CN114921469B - Application of silkworm olfactory receptor gene BmOR56 - Google Patents

Abstract

The invention discloses the use of the silkworm olfactory receptor gene BmOR56, which is used for silkworm breeding and can increase the feeding range of the silkworm.

Description

The purpose of silkworm olfactory receptor gene BmOR56

Technical field

The present invention relates to the technical field related to silkworm breeding. More specifically, the present invention relates to the use of a Bombyx mori olfactory receptor gene BmOR56.

Background technique

Bombyx mori is a typical lepidopteran insect that mainly feeds on mulberry leaves, but can also feed on other plants such as Moraceae, Asteraceae or Ulmus. Studies have shown that through functional analysis of olfactory receptors expressed by all larvae in Xenopus oocytes, it was found that one of the olfactory receptors, BmOR56, is highly sensitive to cis-jasmone, and cis-jasmone is One of the main volatile substances in mulberry leaves can specifically attract silkworm larvae. Therefore, using the BmOR56 gene for silkworm breeding may increase the range of feeding habits of silkworms.

Contents of the invention

One object of the present invention is to provide a use of the silkworm olfactory receptor gene BmOR56, which can be used for silkworm breeding and improve the feeding range of silkworms.

In order to achieve these objects and other advantages of the present invention, according to one aspect of the present invention, the present invention provides the use of the silkworm olfactory receptor gene BmOR56 for silkworm breeding.

Furthermore, the silkworm olfactory receptor gene BmOR56 was deleted, and the BmOR56-deficient silkworms were used for silkworm breeding.

Further, according to the BmOR56 gene CDS sequence information, the gRNA target site is designed, and the target site sequence is as shown in SEQ ID NO.2~3; primers are designed according to the target site sequence and sgRNA is synthesized, and the primer sequence is as SEQ ID NO.4~ As shown in 6; sgRNA and cas9 protein are injected into silkworm eggs; after hatching, the injected silkworm eggs are raised and the required silkworm strains are selected for silkworm breeding.

Further, it is used to cultivate silkworms with different feeding behaviors.

Further, it is used to cultivate silkworms that feed on lettuce.

Further, it is used to breed silkworms that feed on cabbage.

Further, it is used to cultivate silkworms with different growth rates.

The present invention at least includes the following beneficial effects:

The present invention provides the use of the silkworm olfactory receptor gene BmOR56, which can be used for silkworm breeding and improve the feeding range of the silkworm.

Other advantages, objects, and features of the present invention will be apparent in part from the description below, and in part will be understood by those skilled in the art through study and practice of the present invention.

Description of drawings

Figure 1 shows the mutant obtained through CRISPR/cas9; (A) BmOR56 gene structure and gRNA site. The BmOR56 gene is located on chromosome 28 and has 6 exons. The black line represents the intron, the rectangle represents the exon, Met Indicates the start codon, stop indicates the stop codon; the two gRNA target sites are both located on the first exon, the red background sequence is the gRNA target sequence, and the blue background sequence is PAM (protospacer adjacent motif); (B) Generation of homozygous mutants; inject a mixture of sgRNA and cas9 protein into silkworm eggs. After developing into moths and laying eggs, their genotypes are identified, and heterozygous mutants are retained for seeding, and finally in the G3 generation Homozygous mutants were screened;

Figure 2 shows the gene structure analysis and amino acid structure analysis of BmOR56 in the mutant line; (A) The changes in the genome structure of the two mutants at target site No. 1; (B) The changes in the genome structure of the two mutants at target site No. 2; ( C) nonsense mutation occurs in the gene in the mutant;

Figure 3 shows the expression analysis of BmOR56 and related genes; (A) Expression analysis of BmOR56 at different developmental stages; (B) Expression analysis of BmOrco at different developmental stages; (C) Expression analysis of ionotropic receptors; * indicates T test The result is P<0.05, ** indicates the T test result P<0.01, *** indicates the T test result P<0.001;

Figure 4 shows the anatomical model of the mutant midgut; the fifth instar larvae after starvation for 12 hours were fed apples (A), pears (B), corn (C), lettuce (D) and cabbage (E) for 12 hours. The rear silkworm midgut is shown as apple (F), pear (G), corn (H), lettuce (I) and cabbage (J);

Figure 5 shows the detection and analysis of the polyphagous nature of mutant silkworms; A: Changes in body weight; B: Pattern of body changes; C: Number of silkworm pellets; D: State before feeding; E: State after feeding; statistical analysis of the average data of each silkworm .

