Application of BmTrh, a gene determining the cocoon shape of silkworm cocoons

Abstract

The invention discloses the application of the gene BmTrh determining the cocoon shape of silkworms, and belongs to the field of silkworm technology research. The invention targets the silkworm BmTrh gene, performs genetic modification operations on it, changes the gene expression amount, or destroys the BmTrh gene structure, thereby changing the morphology of silkworm cocoons, from normal cocoon shape to cotton cocoon shape, and obtains improved mechanical properties of cotton cocoon silk. The invention provides the gene target BmTrh gene of the silkworm cotton cocoon shape, provides a potential molecular target for silkworm breeding and selection and the utilization of economic insects, and provides a new idea for the development of new silk materials.

Description

Technical Field

[0001] The invention relates to the technical field of silkworm breeding, and in particular to the application of a gene BmTrh that determines the cocoon shape of silkworm cocoons. Background Art

[0002] Insects have become the most prosperous animal group on Earth, thanks to their ability to adapt to the natural environment and their excellent reproductive ability. Lepidoptera insects are typical holometabolous insects, and one generation needs to go through four stages: egg, larva, pupa, and adult. Silk secretion and cocooning are one of the effective strategies for holometabolous insects to protect pupae from transforming into adults. Cocoons are not only a place for insects to pupate, but also can protect pupae from mechanical damage from the external environment and from natural enemies, protecting them from the metamorphosis stage.

[0003] The silkworm is not only an important economic insect and a model organism of Lepidoptera, but also a typical insect that produces silk and cocoons. The shape and size of its cocoons are directly related to its economic traits. The cotton cocoon is named for its loose cocoon layer and wide cocoon shape, which provides an important material basis for identifying the genes that control the cocoon shape and cocooning behavior of the silkworm, and for explaining the association mechanism between the cocoon shape and the quality of the silkworm silk. So far, the gene that determines the shape of the silkworm cocoon has not been revealed.

[0004] Therefore, it is urgent to identify the genes that control the cocoon shape of silkworms, provide potential molecular targets for silkworm breeding and selection and the utilization of economic insects, and provide a new idea for the development of new high-performance silk materials. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a use of a silkworm cocoon shape determining gene BmTrh as a molecular target for silkworm breeding.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] 1. Application of the silkworm cocoon shape determining gene BmTrh as a molecular target for silkworm breeding, wherein the nucleotide sequence of the BmTrh gene is shown in SEQ ID NO.1.

[0008] Preferably, the application of the present invention is to prepare a cotton cocoon-shaped silkworm variety by knocking down or silencing the expression of the BmTrh gene.

[0009] Preferably, the application of the present invention is to improve the mechanical properties of silk fibers by knocking down or silencing the expression of the BmTrh gene.

[0010] Preferably, in the present invention, the method for knocking down or silencing the expression of the BmTrh gene is achieved by gene mutation, gene silencing and gene knockout.

[0011] Preferably, the gene knockout is gene editing using the CRISPR / Cas9 system to knock out the BmTrh gene.

[0012] Preferably, the sgRNA target sequences of the CRISPR / Cas9 system are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0013] Preferably, the method for knocking out the BmTrh gene by gene editing with the CRISPR / Cas9 system is to anneal the sequences shown in SEQ ID NO.5 and SEQ ID NO.6 to SEQ ID NO.7 to form double strands, and then in vitro transcribe and synthesize sgRNA using a T7 in vitro transcription kit, mix the transcribed sgRNA and Cas9 protein, and microinject them into large silkworm eggs, accelerate greening, hatch, and screen transgenic silkworms to obtain silkworms with the BmTrh gene knocked out.

[0014] Preferably, the gene silencing is achieved by using dsRNA, antisense nucleic acid, small interfering RNA, micro RNA of the silencing target, or a construct capable of expressing or forming the dsRNA, antisense nucleic acid, small interfering RNA, micro RNA.

[0015] The beneficial effects of the present invention are as follows: the present invention discloses the application of the silkworm cocoon shape determining gene BmTrh as a molecular target for silkworm breeding, and by targeting the silkworm BmTrh gene, reducing its expression level, or destroying the BmTrh gene structure, the shape of the cocoon produced by the silkworm can be changed from a normal cocoon shape to a cotton cocoon shape, and the mechanical properties of the silk fiber can be improved. This invention can be used to cultivate silkworm materials and varieties of cotton cocoons in silkworm breeding and selection, and at the same time provide a new idea for the development of new silk materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0017] Figure 1 The schematic diagram of the structure of the silkworm BmTrh gene and the sgRNA target sequence;

[0018] Figure 2 This is the mutation type of BmTrh knockout silkworm;

[0019] Figure 3 The BmTrh gene knockout showed a cocoon shape, with a wide cocoon shape and loose layers inside.

[0020] Figure 4 After the BmTrh gene was knocked out, the mechanical properties of silk fibers changed, and the Young's modulus and elongation were significantly improved. DETAILED DESCRIPTION

[0021] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0022] Example 1. Construction of BmTrh gene knockout system

[0023] Knockout of BmTrh gene by CRISPR / Cas9 gene editing system based on ribonucleoprotein

[0024] Selection of sgRNA binding sites and synthesis of sgRNA

[0025] The website CRISPRdirect: (http: / / crispr.dbcls.jp / ) was used to design the sgRNA binding sites in the BmTrh gene sequence (including protein coding sequences and cis-regulatory element sequences), and two sgRNA binding site sequences with high specificity were selected. The nucleotide sequence of the BmTrh gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. The schematic diagram of the structure of the silkworm BmTrh gene and the sgRNA target sequence are shown in Figure 1 shown.

