGTCA

[0120] GGTTTTGCACAAAAATTAGACAAAATGATTTTTATCTCGAAAATCGTCACAGATATGTATAGGGAATTA

[0121] TGTGAGAAAGTTGTGAGGAGTCCTATAAGGATTCCAAAAATGGTATAATATACGAGGTGTTTCATTTGA

[0122] AATAACAAAGTATTTACTACTTTCTGATACAAGCGGCAACACTGTATTGATAAAAATATTTTAGATGGAT

[0123] AGAGCAAGTTTGATTACCCACGCCCACAAATTTTCGAAATGATCTGTCTTTCTGTTCTCCAGATACTTC

[0124] TAATGTTCCATCCACTGTGAAACACCCGGTATAAGTATGTTGCATAGCTGAATATGATTTACTAGAATCC

[0125] AAAATTAGTTACTAGGGACTAATGGAATCTTACGGATCTGCAGTCAAGACAACCTCCCTAAAAATTAG

[0126] TTTCAACAGAAAATCAATCAAATTTTGCAGATTTTTTATAAAACGTAATGTAGTCTGAGGTGAAATCA

[0127] TATTGAAATTGTTTACAAAACGTAATTATATATTGTTTGCAGATATTTCTTCTATAACTTCTCAACTAATG

[0128] TTTCCCAATTGATGAGCACAAATTCAAATTATGAAACTGTTATTCTAGAAGTGTCTTCTGATGATGATAT

[0129] TTGTATGACAGTAAGCATACAGAATATTAGTGTGAGTATATTAATCATCAAACATATAATATTTCTAGTAA

[0130] ATGAAGATGTTTATAAGTTCATCAGATTAGCCAATTTCCTTTTATTTGTCTTATAATTTTAATAAGATTATG

[0131] CAAAGCCATACCAAACCCGCCATTTCATTACTGCATTTATTAAACGTACGATTCAACTTTTCCTATCGCA

[0132] TGTTATCACGAGGAATGTAAATAATCATTTATTTCTGCTATCAAAAGTATTTCCCTCAGTTGTTCATTCAT

[0133] GAAATTGTGATTCATTTTGATATAAATATGGGTGTATACATTCTAACCGCCACATCATGGGTATTTCATAA

[0134] CCTTTGAAAGATATAGGGTCGCTTAAGGTATAGAAAGGTTGCGTGATTTCTTTCCGAAATTCTTTTAGG

[0135] AATTACAGAATTCTGAATACTTTTTGATATACAGTAAAAATCCAAAATTGGTGATATTTTTATGCTTTC

[0136] CCCCACAAGGTGACGTTTCGTCTATTGTCCAATAATGAGTTTGCGTTACTACTCTTTTGGAAGAGAAT

[0137] CAAAAAACGGCATGCCAAATTCCACATTTTTATTTTTTATAAATTATTTCGAATCATAACCAATCCTATTT

[0138] TTGGAATAATCAATATAATCCAATTAAAATTTCATATTAGATAAATAATATTCCACACATTGTAAGACAAA

[0139] GAAAACCTGGAGCTGGGACATGGTACTCCTGTTAATTTTTGTGCAACTTAACGTAATCCAAAGTTACC

[0140] AAGAATAACGTTGAGTTATTTGAAGTAGCATTACCAGTTTTTTGAATTTTTAAATTTTAAATATAAATATT

[0141] TTCTCCAAGAATTGTTTTGTATGTTTTTTACGCAAGTTGATAATTTATAATTTTTTCTTCGGTTTTTTCATC

[0142] AATTTGTTGACAGCAATTTGCAGAACCTCAATGAATGATTATTTCAGTTTTCTGAATTTTTGAATTTCAA

[0143] ATATGAATATTTGTCCAAGAATTGTTTTGTGTATTTCACGCAAGGTGATAATTTACAATTTTTTCTTCG

[0144] GTTTTATTATCAATTTGTTGTGATGGCAATTTGCAGAACCTCAATGAATGATTATTAATTTTTAGTGTCCA

[0145] GTTTTTGATACCAATCAAGATGTTACATTTCGAGGATAACTATGAAACAGTAAATAGAAAAGGAGGGATA

[0146] ACGATTTCTGTAAGTATATATCATATTTGAAAGAGAAAATCACAGGATAATTTATTAACGAAATTCTTTC

[0147] TACGATAATGGGCGACTGGACTATTTTTTCATTTTGGATTTAATTATTATGCATGATTGAATTTTCATCAC

[0148] TGTCGCGTCCCTACGCTCTGTTTGTGTAAGAGTTTTGTATTGTTTATGAATGTTTCGTATCCAAAAATT

[0149] GGTTGCTCCGGAAATCAAACCCAGACCGCTGGCTGAGAGTCATTTACGTTGCTCACTTTGCCACCACG

[0150] GCAACATTATTTCGAGATCAATCAAACAATTATCATTTTGTCCAAATTCTTCATCCATGTTGTATTTGAAT

[0151] TTTGCTAGATATAATATTTCAGTCATTGATTTTATTCTCACGTGATCATAACCCAGGACTACTCTCCTAGC

[0152] AGATAAGTATGGCAATTTATTCTTCGAAAATAGTGACACCCTCTCACTCAAAAGTGCAAGAGTTGCAT

[0153] AAAAACATGAGTCTTAGCTTGAAGTTCCTGATATTAGTGGCTACTGAAGTATTGGCACTTGACTATTTT

[0154] TTGCATTGGAGAACTACCAAATATATATTCTCGTGCATTTGTTTTTCAGAAATACAAGTTCCCACATGGA

[0155] TTTTATGTAGTGCTTGTGGCCAAAGCCGATGATTACCAATGTGATAGACTGGCAAGATCTACATCGTCA

[0156] TCAAATCCTAACAGAATAAAACACTTAACTCTCAAAATAAAACCCAGTATTACTTATGCAGATTATATA

[0157] ACAGCTGTAATTCTAACATTATGTTGCATAGGTAAGGTTTTCACATCATTTAATTTATTATCAATTATTTC

[0158] CAAACAGCATACATTGATTATACAGGGTGCGCCAGAGAAACTTACTCATTTGAAAAAATTCCCGTGCG

[0159] GTGAGTTGGGCGGGTAGAGGGGCAGAGGGGGCGCATCTGGAGGGGGAAGTTCCCAAGTTTGTTTCT

[0160] CTCTCCAGTCAGTTGCCATCATGCTCTGGTCTAGAGAGCAGCGGGCCTTCGCAGTTGAGAGCTTCTTT

[0161] TCTAACGGTCGCTTACTTGTTGCTACGCAACGTGCCTTCCGCGCTCGCTTTGAAATTCCTCCTTACAGA

[0162] CTCGTTCCTGGCCGTAATTCGATTCTGTCATGGGTTAACTCATTTCGGGTGTGTGGAAGTGTCGCTAAA

[0163] CCCAGAAATGGACTTCAACGGACCATCAGAACTCCGGAAAATATCGAAAGAGTGAGGCAGTCAGTTG

[0164] TGCGTTCTCCCCGGCGTTCGGCTCGCAAACACGCAGCTGCTATGGGAATTTCTGATACCACTGTTCGA

[0165] CGAATTCTCCATGAAGACCTTCAATTTCATCCCTATAAATTGGCTGTTGTGCAAGAATCAACTGAACGC

[0166] GATTTTGTTGCCCGTCAAAATGCATGTGAAATGCTGATTGACAACTTGCCTGAAGACGCGGTCGTTTT

[0167] TTTCAGTGACGAGGCTCACTTCCACATTTCCGGCTGCGTCAACAAGCAAAACATGCGTTATTGGTGTG

[0168] ATGTAAATCCGCGTGATCCACTTAATTGTGAACGAGTTACTGTCTGGTGTGCGATGTCGGTAATGGGAA

[0169] TAATTGGTCCTTATTTTTTTGAAGAAAACGAACGCGCAGTTACCGTGAATGGAGCTCGTTACAGGAAC

[0170] ATGATCGAGGAAGTTTTTCTTCAAAGACTCGAAGAAATGAACGTTAGGAACGTGTGGTTTCAGCAAG