Detailed ways

The present invention will be further described in detail below with reference to the accompanying drawings, so that those skilled in the art can implement it with reference to the text of the description.

It should be understood that terms such as "having," "comprising," and "including" as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

The embodiments of the present application provide the use of the silkworm olfactory receptor gene BmOR56 for silkworm breeding; the BmOR56 gene is located on chromosome 28. After BmOR56 is knocked out, the olfactory sensitivity of silkworms is reduced, and the food range of the silkworm is expanded to a certain extent. The mutant Compared with the wild type, it can better feed on foods such as apples, corn, lettuce, etc., and can be used in the breeding of polyphagous silkworm varieties.

In other embodiments, the Bombyx mori olfactory receptor gene BmOR56 is knocked out, the BmOR56-knocked-out silkworms are used for silkworm breeding, and CRISPR/cas9 gene editing technology is used to knock out the cis-jasmonone receptor BmOR56 in the silkworm.

In other embodiments, the gRNA target site is designed based on the BmOR56 gene CDS sequence information, and the target site sequence is as shown in SEQ ID NO. 2-3; primers are designed according to the target site sequence and sgRNA is synthesized, and the primer sequence is as SEQ ID NO. As shown in ID NO.4 to 6; sgRNA and cas9 protein are injected into silkworm eggs; after hatching, the injected silkworm eggs are raised and the required silkworm strains are selected for silkworm breeding. Specifically, (1) use gRNA to design the website in Design 2 gRNA target sites on BmOR56 and use sgRNA in vitro synthesis kit to synthesize and purify sgRNA; (2) sgRNA is mixed with cas9 protein, and silkworm eggs are injected using the silkworm microinjection system; (3) Silkworm eggs hatch normally after injection Molecular identification is carried out to screen out positive individuals for seed production; (4) Homozygous mutants are obtained through continuous hybridization and molecular identification; (5) Homozygous mutants are used for feeding habits identification, fed with different foods, and compared with the wild type Differences between mutants; (6) Use homozygous mutants for cross breeding.

In other embodiments, silkworms with different feeding behaviors are cultivated so that the silkworms can feed on foods such as apples, corn, lettuce, cabbage, etc., thereby reducing their dependence on mulberry leaves.

In other embodiments, the method is used to breed silkworms that feed on lettuce.

In other embodiments, the method is used to breed silkworms that feed on cabbage.

In other embodiments, it is used to cultivate silkworms with different growth rates. After eating lettuce and cabbage, the weight of silkworms increases rapidly. Other genotypes can be used to breed silkworms with faster growth rates.

The following is a detailed description with specific examples.

(1) gRNA design and sgRNA synthesis

According to the BmOR56 gene CDS sequence information (SEQ ID NO.1), the gRNA target site was designed on the sgRNA design website (http://crispr.dbcls.jp), and 2 target sites (SEQ ID NO.2 and SEQ ID NO.3) are all located on the first exon of the BmOR56 gene (Figure 1A).

Design primers sgRNA_OR56_1F (SEQ ID NO.4), sgRNA_OR56_2F (SEQ ID NO.5) and sgRNA_R (SEQ ID NO.6) according to the target sequence. Refer to the EnGensgRNA synthesis kit to synthesize sgRNA in vitro. After measuring the concentration, dilute it to 1000ng/μL for later use. .

(2) Microinjection of silkworm eggs

Mix the two sgRNA and cas9 proteins in a volume ratio of 2:2:1, and put 5 μL of the mixed solution into the glass needle of the microinjection system for later use. Use non-diapause eggs that have just been laid within 2 hours, stick them on a glass slide, use a steel needle to poke a small hole in the silkworm egg, then insert the glass needle containing the mixed solution into the small hole, and inject 10-15pL solution , then the small holes are sealed with non-toxic biological glue, and after solidification, they are fumigated and disinfected with formalin solution, and then moisturized and greened.

(3) Homozygous strain screening

The injected silkworm eggs will be greened normally at 28°C. After hatching, they will be raised with fresh mulberry leaves under standard conditions (temperature 25±2°C, humidity 75±5%, 12 hours dark/12 hours day). After hatching, random mating will be carried out. After laying eggs, perform molecular identification on male and female moths respectively. Design primers BmOR56_test_F (SEQ ID NO.7) and BmOR56_test_R (SEQ IDNO.8) at both ends of the target site. Use male and female moths to extract genomic DNA. After performing PCR, perform PCR The products are sequenced, and when overlapping peaks appear, they are considered positive individuals. The silkworm eggs produced will continue to be raised, and the offspring will be randomly selfed in the area. After the eggs are laid, the silkworm moths will continue to be identified by PCR, and the hybrid combinations in which both male and female moths are positive will be selected and retained. After 2-3 generations, homozygous mutant lines can be obtained, and the mutation type can be finally confirmed through cloning and sequencing (Figure 1B).

