Bombyx mori silk fibroin heavy chain gene mutant obtained by utilizing CRISPR/Cas technology and mutation method and application

A silkworm, heavy chain technology, applied in genetic engineering, using microinjection method, recombinant DNA technology, etc., can solve the problems of construction difficulty, time-consuming zinc finger nuclease technology, and complicated operation.

Inactive Publication Date: 2016-12-14
云南纳博生物科技有限公司
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Problems solved by technology

The zinc finger nuclease technology used in this patent is time-consuming, complicated to operate, and difficult to construct; from the mutation sequence diagram

Method used

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  • Bombyx mori silk fibroin heavy chain gene mutant obtained by utilizing CRISPR/Cas technology and mutation method and application
  • Bombyx mori silk fibroin heavy chain gene mutant obtained by utilizing CRISPR/Cas technology and mutation method and application
  • Bombyx mori silk fibroin heavy chain gene mutant obtained by utilizing CRISPR/Cas technology and mutation method and application

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Embodiment 1

[0033] The partial nucleotide sequence (base +1201 to +1600) of the silk fibroin heavy chain gene is shown in SEQ ID NO:1.

[0034]Bombyx mori silk fibroin heavy chain gene (gene number AF226688) was obtained from the ncbi database, and Nistari silk fibroin heavy chain genotype was obtained after designing primers to verify the gene sequence. The 20bd sgRNA core sequence was designed using the online software CRISPRdirect, and the synthetic sequences of three base sites (CAAGACGTTCGTTATAACCAcgg, CTCATGAAGACACTTTCCGAtgg, GGGCCATACGTATCAAACAGtgg) were selected in front of +1213~+1236, +1274~1297 and +1349~+1372, respectively adding gaaattaatacgactcactata The T7 promoter sequence, and the fragment gttttagagctagaaatagc complementary to crRNA / tracrRNA, are artificially synthesized as a 62bp front primer, and the back primer crRNA / tracrRNA undergoes PCR procedures to obtain the complete sgRNA sequence, see Figure 1 ~ Figure 3 .

[0035] The reagents used are listed in Table 1.

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Abstract

The invention discloses a bombyx mori silk fibroin heavy chain gene mutant obtained by utilizing CRISPR/Cas technology and a mutation method and application. The mutation method includes: mixing cas9mRNA with sgRNA, micro-injecting a mixture into a bombyx mori egg, identifying genotype through PCR (polymerase chain reaction), screening out heterozygotes, and mutually mating the heterozygotes to obtain F1 generation in which pure heterozygotes appear; performing genotype detection on bombyx mori with phenotype, mating the bombyx mori with same genotype, screening out homozygotes which can be stably inherited to next generation, and then screening out the bombyx mori silk fibroin heavy chain gene mutant, wherein a core sequence of sgRNA is designed aiming at 1213-1236, 1274-1297 or 1349-1372 loci of bombyx mori silk fibroin heavy chain gene. By the method, an experiment material having economic value and scientific value is obtained quickly and efficiently, and a new material is provided for large-scale production of bombyx mori sericine cocoon and enabling the bombyx mori to serve as a bioreactor to express foreign protein.

Description

technical field [0001] The invention relates to the technical fields of silkworm breeding and genetic engineering, in particular to a method and application for obtaining silk fibroin heavy chain gene mutants of silkworms by using CRISPR / Cas technology. Background technique [0002] Silkworm (Bombyx mori), Lepidoptera Bombyxidae, is a silk insect domesticated from the ancient wild silkworm. Silk, which is made of silkworm silk, is a symbol of ancient Chinese culture and has made an indelible contribution to the development of human civilization in the world. Today, the silk industry is still an economic industry with Chinese characteristics and occupies a very important position in the world silk industry. Silk is strong, soft, smooth, elastic, and has good hygroscopicity and air permeability. Therefore, most of silk has been used as textile materials all the time. With the continuous innovation of technology, besides being used as textile raw material, silk can also be us...

Claims

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Application Information

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IPC IPC(8): C12N15/89C12N15/113C07K14/435A01K67/04
CPCC12N15/89A01K67/0333C07K14/43586C12N15/113C12N2310/10
Inventor 朱亚楠相辉王文陈垒陈安利董扬
Owner 云南纳博生物科技有限公司
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