[0171] ATGGAGCCACAGTACACACACAGCACGAGACTCAATGAATTTGTTGCGACAACATTTCCCCGGACCGCCT

[0172] CATCTCTCTCAGAGGCGATCTCCAATGGCCGGCACGCTCTCCAGATTTAACTCCATGCGATTTCTTTTT

[0173] ATGGGGTTATTTGAAATCCCTGGTTTATGTCGATCGACCACGGACCATAGCACACCTCAAGAACAACA

[0174] TTCGCGACGCCATTGCCGATTGATATGCTGCGAAGAGTGGATACGAATTTCAAAAATCGACTTCATCA

[0175] GTGTATGCGCAATGGGAGTCGCCATTTATCCGATGTCATTTTTAAGACTGCATAAAAAAAATTGGGATA

[0176] TTGTATTCTCAAATAAAAAAAAAATTAAAAAAATAACTGAAGTACTTTTTTTTTATTGATCTTTCAAAT

[0177] AAGTTTCTCTGGCGCACCCTGTACATACAAAATTTAATTTTTGTGCTCAAAAGTGGTGCTCATTGATAG

[0178] GAAATGCCAGTTTCGGAAGAACGTGAAAAGAAATCAACTTGCATATAATAATATTTTCTAGATGCATTTT

[0179] CATCCCAGTTCTGAAAATCATACATTTTACACTTTATAGAAAGGAATTCAATGGTGTTTCTATTTTTTTA

[0180] AAGCCATTTTGGAGTCATAAGCCATGCTTGTATTTTTTCATGGACCGTTTATTTGCATTCAAATTATATCA

[0181] GACGAATACTTCTGAATTTGGCGAATTGATTCAATACATTCGACAAAAAAATAGGCAAGGTCGTGAAA

[0182] TCGGGAGTCTTTCCGGCCAATGCTTTTGATTACTCGTAACATCAGGATTGTTTTTTTTTCTTCAGAAAT

[0183] CAGCTTTTCCTATCAAAAATTTCCAATTTCGATACAAAATGATTATGTCAACAACATGAGAGTGTTGT

[0184] CTCTTGAACATCATGCCTTCAATGGAATAAACTGCGTATAATTATTCTGGAGAACTTCCTTTTATATAA

[0185] TCACAACATTAATTATTTCGATGTCTTTATATACGTTTTATATTATCTTCTAGGTGGATTCTATGTTGTTTT

[0186] TGGATTATCATATTTTATTTGTTCTGTTCGTGGTACAATTCCGAGACAAATGGCTTATGTAGATGGAAAT

[0187] ATCAACTCCACACCGAATACAAGTGAGTTGCGTGAAATCTTTAAACTTGTTATTCACAGAATGATGTTT

[0188] ATGTGGCTCGACGCTCTATTGCACAATGGAGTCCAAATTATCTCGCTCAAATTATCAGTCTGAGAGAAG

[0189] TTATTAGGCATTCCAAATCACGTGACATTCAATATGGCGAGAGTAGGCACACAGCTGTTTGAAGTGAC

[0190] GTCATTACGGCATTATTTCCGAAGTCGTGTGAGTTTGTTGTTGATGATAGAAGTTTTAAGATGCAGAGT

[0191] GTATATTTAAGGTAAAGGTAGAAAGGTAAGCTGAAAACCCAATTTGCTTTTTGTTTACACAACATTTTG

[0192] TTTCCCATATGTGAAGTGGAATGTTTATTTACAGATACCGAATGAAGCTGAAGATAATAAATGATGTAG

[0193] ACCCTTTTACATTGAACATCCAGGAATTGGATAGTTCAATTCATGGAATTCCACCTATTACAAATATAGA

[0194] TATTGTTTCGTATTTAGTGCTAACCCATAGCTATTGCACCAAAGAACAAATGAAGGCCTACAAAAGCTT

[0195] GCATTCATTTAAGTATTTTGAGTCAGGCTTTGTTGTGAATGTGGGGACAAAAGTGGTGAATGGTTTCTA

[0196] CATTGTAGTGGGAAAGGTGAGGTTTGTTCATTTCTATTGACTTTTGTTTATTCAGCAATTCTTTTAGGTC

[0197] AAACATTCTCAGAGAGGAAATGAGAAACCATTAGACGTTTGGTGCATTGTGGCCAAAGATGAAGGTG

[0198] TATTAACAGCACATTGCCCATGAATGTCTGGACACAGTGAAGTTTGCAGTCATGTGGGTGCACTTTTA

[0199] GTTGCAGCTGAATAAAATGTACAATGTAAATACAATAAAAGTTATAAAATGTTTACTGCAGACTCTCTG

[0200] ATTTTTTAGTATCGATTCAGTAAGATCGTCCCTCTTAATTGCTGCTATCCATAGCCTTTCAGACAATTCG

[0201] TTTTTATCCGTAAGAATGAACCTGTGGCTTAAAACCGCAGGCACTAAAAAAAATTTTACATCATCCTTG

[0202] GAACGACTTCCACAGTTGACTAAAAATCACGGTCATATAACATAAGGTCTGGAATCTACGGCAATTTT

[0203] GAGTGGTGGGGGGTGGCACCAGTTTTGAAGAAAACGTTTCCATAATTATCCGCTAGGCGGACATAGG

[0204] GGTATAATCACGTGATTAATCTTCGACAAAAAAACAAACGGTTAGGACTTGAATGGTGAATAAAATCA

[0205] ATGACAGAAATATTATATCTTGCAAAATTCTCTCGAATAAATAAAAATCATTCCTACTTCCAAAAAGCT

[0206] GATCGGATCTGATTTGTGATTGGCTGACAAAGTGACAATAGAGGACAGCGCTGCAGTTGACGAAACA

[0207] TGAGAACCACGAAAATTTCCCCAATGTTTGGGTCACCCCCCACCACCCGAGTTGCAAGACCTATGTAT

[0208] ACGATCGTGCTAAAAATTATGTCATAACCTCACATTATCAATAGGGCATTCCAACGACCTGTCAAACTA

[0209] ATTCAATATGGTGGGAGTAGGCACACAATGCTTTGTTTACATATTGTTATGTTATAAATTGATCTAAAAG

[0210] TTGTAAGATTTTTGTTTTCGTGACTGCATCAAATGATTTTTTATATTTTTTTAGAGCTTATTAGTGGTATT

[0211] CTCTCTAAAAAATGCCTGTTACATGCCTTTTAGTCAACTGTGAAAGTCGTTCCAAGGATGATGTAAAAT

[0212] TCTTTTTAGTGCCTGCGGTTTTAAGCCACAGGTTTCTAACGGATAAAAACGAATTGTCTGAAAGGCTAT

[0213] GGATAGCAGCAATTAAGAGGGACGATCTTACTGAATCGATACTAAAAAATCAGAGAGTCTGCAGTAAA

[0214] CATTCATAACTTTTATTGTATTTACATTGTACATTTTATTCAGCTGCAAATAAAAGTGCATCCACATGAC

[0215] TGCAAACTTCACTGTGTCCAGCCATTCATGGGCAATGTGCTGTTAATACACTTTCATCTTTGGCCACAA

[0216] TGCACTAAACGTCTAATGGTTTCTCATTTCCTCTCGAGAATGTTTGACTTGAAAAAATTGCTGAATAA

[0217] ACAAAAGTCAATAGAAATGAGCAAACCTTACCCCACTACAATGTGGAAACCATTCACCACTTTTGTCC

[0218] CCACATTCACAACAAAGCATGACTCAAAATACTTGAATGAATGCAAGCTTTTGTAGGCCTTCATTTGTT

[0219] CTTGGTGTAATAGCTAGGGTTAGCACTAAATACGAAACAATATCTATATTTGTAATAGGTGGAATTCC

[0220] ATGAATTGAACTATCCAAATCCTGGATGTTCAATGTAAAAGGGTCTACATCATTTATTATCTTCAGCTTC

[0221] ATTCGGTATCTGTAAATAAACATTCCACTTCACATATGGGAAACAATTGTTGTGTAAACAAAAAACA

[0222] AATTGGGTTTTCAGCTTACCTTTCACCAATATCTGGATTTTCCAACAATAATGTCCTTAAATCTGCATCT

