CN114921469A

CN114921469A - Application of bombyx mori olfactory receptor gene BmOR56

Info

Publication number: CN114921469A
Application number: CN202210380688.5A
Authority: CN
Inventors: 王平阳; 赵巧玲; 李聪; 刘艳伟; 张桂征; 闭立辉; 蒋满贵; 潘志新; 韦伟; 崔秋英; 张雨丽; 王霞; 黄旭华
Original assignee: Sericultural Research Institute Chinese Academy Of Agricultural Sciences; Guangxi Zhuang Autonomous Region Sericulture Technology Promotion Master Station
Current assignee: Sericultural Research Institute Chinese Academy Of Agricultural Sciences; Guangxi Zhuang Autonomous Region Sericulture Technology Promotion Master Station
Priority date: 2022-04-12
Filing date: 2022-04-12
Publication date: 2022-08-19
Anticipated expiration: 2042-04-12
Also published as: CN114921469B

Abstract

The invention discloses application of a silkworm olfactory receptor gene BmOR56, which is used for silkworm breeding and can improve the feeding range of silkworms.

Description

Application of bombyx mori olfactory receptor gene BmOR56

Technical Field

The invention relates to the technical field related to silkworm breeding. More particularly, the invention relates to an application of bombyx mori olfactory receptor gene BmOR 56.

Background

Silkworm is a typical lepidopteran insect, and mainly feeds mulberry leaves, and can also feed other plants of Moraceae, Compositae or Ulmaceae. Research shows that through functional analysis of olfactory receptors expressed by all larvae in Xenopus laevis oocytes, one of the olfactory receptors BmOR56 has high sensitivity to cis-jasmone, which is one of the main volatile substances of mulberry leaves and can specifically attract silkworm larvae. Therefore, the BmOR56 gene is used for breeding silkworms, and the feeding range of the silkworms can be improved.

Disclosure of Invention

The invention aims to provide application of a silkworm olfactory receptor gene BmOR56, which can be used for silkworm breeding and can improve the feeding range of silkworms.

To achieve these objects and other advantages of the present invention, according to one aspect of the present invention, there is provided a use of a silkworm olfactory receptor gene BmOR56 for silkworm breeding.

Further, a knockout of the silkworm olfactory receptor gene BmOR56 was performed, and silkworms with the BmOR56 knocked out were used for silkworm breeding.

Further, designing a gRNA target site according to CDS sequence information of the BmOR56 gene, wherein the sequence of the target site is shown as SEQ ID NO. 2-3; designing a primer according to a target site sequence and synthesizing sgRNA, wherein the primer sequence is shown in SEQ ID NO. 4-6; injecting the sgRNA and the cas9 protein into silkworm eggs; after the silkworm eggs are injected for incubation, the required silkworm strain is screened out for silkworm breeding.

Further, the method is used for breeding silkworms with different feeding behaviors.

Further, the method is used for cultivating the silkworms which take the lettuce.

Further, the method is used for breeding silkworms for eating cabbage.

Further, the method is used for breeding silkworms with different growth speeds.

The invention at least comprises the following beneficial effects:

the invention provides application of a silkworm olfactory receptor gene BmOR56, which can be used for silkworm breeding and can improve the feeding range of silkworms.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

Fig. 1 is a mutant obtained by CRISPR/cas 9; (A) the BmOR56 gene structure and gRNA locus, wherein the BmOR56 gene is positioned on the 28 th chromosome, 6 exons are arranged, a black line represents an intron, a rectangle represents an exon, Met represents a start codon, and stop represents a stop codon; 2 gRNA target sites are all positioned on the first exon, a red background sequence is a gRNA target sequence, and a blue background sequence is PAM (promoter ad jacent motif); (B) generation of homozygous mutants; injecting a mixture of sgRNA and cas9 protein into silkworm eggs, identifying the genotype of the silkworm eggs after the silkworm eggs develop into moths and lay eggs, reserving heterozygote mutants for seed reservation, and finally screening out homozygous mutants in G3 generations;

FIG. 2 is a structural analysis of BmOR56 gene and an amino acid structure analysis in a mutant line; (A) the genome structure of the two mutants at the No.1 target point is changed; (B) the genome structure of the two mutants at the No.2 target point is changed; (C) nonsense mutation occurs in the gene in the mutant;

FIG. 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) analysis of the expression of ionotropic receptors; denotes T test result P <0.05, denotes T test result P <0.01, denotes T test result P < 0.001;

FIG. 4 is a plot of the anatomical pattern of the mutant midgut; feeding apples (A), pears (B), corns (C), lettuce (D) and cabbages (E) to five-instar larvae after starvation for 12H respectively, wherein the midgut of the silkworm fed for 12H is shown as apples (F), pears (G), corns (H), lettuce (I) and cabbages (J);

FIG. 5 is an analysis of the mutant bombyx mori euryphagous test; a: a change in body weight; b: a figure change pattern diagram; c: the number of silkworm excrement; d: pre-feeding state; e: after the addition of food; the average data of each silkworm was statistically analyzed.