[0026] The sgRNA recognition target sequence is as follows:

[0027] sgRNA1 target: 5′-ACCCGGCCTTCACCCATTCC-3′ (SEQ ID NO. 3);

[0028] sgRNA2 target: 5'-CATCACTCCGGGCAAACCAC-3' (SEQ ID NO. 4).

[0029] According to the selected sgRNA target sequence, synthesize the following primers (sgRNA target sequence is underlined):

[0030] BmTrh-sgRNA-F1: 5'-TAATACGACTCACTATAGGACCCGGCCTTCACCCATTCCGTTTTAGAGCTAGAAATAGC-3' (SEQ ID NO. 5);

[0031] BmTrh-sgRNA-F2: 5'-TAATACGACTCACTATAGGCATCACTCCGGGCAAACCACGTTTTAGAGCTAGAAATAGC-3' (SEQ ID NO. 6);

[0032] sgRNA-R (Universal): 5'-AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3' (SEQ ID NO. 7);

[0033] Prime STAR GXL DNA Polymerase produced by Takara was used for annealing and extension. After PCR, the template for in vitro transcription was obtained by gel excision and recovery. Then, the sgRNA was synthesized by in vitro transcription using the T7 in vitro transcription kit produced by Promega and purified. The synthesized sgRNA was transcribed from SEQ ID NO.5 and SEQ ID NO.6.

[0034] Example 2: Silkworm embryo microinjection and screening of knockout strains

[0035] The sgRNA and Cas9 proteins were mixed, with the final concentration of sgRNA being 1000ng / uL and the final concentration of Cas9 being 500ng / uL. The mixture was incubated and reacted at 37°C for 15min. The mixture was injected into wild-type silkworm eggs within 3h of laying using a microinjector. After injection, the eggs were sealed with instant adhesive and placed in an artificial climate incubator. They were incubated at 25°C and 80% humidity and the development of the eggs was observed every other day.

[0036] After the silkworm eggs hatched, they were fed with fresh mulberry leaves and raised under suitable temperature and humidity until the moth stage. The wings of the silkworm moths were clipped, and genomic DNA was extracted. PCR amplification was performed using the primers BmTrh-F1 and BmTrh-R1 upstream and downstream of the two sgRNA target sites, and sequencing was used to detect the mutation type.

[0037] BmTrh-F1: 5'-ACGGATAAATTGATCGCGGC-3' (SEQ ID N0.8);

[0038] BmTrh-R1: 5'-GCACACTTCGCATCTGTAGG-3' (SEQ ID N0.9);

[0039] The G0 generation silkworm moths containing sequence mutations are screened for mating, and the silkworm eggs produced are continuously raised for several generations. The BmTrh coding region sequence containing mutations other than three base multiples is screened, and silkworms that can be stably inherited are obtained. The mutation type is Figure 2 shown.

[0040] Example 3: Observation of cocoon shape of BmTrh gene knockout silkworm

[0041] The cocoon morphology of the BmTrh gene knockout silkworms was observed, and the wild-type Dazao strain was selected as a control. The results showed that the BmTrh gene knockout silkworms showed a cocoon shape, with a wide cocoon shape and loose layers inside; while the wild-type Dazao cocoon was smaller in shape and had no layers inside. Figure 3 shown.

[0042] Example 4: Mechanical properties test of BmTrh gene knockout silkworm

[0043] The mechanical properties of silk fibers were tested by static tensile tests on wild-type Dazao and BmTrh gene heterozygous knockout lines. Figure 4 The results showed that the Young's modulus and elongation of silk fibers in the BmTrh gene knockout line were significantly improved.

[0044] In Example 1 of the present invention, the BmTrh gene is knocked out by CRISPR / Cas9-mediated gene editing technology. It is well known to those skilled in the art that any means capable of knocking out or knocking down the BmTrh gene can achieve the purpose of the present invention. It should be understood by those skilled in the art that various modifications may be made to the present invention without departing from the scope defined by the claims of the present invention.

Claims

1. Application of the silkworm cocoon shape determining gene BmTrh as a molecular target for silkworm breeding, characterized in that: The nucleotide sequence of the BmTrh gene is shown in SEQ ID NO.1; the application is to prepare a cotton cocoon-shaped silkworm variety by knocking down or silencing the expression of the BmTrh gene.

2. The use according to claim 1, characterized in that: The application is to improve the Young's modulus and elongation of silk fibers by knocking down or silencing the expression of the BmTrh gene.

3. The use according to claim 2, characterized in that: The method for knocking down or silencing BmTrh gene expression is achieved by gene mutation, gene silencing and gene knockout.

4. The use according to claim 3, characterized in that: The gene knockout is to use the CRISPR / Cas9 system to perform gene editing to knock out the BmTrh gene.

5. The use according to claim 4, characterized in that: The sgRNA target sequences of the CRISPR / Cas9 system are shown in SEQ ID NO.3 and SEQ ID NO.

4.

6. The use according to claim 4, characterized in that: The method for knocking out the BmTrh gene by gene editing using the CRISPR / Cas9 system is to anneal the sequences shown in SEQ ID NO.5 and SEQ ID NO.6 to SEQ ID NO.7 to form double strands, and then in vitro transcribe and synthesize sgRNA using a T7 in vitro transcription kit, mix the transcribed sgRNA and Cas9 protein, and microinject them into large silkworm eggs, accelerate greening, hatch, and screen transgenic silkworms to obtain silkworms with the BmTrh gene knocked out.

7. The use according to claim 3, characterized in that: The gene silencing is performed by using dsRNA, antisense nucleic acid, small interfering RNA, micro RNA of the silencing target, or a construct capable of expressing or forming the dsRNA, antisense nucleic acid, small interfering RNA, micro RNA.

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