[0223] GCATCTTAAAACTTCTATCATCAACAACAAACTCACACAACTTCGGAAATAATGCCGTAATGACGTCA

[0224] CTTCAAACAGCTGTGTGCCTACTCTCGCCATATTGAATGTCACGTGATTTGGAATGCCTAATTACACTG

[0225] TTCACATTCAGAAATGACAGCTCTAGGTACGGCTGCCCTTGAAGAAACCACCTCCATCGACAGCGACT

[0226] ACGACGTCATCAGCGAAGTCGAGAATGACAGAAGTCTGCGTCTCGGTAGGGGCAACCCTTGTCTGAT

[0227] GGACCTTGCTAGAAGACATCCCAGTGTGCTCAAAGAAGTCCTATCTTTACCTCTATAACGTGTTGA

[0228] CGGTGGCGTTGTTTTTACGGACTTCCTGTGATTCAACTAGTCGTCACTTACCAAAGGGTATGAGTTTCAT

[0229] CTTGCAACGCTAAATAATCATCTTTCTATGATGTTTCATAAAAATTGTCAGTTGGTAGTCGCTCTCTTTA

[0230] ATTTCCTAATTTTTTTCATTCAGTTTTCAGGATTTGATCAAGTTTTGCTTAAATTTTCAACATACTGTACA

[0231] GGGCGGTCCAGTTCAAATTGGACATTACTGAAAGCAAATACCGATTCCTTATTCATGTTACTCACCCTT

[0232] GATCTCACTTGGTTATTATATTTTCTTGTTCCTGTATAAACATTCTTTAGATACCGAATAACAGTGGGGTA

[0233] TCTAGTTTTTTTTTTACTGGTTTCGTAGTCATGACATAATCTAGTTATTTTTCTTCTGGGCCCCTGTTTATTT

[0234] CTGCACATTCCTTTGAGAATTAAATCATATATTCAAATACAGTTGAAACTTTTGTCCTATTGATGTTTTC

[0235] AGATTTTTTCAAATATTGAACAATTTGACTTTTCATGTTTGAAAAAAAAAAACTTGAGCAACCAACCGT

[0236] CACTTTCGAATTATTGCTTTATTTGAATGAAATTTGATTTCTAAGTTTGCCTTCAAGTATATGACATGTTG

[0237] AACTAGTTTTTTAATATCATGACAATCATTTTCTAAAATTTGCTTTCTTTCTTGGCTTGTGCTCTGACCTA

[0238] TTGAATTTGTCGTTTCGGATTTTTATTCAGAATTACAATAGAAAGTGAATGCATTTGGATGACTGAGA

[0239] AAAACGAAAATATTGTGTTCAATAAAATCGTCTTGAAAGTACGAAATTAGCGAAAAAGTCCAAACTTC

[0240] GTTATCGTTTATTTCATTCAATTTATATTCATTTTCTATTGTTGATTCTCCTCATGACCCAAGGAAAATAAT

[0241] GTCCTCTTGAAGAAAATATGAGTTATTGATTCACTAGGGATCCTTAGGTCATAATGTCAACCATTTGAAT

[0242] AATTCATTCCAGTTTAAATAAGACCTATATGTATTATTGTCAAGACTATATGACATTTTATAAATATAGTCT

[0243] ATTTCATCTGGGTTGATACCTGTGACGCGTCGCCAGATTTAGCCGTGATGCCACATGTTTTTCACTTTTT

[0244] CAGCGATGCTCCGAATTAACTCCGAATAAAAACTTGGGTTAGGTTGACAGTTTGTTTTGTTTTGAAAG

[0245] TGATTTCGACTGACGAAATATATTCATTATATTTGGATCACTATTGATTGTGGATAACCGTTGAATGGTTC

[0246] AAATGATTTAATGGATTGTTCTCATGCTAGGAATCGAATATTCAAAGGAAGTACATAATAACTACATAAT

[0247] TATATCTAAAAATGTTCATCTTTCAGTATCAAATCTGTTCTTTCTTTCTCTTTTGATGGTTCTATATTTCAA

[0248] AACCAATTACTGTCCAATTCTAATAGGTGTGTTTGAGAAAAAATCCAGAAGTTATAGATTCATTTATTT

[0249] GTGGGACAAAAATATTTGTGCATTTGCTCATTATGTGAACGCTACACATCATTACATTTCAACAAAAAT

[0250] AACAGTACAGATTCAAATTCAAATACCTCTCAATTCACTAAGTACCCACGTTTTGATATCCCACATCAA

[0251] TTGGATAAAAAAACACAAATGTAAGGTGGGAGTCAAAGATCTTGATATCCATATCTTTCTGCTATTATTTA

[0252] TTAGGGCTGGTTTGTTTCTAAAAATCAAAGAATATATTATATGTATCATAAAGTCTATGGCGTACAATTGT

[0253] ACTAAAATTAGTAACTGCAGCTTCAACATTCCATAAACAAAATAAATTACATTGTTAGTAAGTCATTCTT

[0254] GAATTGCTGCCTTATTAGAAGCTGAGGTGGGAACTTTGGTTAATAATTATGCTGTGATAAGTAGCATTA

[0255] CCCATACATAACAATTGTAGAAGGATTTTCTAATTTTGGCAGCAGTTGTGTATCGAATCAGTGGAGAAC

[0256] GTTATCCTGCTTCATTTCTCTATGGTTAACTTTGTTAGCAGTTTCAGGAATAAAACAAATTTTGTCCCA

[0257] GGAATGAATCCATCCGAACAGCCAGCATGCAAAACAATGAATCTTATAAACAACATAATTCCAATACA

[0258] CCATCTAATGATATTCAGATTCTAAAAACCTTGAATCTCATATACTTTATATTTTCACACTTTTGACACTA

[0259] TAATCTTGCCAAGAATGCCCTAGAGTTCCATTCTGAAATATCCAGATCTCATCAAAGAAAACAAATTGC

[0260] CTCAAATCTTCATTTTTCAACTGAACGTACTGTGGAGGAATGTATTACGTTTCATAGCAACATTAGGTA

[0261] ACTCCACCAATGTTTTCCATAAAGATGTGAGGGCAAATGTTGAGAAAGCTTTCAATTTAAAGACTTCAA

[0262] TGTTATGGTTCCTCTGCAATGAAGAATGTTCATCAACTGATAATATGTGATACGCACTAAATACTCACT

[0263] GTTCCTGTGTACCTATTGTAAATACAGTTCCTTATTTGATATTGTTGAGGTTGTTCAATATCCACAACTG

[0264] ATTTCTTCCATTTTGTAGTTCTGTAGAAGTCCTTCGACGGTTTAGTTCTTAGCTTACCTTCTGTGAAAGT

[0265] ATGCAGATATTTCCTACACAGCTCAATTCCTACATTTAAAGCAGCAATCACCCTCTGCAATAATCATATT

[0266] TCATAAAAACTATAAAACCATTGTAAGATCATGGTGAATGAATATTCATGAAGATTCTTTACTTACCTTT

[0267] CAAATAAAATCCAATGAAAGTAAGGGGCTCCTATTATCCCTTTCTCGTTGAAAATGTTCTAATAAAGAT

[0268] GCAACCATTTTCTGGGCATCTCCACTCAATCCTCTCAGCATTTTGATATACGATGATATTAAGCTCTCAA

[0269] AAGAACAAATTCAAAATCGCAATAAAAGAACAATTCGTATGTAAACAAACGATTGTAAGGATTCG

[0270] CTCCAAACAAATTGGCATCGTTGTGAAGTGACATTTAAGTGTTGCCATAATTGTTGATGCTTTGATGTT

[0271] CCCAGCTAATGCATAAACCTATTGAGACGTCTGTCATTGCTAGATACTTTTTGTTTTGTACCAATGGCAT

[0272] TGAAAAGTTACCAAATCTGACACAGGTATCAACCCAGATGAAATATACTATAGTTATGGTGCTCCAAAG

[0273] TAAAAATAACACCTAAGACTGGTGCAGAAGGGTGGATATCTTTTTTTTCTATTAGATTTTACCCCTATTA