Detailed Description

The present invention is described in further detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.

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

The embodiment of the application provides the application of the silkworm olfactory receptor gene BmOR56, which is used for silkworm breeding; the gene BmOR56 is located on the 28 th chromosome, after BmOR56 is knocked out, the olfactory sensitivity of the silkworm is reduced, the feeding range is expanded to a certain extent, and compared with a wild type, the mutant can better eat foods such as apples, corns, lettuce and the like, and can be applied to breeding of euryphagous silkworm varieties.

In other embodiments, the knockout of bombyx mori olfactory receptor gene BmOR56 is performed, bombyx mori with BmOR56 knocked out is used for silkworm breeding, and cis jasmone receptor BmOR56 is knocked out in bombyx mori using CRISPR/cas9 gene editing techniques.

In other embodiments, a gRNA target site is designed according to CDS sequence information of a BmOR56 gene, and the sequence of the target site is shown as SEQ ID NO. 2-3; designing a primer according to a target site sequence and synthesizing sgRNA, wherein the primer sequence is shown in SEQ ID NO. 4-6; injecting the sgRNA and the cas9 protein into silkworm eggs; after the silkworm eggs are injected for hatching, required silkworm strains are screened out for silkworm breeding, specifically, (1) 2 gRNA target sites are designed on BmOR56 by utilizing a gRNA design website, and sgRNA is synthesized and purified by utilizing a sgRNA in-vitro synthesis kit; (2) mixing the sgRNA and cas9 protein, and injecting silkworm eggs by using a silkworm microinjection system; (3) normally hatching the silkworm eggs after injection, performing molecular identification, and screening out positive individuals for seed production; (4) obtaining homozygous mutant through continuous hybridization and molecular identification; (5) homozygous mutants were used for feeding sex identification, fed with different foods, and compared for differences between wild type and mutant; (6) homozygous mutants were used for cross breeding.

In other embodiments, the silkworm breeding method is used for breeding silkworms with different feeding behaviors, and the silkworms can feed foods such as apples, corns, lettuce, cabbages and the like through breeding, so that the dependence on mulberry leaves is reduced.

In other embodiments, the method is used for breeding silkworms for eating lettuce.

In other embodiments, the silkworm is cultivated for feeding cabbage.

In other embodiments, the silkworm breeding method is used for breeding silkworms with different growth speeds, after the silkworms are fed with lettuce and cabbage, the weight of the silkworms can be increased quickly, and the silkworm breeding method can be matched with other genotypes to breed silkworms with higher growth speeds.

The following examples are given for illustrative purposes.

(1) gRNA design and synthesis of sgRNAs

Based on the CDS sequence information (SEQ ID NO.1) of the BmOR56 gene, a 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) were all on the first exon of the BmOR56 gene (FIG. 1A).

Primers sgRNA _ OR56_1F (SEQ ID No.4), sgRNA _ OR56_2F (SEQ ID No.5) and sgRNA _ R (SEQ ID No.6) were designed based on the target sequence, sgrnas were synthesized in vitro with reference to the engsgrna synthesis kit, respectively, and the concentrations were determined and diluted to 1000 ng/. mu.l for use.

(2) Microinjection of silkworm eggs

2 sgRNA and cas9 protein are mixed according to the volume ratio of 2:2:1, and 5 mu L of mixed solution is put into a glass needle of a microinjection system for standby. Sticking the non-diapause eggs which are just laid for 2 hours on a glass slide, pricking a small hole on the silkworm eggs by using a steel needle, then inserting the glass needle containing the mixed solution into the small hole, injecting a 10-15pL solution, then sealing the small hole by using non-toxic biological glue, fumigating and disinfecting by using a formalin solution after solidification, and then preserving moisture and hastening the youth.

(3) Homozygous line screening

Normally hatching the silkworm eggs at 28 ℃, breeding the silkworm eggs by using fresh mulberry leaves under standard conditions (the temperature is 25 +/-2 ℃, the humidity is 75 +/-5 percent, and the time is 12h dark/12 h day) after hatching, randomly mating the silkworm eggs after moth formation, respectively performing molecular identification on male and female moths after spawning, primers BmOR56_ test _ F (SEQ ID NO.7) and BmOR56_ test _ R (SEQ ID NO.8) were designed at both ends of the target site, genomic DNA was extracted from male and female moths, and after PCR, sequencing the PCR product, namely, obtaining a positive individual when an overlapping peak appears, continuously feeding the produced silkworm eggs, randomly selfing the offspring, continuously performing PCR identification on the silkworm moths after spawning, selecting a hybridization combination with positive male moths for seed reservation, obtaining a homozygous mutant strain after 2-3 generations, and finally confirming the mutation type by a cloning sequencing mode (figure 1B).