Through screening, two mutant lines were obtained. In the mutant line M1, 2 bases were replaced by 113 bases at target site 1 (Figure 2A), and target site 2 had no change (Figure 2B). The mutant line In M2, 4 bases are deleted at target site 1 (Figure 2A), and 25 bases are deleted at target site 2 (Figure 2B). Both mutants have nonsense mutations, resulting in complete loss of gene function. (Figure 2C).

(4) Gene expression analysis and identification

After the BmOR56 gene was successfully knocked out, the expression levels of BmOR56, BmOrco and some ionotropic receptors were analyzed to further confirm that the expression levels of genes related to feeding habits have changed after BmOR56 knockout. The results showed that the expression of three types of olfactory receptors The expression was up-regulated and significantly affected (Figure 3)

(5) Identification and analysis of mutant feeding habits

When the silkworms were at the fifth instar, the two mutants and wild-type silkworms were fed with apples, pears, corn, lettuce and cabbage respectively. There were 20 silkworms in each group. After 12 hours, the feeding effect of the 20 silkworms was averaged. The weight changes of silkworms and the amount of silkworm excrement were statistically analyzed. Dissect the silkworm and check the midgut tissue to confirm that the silkworm has eaten and digested the food, and the midgut is full of corresponding food residues (Figure 4). Statistics on silkworm weight and silkworm excrement show that silkworms feeding on lettuce and cabbage have better results. The weight of wild-type silkworms increases by 0.111g/head and 0.043g/head respectively, while the weight of M1 silkworms increases by 0.241g/head and 0.114g/head respectively. M2 increased by 0.226g/head and 0.047g/head respectively. The results showed that the mutant gained faster weight after feeding on lettuce and cabbage (Figure 5A and 5B). Counting the amount of silkworm excrement, it was found that the overall amount of silkworm excrement in the mutant was more than that of the wild type. Among them, the lettuce and cabbage groups had the largest changes. The wild type had 2.47 grains/head and 2.45 grains/head respectively, and the M1 had 11.67 grains/head and 11.67 grains/head respectively. 9.75 grains/head, M2 were 5.91 grains/head and 4.05 grains/head respectively (Figure 5C).

In summary, the above analysis shows that the BmOR56 deletion mutant has a significantly better feeding effect on lettuce, cabbage, etc. than the wild type, indicating that BmOR56 can be used for silkworm breeding, especially the selection of polyphagous silkworm varieties. After the mutant feeds on lettuce and cabbage, the mutation The body weight increased faster, indicating that BmOR56 may also be used in the breeding of silkworm varieties with different growth rates.

The equipment numbers and processing scales described here are intended to simplify the description of the present invention. Applications, modifications and variations of the use of the silkworm olfactory receptor gene BmOR56 of the present invention will be apparent to those skilled in the art.

Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and embodiments. They can be applied to various fields suitable for the present invention. For those familiar with the art, they can easily Additional modifications may be made, and the invention is therefore not limited to the specific details and illustrations shown and described herein without departing from the general concept defined by the claims and equivalent scope.

SEQUENCE LISTING

<110> Guangxi Zhuang Autonomous Region Sericulture Technology Promotion Station, Sericulture Research Institute, Chinese Academy of Agricultural Sciences

<120> Use of Bombyx mori olfactory receptor gene BmOR56

<130> CN21NN13180I

<160> 8

<170> PatentIn version 3.5

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<213> BmOR56 gene

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Claims (1)

1. A method of rearing silkworms feeding lettuce and cabbage, comprising:

designing a gRNA target site according to CDS sequence information of BmOR56 genes, wherein the sequence of the target site is shown as SEQ ID NO. 2-3; designing a primer according to the target site sequence, and synthesizing sgRNA, wherein the primer sequence is shown as SEQ ID NO. 4-6; injecting two sgRNAs and cas9 proteins into silkworm eggs; after the injected silkworm eggs are hatched, the silkworm strains are selected for breeding the silkworm.