[0274] GATAATGATAATCTCTCATAAATACACTACATTCCGTAATTATTTTATTCAATATTATACAGGGTGTCCAG

[0275] AAACTCTCCTGCCAAACGAAAACTGCAGTTAGTAGAGGTAATTTTAAGTCGATTTAGTTCAAACTACT

[0276] TTGATCCGAAAATGCTTCCTAAGGGAGCTACAGCCCTTCGAAGAAAATGTCTCGAAATTAGTTTTCTT

[0277] CAAATTATATAATTTTCATAAAAAATTCTCTTTCAATTCGATATCATGAACCGTTTTTCGAGAAAAACGA

[0278] ATTTAAACTTTTTGTATTTCTTACATAAAATCATGTTGTATTATAAAATTGTATCTGTAATATCAAGTAGG

[0279] CTTTGAAAAATCTACAAAATCTATTTGTAACTGTGTATTGATTATACAGTTTGAATGTTTCAAGTAACGG

[0280] CATTTTTAAATTATAGGAAAACTGTATAATGATGTCTTCTTCAAAGAGCTGAGTTAATTTGAGCTTAATC

[0281] GACTCGAAATCACCTCTAGTAACAACAGTTTCCGTTTAGCAGGAGAGTTTCTGGACACCCTATACAGG

[0282] GTGTCCGTAATTTGAGCTTAAGCATGGGGATATTGGTTACCTTGAGAGAGATACGATGTTGCTCAAATTAG

[0283] GGCAAAGTTGCGCATTCTGATTCATAATCTGGAAAGAAACCTGACCTTTCTTCCTTGATTTTCAAGATA

[0284] TCCGTAAAAACGGAATTTTCGATTTTCGGTTTTTATTTTTTCATGGGAAAACCAATCAAGTTATCCATTT

[0285] TCAACCGTAACATTCTCAAAGCAGTTTTTTGCGAGGAATTAGGAAATGACATTACATTTTTTCTAACAC

[0286] ACGAAGAACGAAACAAATGACTGTCATTTTTTTCTTATGGACTCCATATTGAATTGTAATATTTCTGAA

[0287] TGCAGTTTTTTTTTCTGATTCCGAACATGTTTCGATTTCAAAGTCGAAATTTCCTAATAAACATTTTTT

[0288] CGAAGATAGCCGTGGAAAAAAACTCGCTAGATTTGTTCAATTCGAATTTTGACAGTTCAGTAGTTTTT

[0289] ACGCTTGGACTCCACTCTTGTGAATTGAAATATGCCATTTTCCAATGAAGAACAGAGGGATATGTGATA

[0290] TGTTGGAAATTTATTATGCTTGTCATCGAAATGTTAAGTAACAAATGAAACATATCTACAACGTTTTCCT

[0291] GAAAGACAGCAACCAAATACTGGATATTTCCTAGGTTCCCATAGAAATCTGAGCCAATATGGTTCATTT

[0292] CGGCGACCCAGACATAAATATCGCGAGAGAGATAATAAATATGAAGAAAACATATTGCAAGCGGTATG

[0293] TTCTAATTTCAGATTGGATTTCAGTTTTAATATCGCTTAGGTCCAAGAAGATCACTACATATCAACGAG

[0294] ACAAATTGGAGCGCAGTTGAATTTACAACACTAGTTGGTATGGAGGACACTCAGAAAGAATCGGTATC

[0295] ATCTTTACGAAATTCTTCCTCACAAACTCTTCATCCTGGAGACAGGCAACGCAGATTAGAATTTTTTAG

[0296] GTTCTTGTTGGATCGGATACGTGAAAATCCAGATTTCTTTCGCAACATAATATGAAGTGACGAGTCGA

[0297] ATTCACGAATTTAGGGATGTCAATAGACATAATGAACATGTGTGGTCTATCGAGAATCCCCGTGTGAA

[0298] CCAAGAACGGCGACCTCAGATACGCTTTGGACTGAATGTATGGGTTGGTTTACTAGGTGGTACAGTTA

[0299] TTGGTCCATACATTTATGAAGATAATTTGAACGCAGCGAGATATGCTCATTTTATGAGGACTTTTTTGAC

[0300] GGATTATATCGATAACAATATTAATCTCAACCGCCTTTGTGATTTATGGTTCAGCAATATGCCAGAAGG

[0301] GTTACAGAATATTTGTCGGAAATGTTTCCTAATAGGGTTATTGCGAATAATGGTTCGTTGGCCAGCGCG

[0302] TTCTTCAGACTTATCACCCTTGAACTACTTTTTATGGGGAACAATGAAAAATGAAGTTAAAACTGTACC

[0303] AGTCGATATAAATGACCTTCGGCAAAATATTATCGACTCTTTGAGACAGATCAGGAGACGAAGTATCG

[0304] AAGAGCAATTGCAAATGTAAAAAAACGTGTAAAATTATGTATACTGGTGGAAGGTGGACAGTTTGA

[0305] ACAGCTTCTCTGATTATTCATATTTTGCTTTCGTTTTTCTGTTTGGTTCACATGATATATCTGTTTTATTAAA

[0306] AATATAAATTTCTAACATATCCCTCTGTTCTTCATTGGAAAATGGCATATTGCAATTCACAAGAGTGGAC

[0307] TCCAAGCATGAAAACTACTGAACTGTCAAAATTCGAATTAGGAAAATTTCGACCATAATTTGAAATCAA

[0308] AACATGTTGTGTGTAGTACATCGTCGAAATCAAAAAAAAACTCCAGAAATATGAAAATTCAATA

[0309] TGGCGTCCTTAAGAAAAAAAATTGACTGTCATTTGTTTCTTTCTTCGTGTGTTTAGAAATAATGTAATA

[0310] TCATTTCCTAATTCCTCACAAAAAACTGCTATGAGAATGTTACGGTTGAAAATGGATAACTTGGTCGGT

[0311] TTTCCAATGAAAAAATAAAACGAAATTACGTTTTTCAGCGATATTTTGAAAACCAATGAAGATAGGT

[0312] CAGATTTATTTTCATATTGTTGATCAGTACCAGAATGCGCAACTTTGCCCTAATTCGTATCTCTCAAGGT

[0313] AACCGAGATACCCATGCTTTAGCTCGAATTACGGGCACTCTGTATATTATTAAATAGTTATGATGGTGAA

[0314] TAATTTCAAGGTTCTGAACGAAACTGGAGAACAAGACTTGTGTTATTACAACTTCCTATGCGCTCATC

[0315] CATTAGGGTTTCTAAGTGACTTCAATCATCTGTTTTCCAATATTGGGTATATCTTGTTGGGCTTGCTATTC

[0316] CTCATCATTACTTATTTGAGAACACTGACCCACGGAGACTTCGATTTTGACAGGGTAAGTTTCCAAAGT

[0317] TGAATTCGTGTAACATCGATTAAGAATACTGATGAATCTGTGAAAACTGGCACTTATGAGATGGGTGTT

[0318] TATGATTTTGTTCCTTCAACTATAGATTTCTTTCATCGATTACAGCAGAATATTCCTAAATATAAAGAAAA

[0319] TTTCCTTATGATGATATGCTATATGAAATATATTCTTGGAAATTCTCCTGAAATATAGTATATCTTTTGTA

[0320] GCAATATGGTATACCGCAACACTATGGATTATTTTATGCGATGGGAGTGTCCTTAACGATGGAGGGGAT

[0321] TTTGAGTGCATGTTATCATATTTGCCCGAATCAACTAAACTTCCAATTTGGTATGTATTCGGAAGCATTT

[0322] CAACGCTTTGATATTTCGTATCCACTTTTGGTGAAGATACACATGAAAAAATGTTTTCTTTAATTTCAG

[0323] ATTCAAGTTTTATGTATGTGATGGCAGTATTATGTATGGTGAAACTCTATCAAAACAGACATCCTGATAT

[0324] TAATGCGAGTGCTTATAGCACATTTGGTGTATTAGCTGTTGCTGTTCTGTTGGGTGGGTATTTATTGTCA

[0325] AATTAAATTTCCGTCTTGATAAAAATTACGTGTTTTAGGTATGATTGGCATGCTGCAAGGAACGACA

[0326] AATTTCTGGATACTTTTTGTTGTTTTATATATTATAATGTGTTTGTATATTAGTATCAAAATTTACTATATGG

[0327] GATGCTGGAAAATCAGTAAGTCAAAATTCCTACTAAATTAATAATAGTTGAATTCTTGATTTCAAGGAT

[0328] TTTATATTATTTCATAATTGATTGTTCTTATATTGTATTTTTTAGATGACATTTCTGTGCAGAGGTTGAAAC

[0329] AATTATGGAATAACGATTTTCGGGCTGGTCCAATAAATATTTTGAAGCCTTGTCATAAAGGTGAGTAGG

[0330] ATTACAGCAAAAGAAATATGGAAAATATGTGAATAATTTTTTTCAGCTCGATTTATTCTTCTGTTCTTGG

[0331] GTAATATTTGCAATTGGACTCTCGCTGCTTTTGCATTATATTACCATCCAAATAATTTTTGCTGTTTTTTTAT

[0332] TGGCTATATTCATGGCCAATACGCTATTATATTTCTTCTTTTATATAATCATGAAAGTAAGTATAAGAAATT

[0333] TTTTATGATATTGAGTATGTGATAATAGGAGAAATTCACAAATCTTTGATATATCACAACGAAATTTTATTA

[0334] TCATATATTAATTATACTTATAATTTTTTGATACCGATTCGGAAATAATTCATCTTCCATATAAGAATTTGA

[0335] TCAGATCTAAAAGAATAGCCTGTATTAATTTGAGATAGCTTGGATAGAGCTTCTGCGGCGCGTCACAAA

[0336] TTGGCGTTTCTCTCCTGGAAGAATAAAAAACTTCAAACAATTCTGTTCCGTTCATTTGTTTGTGTTCGTT

[0337] GAACGTTTACTTTGATACTTCGTATTGTATTTCGAAAGGCCAGTTTATCGAATTTTTCAATAACTTTTTT

[0338] CACAATTGAACGTTTGACATTCGATGCTGTGTGAAGTCAGGTGCAGAAGCTGTTCACATGGGCGTA

[0339] GCACGCACTTTAGCGGTTTTGGAGAAATCAATATCGCTTGATTATTTGGGATTCAAATACTCTTGTTTTT

[0340] CTTTTTGGTTCAATAGATTTCTATAGTAGATATATACCCAAAGATTTTTTTCCGAATCAGAGGTTTCAAC

[0341] AAAAATAGTAATATTTATCGTAAGATTGCAGATTCACACATAAGTGTTTTATCCTTTATGGATAAGTAAT

[0342] AGAAAATGTGTAATTGAATTATCACGAACTTTTCATAACAATAATATTTAGTAGACATTCTGAAGTGTTT

[0343] GATTTTTTTTTTCAGCATATAAATAGAGAAAGAGTTAAAATAGCGACATGGATCTACTTATTCTTCTCGA

[0344] CTATATGTGCTATATCAGCCATGTATTTCTTTTTACACAAATCGATTCCTGGTCGGTAAGTTGAAGAAA

[0345] AGTGATAAATTTATATGTCTGTCTAAACAAATCCATCCAAGGGGAAAATACGTATCTAATAGACACATA

[0346] AGACAACGCACATTTATCCTGGTAGGACATGTGAGTAACTGTGTCTGTTTCAACATTTACCATGACATT

[0347] GTATTTCAAGCAGCCAGAAATATAAAATACTGAAGAAGTCAGTATTTATAATAGTTATTATCAAAACTAC

[0348] TCATAACCAATATTTGTTTGAAGGCTGCAATTCCATTTTTCCAAATTTCATCAATATTTCAGGACCAATT

[0349] TTTCGGTATTTACGTCGAAATTGTTCGAATTCCCCTTAGAACATGTTTTTCCAGTGGATCATTTGCTTGC

[0350] CATATGAGCGTCTTGAGAAAGTTCCTATAACTATTTATTTTTTGAACTATTTTGTTTTCCATGTACAAGGT

[0351] ATATTAATATTATCCGAAGTTTTCGTTCTCTCAACCAGTAAGCTAAGATAACGAAGTTAATGATTACTAT

[0352] TGATTTTACGAAAGACCAAATGATGAATAATTTGAACAAAAAAATCGGCATTTGATCATATATTTTT

[0353] CTAATATCGAATCTATTGAAAATTGGAGTAGTCATATTGAAGCTTTATATAATATGGCTGAAATAATTTTC

[0354] ATCATTTCAAAGATTATGACCAACAGTACGTGGCAATTTCTTTGAATAATCTTTGAAACTTGAAGACTT

[0355] AGGAGGAATCACAAACGACGCCGCATATTCGAGAGAGCTTTATGACAACTACTTTCAGGCCAACAA

[0356] CCCTGTCAAATATGTCAATTCTGTTGATAATAATCGCTTTACAGTTGATGAATTTGAAATTTGAAATTTTC

[0357] ATTCCTCAAACCTGAATAGCAATTTATGTCGTTACCTTGTTCGTCGGTTATGATCATTTATAATTCTTTGT

[0358] TTTTAATTACTATAAAATGATAAAATGATTGTCTTCCAATTATTTTGCAGAAAACTCCGGCCCAATCGAG

[0359] GCAGTTCAACATGGAATGTAAGCTGTTTAGGTTTTATGATTTTCATGACATTTGGCACTTTTTAAGTGC

[0360] AATCGGAATGTTTTTTACATTCATGGTATTACTGACTATGGACGATGATCTGTCTCATACGCACCATTCG

[0361] CAGATTCCGGTATTCTGAGGGGAATGTGGAAATATATTGAAGTTGAAACGCTCAAACGTACAGGTTAG

[0362] AATTCGGTGGAATGCAACAGAAATGTAAATTCCCGACAATAGAAATGGTAATTTTTTATTAGTCAATTT

[0363] ATATTTTTGTTGTACTTCTTCCGATCAGATGTTTCATAGAAATAGGATAGAACTCTATCAAATAACGTC

[0364] CCGGACAAATTCTTCACGGTATTTGAATGAATTCGTAATGTGATGTGATAACCCCAACTGAAGTTTCCT

[0365] AATGAAATAGGATGAAAAAGTACTTGAGAGTGCTTGATTATTCCAAAACAAGTAGAGTAAAATGTTAT

[0366] GATTATTTGAAAAAAATTCTACGCAAGAGGTTACGCAGTCAGATATTCTCTAAGAACTCTTTCACACGC

[0367] ATATCCGAGATAAAGATTGTATGGGATTCACGAATTTTTCAATCAGGACTGAAAT

[0368] In a second aspect, the embodiments disclose a CRISPR / Cas9 guide sequence, including an RNA or DNA sequence as shown in SEQ ID NO: 5 or 6.

[0369] Molecules, and RNA or DNA molecules as shown in SEQ ID NO: 7 or 8. The guide sequences as shown in SEQ ID NO: 5 to 8 guide Cas9

[0370] The transmembrane protein-related coding gene SIDT2 of the blue leaf beetle is cut to affect the expression of related genes.

[0371] The DNA molecule can be transcribed to obtain the RNA described in SEQ ID NO:5, and the DNA molecule described in SEQ ID NO:8 can be transcribed to obtain the RNA described in SEQ ID NO:7.

[0372] The nucleotide sequence of the SIDT2 gene is as follows:

[0373] AAATATTTGAGATAAGCCAGGAAATGAGATAAATCATTTCATATGTGTCTATATTATTTTTATGTTGAGG

[0374] GTATGGAATGCATCGTATAGATATCGAAAGTAGACAGGTGAGTCGATGATAATGGTACAGAATAATAAT

[0375] TTTTGTAATAAATGTATAAATGACCAGGAATGAGCGAACGATTTATAATCAAATTCCAAAAGAGAGTTG

[0376] AATTGGTTTCTTAGCTGAATTCAACTCGACTCTTCTTCAATTGTCTACTATTCAGTCTTCAGAATTATTG

[0377] TTATCAGTGTCTTTCTTCAATAATTTTTTATACCAAGTTTGTTAAAAAAATTTAATTTATGTAGTGACAA

[0378] TGATAGCAACTGCGACAGGTATCGATTGAATTACGATAATGATTACGAACACAACCTAACGAATAAATg

[0379] gGGTGTATAATTCATTTGAATCCGTTTTCTTTCTCCGATAATTTTCCATTCGGCAAATCAGTCATTCTTTTC

[0380] AGAAATGCTTGTGGTTGCTAAATGGTTTCCATTACTGTTCCTGTTATTTCATGTTCGGAGTCAAAATATT

[0381] TCGATAGAACAAATTATACCTGACAAAATGGAACCCCAATCACTCCGACTGAACGCTTTTCGGGAGAT

[0382] TGTTCTAATTTATTCTTCTCAGCCAGGATTGAAGGTGAGTGCTTGATCTTTCATACACATAGATTTACTT

[0383] ATTGTGTAAACATCGATGATTTATCATTTCAGACTATCAATCCATTTCGAATAAAGACATCAACAAATAT

[0384] GACTCATCCCTCACTGCCAATTTTGGTAGTAGCCAATCAGAAATCGCAGGTTTCGGCTTGGTCTACTCC

[0385] ATTGGCTGTTGAAACTTCGATGAAAGACAGGAAATTGTATTTCCAGGCAAGTTTATTAACTTTCTGAAT

[0386] CTGAATCTTCTGAAATTCTGAGCATATTTCGGATAAATGGATTGATTTGAGTAAGAGATTTTGTGGTTGA

[0387] TTTTCAGATTTTTCTGTCGAACTTCCTGATATCTCGCAGAGATTCATAATAGCCTTGACGACGTCATCGA

[0388] AGGAAGACGTTTACGTCACCGTGGAACTGGAGGAACAAAGATTTCTATGTCAAGCAGGATCAGAA

[0389] TTACTCTCTGCTAGTATCGCCTAGCGAACCTCAATACGTATTTTTCAAGTTCAACGAAAACTCTTCAAG

[0390] CACTATCATCATCGAGGTCGATTCCGAAGATGAGATTTGTCTGACTGTATCGGTGCAAGATAGTCAGGT

[0391] GGGTTAATGATGATGGTTGAAGGAATTTGTACTAAACGACATTTGAACGCAAATGAATATGAATTCTA

[0392] CCTTGAGCAGTAATCTAAACTGAGATTGAACGACATAATATGAGAGACGATTTTATAAATTTGAGAAAT

[0393] AGCTTCAGTCAATAATCAACGGAATGCAAAATATGTTTATCAATTTGTTTTTCTTTCTCATTATTTGTCTG

[0394] GTATACAGGATGTCTTAGTAATTATCGAAAATCTATAACACTAATATGTGTTTGTGTCCTTGGAATATATT

[0395] TACCTCGAAAACGGATTGTCGACTATTTACAACTGATCAATTTTCTTCCAGTGTCCCGTATTTGACATG

[0396] AACAAAGATATTAAATACGAAGGTATTCACCAGACTATCAATCTGAATGGGGCTATTACTCTTTCTGTA

[0397] AGTAGAGTTGAAATTCAATTCAGTCATAGTTCAAATCCAGAAATGGGATTTATTTCTGTATATACAAATA

[0398] TTTGTTTACACGGATTTCTGTGAATTCATAAATTCATGCATATAGCTTTCTATCAATCTTATTTTTGATAA

[0399] AAGGATTCGATAAATTTTCAATTTGAACGCATACGCAAATAATTAAATAACATTTCAATATTCAGTTCTT

[0400] CCACTAATTTCTCACTTCTAGAAATCTCAATATCCACAAGGATTTTTCTTAGTCTTCGTAGCTAAACCAG

[0401] ACAACTACGAATGCAGCCAATCCAATTCATTTATACCCAGAATGATGAGAACAGTTTCGATATCTACTA

[0402] GTAATATCACTAGTAAAATTAACTTCAGGATAAGGAACAGCATTAGTAGTTCCGATTATGCTTTTGCTGT

[0403] CATGGGAACATTCTTCACGTTGTTCCTAGTTTGTATGGTAGTCGTCGCAATAGGTTTAGTTTTCCATAGG

[0404] TATGGAACTATTTCCAAAAAATAATACAACGGTAAGGAGAATTCATCGTTATATGACGTTTCATCAAGA

[0405] GTCTAATTAAGCTTGCAACAAAATCACTAATGATAATAAGTAGTGCTTCAATTATAATTATTCTTGTTA

[0406] AAAAAACCAATCATTTTGAGAATGCAATTGATCCAACTGTCCGAATGTTATGAAACGATTCATATTCGAT

[0407] TTTTTTTTTTTAGACGTGGTTGTTACAGATTATTTTGAAGCTCTTTCAGAGGATCAAATTATGAGCATAT

[0408] TAAGAACTGATAACCTCACTGTATCTTACTTTTCAAGACATCCGAAACGAATTTATAGGAGATCATACA

[0409] ATTATCTATCTCATACCATGAGCATCGGCCTATTCTACAGTATTCCTGTTGTACAATTGGTCGTAACATAT

[0410] CAAAGGGTAAGAAACCTGAATTCAGTACTACATGAAAAAAACAAATGAAGGAACATAATCAGTTCGAT

[0411] CACCTCTTTTGCAGATAGTGAATCAATCAGGAAATGAGGACATGTGCTATTACAAACTTCTTATGTGCTC

[0412] ATCCTCTATTTGGGTACAGTGATTTCAATCATATATACTCTAATATTGGTTATGTTTTATTTGGGATTAGCT

[0413] TCATCCTTGTTGTGATAGACAGACACAGAATAATTAAGTTGAGAAAGGTGAGGACAATGATGCAAAAT

[0414] TTTCGAAAGAAAGATTTTTGACTTTTCAGGAGAAAGGAATTCCAGTACATTATGGGTTATTTCATGCGA

[0415] TGGGTGTAGCGTTGATTATCGAAGGATTACTCTCTGCCTGCTACCATATCTGTCCAAGTCAATCTAATTA

[0416] TCAATTTGGTAAGAATTTTTGTTTGAAAAAAATTCAAACGAGGAAATTAATAGAAAGCTCCTAATTTTG

[0417] ACAATTACTCGGCATTCTATTGAAAACATAAAAAACAACAAAATTGATAGCCTTATAGGGTTGATAGTA

[0418] TCCACAAACCCAAACCCTGATTGTTTAACTGGAATTATAGATACGAGCTTCATGTACGTGATGGCAGTA

[0419] CTTTGTATGATCAAATTATACCAAAATCGTCATCCAGATATCAATGCAACAGCTTATACAACATTTGCTA

[0420] TACTTGGAGGTGCAATATTCATGGGTGAGTTATATCATTGGTTGAATGAATTATTAAAATAATATTTAATTG

[0421] TATTTCAGCTATGATTGGAATTTTGAATGGAAGCTTAGCAATCTGGATCGTATTCGCAGTTTCCTATTTT

[0422] GTGTTAGTTGTCGTTCTATCATTCAAAATCTACTACTTGAATCACGTTATAGATGGACTGAAACAGTTC

[0423] AAACAAGAATTTTGGAAGAAAGGATTATGTACCGATATCTTCAAGCCTGTCAGAAAAGTAAGTTTAA

[0424] TTGGTGAAAGACTTTCGAGCCCGTCAAAATACTTTTGAATATATTTTCGAACATATCGTAGCAGCTTTG

[0425] GATCCTTTCACGATTCAGTTGTTATTTCTTTGTTTCAGTGAGATTCGCCCTTCTCGTTTTAGTTAACTTAT

[0426] TGAACTATTTGTTTTTGATAACTGGGATGTTGATGTATAGTTCGAATGTGACAGACTTCGGGACGTTCC

[0427] TTCTGGGACTCCTTTTGGGAAACGCAGTTATTCACACTATTTTCTACACTTGTATGAAGGTAAGGTAGA

[0428] ATTACAGCAAAATATACTTTCTGTGACATTTTTGTTATTTATTATAGTTAATACATAAAGAACGCATTTGC

[0429] TTCGAGGCAATTTGCTATGGAATTATGAGTATAGCTTGCTGGGCTGGTTCTGCAGTTTTCTTTTTAGAC

[0430] GCTGCCACTCTTTGGACAGTGAGTATTCAAAAGTTGATCTTTATAAAATCATTTTCGATCAAATACTATC

[0431] ATCTAGTGATTTTTTAGTAACCCCTGCAGAATCACGTCAATGGAACCAAGCATGCATATTACTTAGCT

[0432] TTTTCGACAAACATGACGTTTTGGCATCTTCTCAGTGCCCCAGGTCTTTATTTCACATTCATGTATTTGAT

[0433] GTGTCTTGATGATGATATAATTGATAAGGATCAAAATGAAATCTCATCATTTTGAAAACGATGTAAAGT

[0434] CTTAGTTGTTTTGACTGCATCTAACCTCTCTATTCGAGTTATGAGTCAACTGTTGACCATCTCGATATAA

[0435] TTTTACGATACCGTCAATTCAAACATTGAAGGGTAATGGCTCTACATTAGAGCTCCCTCGTATACTAGAT

[0436] GTTGACATATATTGATCTATTCAATATAACACGCTGCTTACGACTTTTAAGATATGCACAATATTTGACTA

[0437] TTTATTGAAGAAACAAAAAAGTCGAAAGTCTTTATTCATTGGAATCAAGTTTGGAGAAGTCAGTGAT

[0438] ATAAACTAGATTTATTAATCTATTACAATCAATAATTGAAGAAGTCATTTAAAAAACATATCTAGAGATAT

[0439] CTCCAAAATTTTAAAAGATTGAAATCTAAGAGGATATGTAAAGATTCAATTGGTATGCAGTTGTAATTC

[0440] TTATTAATATTCCTTTGGTATTTTTGAAATTTTGAAATGAATAATACCAAATTTATTCAGGAAATAGAATT

[0441] TATTCAGAAAAAATTCAATACTCCAGTAGTGTTTGTTTGACCACTGTGCTCTGATTCATTCACTGATTG

[0442] TCACTTACTTAAGATAAATTATTATCGAATGTCATCACGTAATCAATTTAATTTATTCATTGGATGAGTAG

[0443] GATAAAGAAGATTTTGGTAAGATATTTGGAACAATGTTGTAAAATTAAACAATTACAATAAGC

[0444] The embodiment discloses a first primer set, comprising

[0445] F1: TAATACGACTCACTATAGTTGGCAGCTGCCTTTAGAAC; SEQ ID NO:9

[0446] R1: TTCTAGCTCTAAAACGTTCTAAAGGCAGCTGCCAA; SEQ ID NO: 10;

[0447] F2: TAATACGACTCACTATAGGCAGTTGCTACAGATACTAT; SEQ ID NO:11

[0448] R2:TTCTAGCTCTAAAACATAGTATCTGTAGCAACTGC; SEQ ID NO:12

[0449] F1 and R1 were used to PCR amplify the DNA molecule shown in SEQ ID NO: 2, and T7 RNA polymerase was used to transcribe the RNA molecule shown in SEQ ID NO: 1 to act as the gRNA (SIDT1-sgRNA1) of CRISPR / Cas9. The specific steps were as follows: GeneArt Precision gRNA Synthesis Kit (Cat. No.: A39377).

[0450] F2 and R2 are used to PCR amplify the DNA molecule shown in SEQ ID NO:4, and T7 RNA polymerase is used to transcribe the RNA molecule shown in SEQ ID NO:3 to act on the gRNA (SIDT1-sgRNA2) of CRISPR / Cas9.

[0451] The embodiment discloses a second primer set, comprising

[0452] F3: TAATACGACTCACTATAGTGCTTGTGGTTGCTAAACAA; SEQ ID NO:13

[0453] R3:TTCTAGCTCTAAAACTTGTTTAGCAACCACAAGCA; SEQ ID NO: 14;

[0454] F4: TAATACGACTCACTATAGATGGCCTCCATCATCGGCTC; SEQ ID NO:15

[0455] R4: TTCTAGCTCTAAAACGAGCCGATGATGGAGGCCAT; SEQ ID NO:16

[0456] F3 and R3 are used to PCR amplify the DNA molecule shown in SEQ ID NO:6, and T7 RNA polymerase is used to transcribe the RNA molecule shown in SEQ ID NO:5 to act on the gRNA (SIDT2-sgRNA1) of CRISPR / Cas9.

[0457] F4 and R4 are used to PCR amplify the DNA molecule shown in SEQ ID NO: 8, and T7 RNA polymerase is used to transcribe the RNA molecule shown in SEQ ID NO: 7 to act on the gRNA (SIDT2-sgRNA2) of CRISPR / Cas9.

[0458] Gene knockout in the blue leaf beetle

[0459] The embodiment discloses a kit, comprising RNA as shown in SEQ ID NO:1, RNA as shown in SEQ ID NO:3, and Cas9 enzyme as shown in SEQ ID NO:17.

[0460] The embodiment discloses a kit comprising RNA as shown in SEQ ID NO:5, RNA as shown in SEQ ID NO:7, and Cas9 enzyme as shown in SEQ ID NO:17.

[0461] The above-synthesized SIDT1-sgRNA1 (SEQ ID NO: 1) and SIDT1-sgRNA2 (SEQ ID NO: 3) were mixed with Cas9 protein for incubation and injected into the eggs of the blue leaf beetle, and then raised to adults. The leg genomic DNA was extracted, and F5 (SEQ ID NO: 20) / R5 (SEQ ID NO: 21) was used as a primer pair for PCR amplification. After PCR amplification of gDNA covering two targets, TA cloning was used to detect its genotype. The amplified fragment of the wild-type SIDT1 was 1026bp (such as SEQ ID NO: 18), and the amplified fragment of the mutant SIDT1 was 199bp (such as SEQ ID NO: 19). After comparison, it was found that the amplified fragment of the mutant SIDT1 lacked 827bp of bases compared with the amplified fragment of the wild-type SIDT1. The obtained mutant adults were hybridized to obtain a large number of homozygous mutant larvae of the blue leaf beetle.

[0462] The above-synthesized SIDT2-sgRNA1 (SEQ ID NO: 5), SIDT2-sgRNA2 (SEQ ID NO: 7) and Cas9 protein were injected into the eggs of the blue leaf beetle and raised to adults. The genomic DNA of the legs was extracted, and F6 (SEQ ID NO: 22) / R6 (SEQ ID NO: 23) was used as a primer pair for PCR amplification. The amplified fragment of the wild-type SIDT2 was 684 bp (such as SEQ ID NO: 24), and the amplified fragment of the mutant SIDT2 was 629 bp (such as SEQ ID NO: 25). After comparison, it was found that the mutant SIDT2 gene had a deletion of 3 bases at the exon 1 target site and a deletion of 52 bases at the exon 2 target site, resulting in a frameshift mutation.

[0463] The reaction system of the above PCR amplification is: 1.5 μL each of upstream and downstream primers, PCR Master Mix 25μL, DNA template 2μL, enzyme-free water to 50μL. PCR amplification program is 94℃ pre-denaturation for 3min; 94℃ denaturation for 30s, 58℃ annealing for 30s, 72℃ extension for 30s, 32 cycles; 72℃ extension for 5min and then stored at 4℃. PCR products were sent to Qingke Bio for sequencing analysis.

[0464] The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.

Claims

1. A guide sequence for CRISPR / Cas9, comprising an RNA or DNA molecule as shown in SEQ ID NO: 1 or 2, and an RNA or DNA molecule as shown in SEQ ID NO: 3 or 4.

2. The guide sequence of CRISPR / Cas9, including the RNA or DNA molecule shown in SEQ ID NO: 5 or 6, and the RNA or DNA molecule shown in SEQ ID NO: 7 or 8.

3. A primer set comprising the DNA molecules shown in SEQ ID NOs: 9 to 12.

4. A primer set comprising the DNA molecules shown in SEQ ID NOs: 13 to 16.

5. A kit comprising RNA as shown in SEQ ID NO: 1, RNA as shown in SEQ ID NO: 3, and Cas9 enzyme as shown in SEQ ID NO:

17.

6. A kit comprising RNA as shown in SEQ ID NO:5, RNA as shown in SEQ ID NO:7 and Cas9 enzyme as shown in SEQ ID NO:

17.

7. A method for knocking out a gene of the blue beetle, comprising the step of introducing and injecting the guide sequence and Cas9 as claimed in claim 1 into blue beetle eggs.

8. A method for knocking out a gene of the blue beetle, comprising the step of introducing and injecting the guide sequence and Cas9 as claimed in claim 2 into blue beetle eggs.

9. Use of the guide sequence according to claim 1 or 2, the primer set according to claim 3 or 4, and the kit according to claim 5 or 6, wherein the use is selected from at least one of constructing a gene-knockout willow leaf beetle strain and preparing a preparation for controlling willow leaf beetles.

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