By screening, 2 mutant lines were obtained in which 2 bases at target site 1 were replaced with 113 bases in mutant line M1 (fig. 2A), target site 2 was not changed (fig. 2B), 4 bases at target site 1 were deleted in mutant line M2 (fig. 2A), 25 bases at target site 2 were deleted (fig. 2B), and nonsense mutations occurred in both mutants, resulting in complete loss of gene function (fig. 2C).

(4) Analysis and identification of gene expression level

After the BmOR56 gene is successfully knocked out, expression level analysis is carried out on BmOR56, BmOrco and partial ion type receptors to further confirm that the expression level of the food-related gene is changed after the BmOR56 is knocked out, and the results show that the expression level of 3 types of olfactory receptors is up-regulated and is significantly influenced (figure 3)

(5) Identification and analysis of mutant feeding habits

When the 5 th silkworm comes up, 2 mutants and wild silkworms are fed with apples, pears, corns, lettuce and cabbages respectively, 20 silkworms in each group are fed, and the feeding effect, the silkworm body weight change and the silkworm excrement amount are counted and analyzed by the average value of 20 silkworms after 12 hours. The silkworms were dissected and the midgut tissue was examined to confirm that the silkworms had eaten and had digested food, and the midgut was filled with the corresponding food debris (fig. 4). Through statistics on the weight of the silkworm and the silkworm excrement, the effect of the silkworm eating lettuce and cabbage is better, the weight of the wild silkworm is respectively increased by 0.111 g/head and 0.043 g/head, M1 is respectively increased by 0.241 g/head and 0.114 g/head, and M2 is respectively increased by 0.226 g/head and 0.047 g/head, and the result shows that the weight of the mutant eating lettuce and cabbage is increased more quickly (figures 5A and 5B). The silkworm excrement amount is counted, and the silkworm excrement amount of the mutant is found to be more than that of the wild type, wherein the change of the lettuce eating group and the cabbage eating group is the largest, the wild type is 2.47 grains/head and 2.45 grains/head respectively, the M1 grains/head is 11.67 grains/head and 9.75 grains/head respectively, and the M2 grains/head is 5.91 grains/head and 4.05 grains/head respectively (figure 5C).

In conclusion, the BmOR56 deletion mutant has obviously better feeding effect on lettuce, cabbage and the like than the wild type, which indicates that the BmOR56 can be used for silkworm breeding, particularly for breeding euryphagous silkworm varieties, and the weight of the mutant is increased more quickly after the mutant feeds the lettuce and the cabbage, which indicates that the BmOR56 can also be used for breeding silkworm varieties with different growth speeds.

The number of apparatuses and the scale of the process described herein are intended to simplify the description of the present invention. Applications, modifications and variations of the use of the bombyx mori olfactory receptor gene BmOR56 of the present invention will be apparent to those skilled in the art.

While embodiments of the invention have been described above, it is not intended to be limited to the details shown, described and illustrated herein, but is to be accorded the widest scope consistent with the principles and novel features herein disclosed, and to such extent that such modifications are readily available to those skilled in the art, and it is not intended to be limited to the details shown and described herein without departing from the general concept as defined by the appended claims and their equivalents.

SEQUENCE LISTING

<110> Guangxi Zhuang nationality autonomous region silkworm industry technology promotion station, and research institute of silkworm industry of Chinese academy of agricultural sciences

Application of <120> bombyx mori olfactory receptor gene BmOR56

<130> CN21NN13180I

<160> 8

<170> PatentIn version 3.5

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Claims

1. The application of silkworm olfactory receptor gene BmOR56 is characterized in that the gene is used for silkworm breeding.

2. The use of the silkworm olfactory receptor gene BmOR56 according to claim 1, wherein a knock-out is performed on the silkworm olfactory receptor gene BmOR56, and silkworms with the BmOR56 knocked-out are used for silkworm breeding.

3. The use of bombyx mori olfactory receptor gene BmOR56 according to claim 2, wherein a gRNA target site is designed according to the CDS sequence information of BmOR56 gene, and the target site sequence is shown in SEQ ID No. 2-3; designing a primer according to a target site sequence and synthesizing sgRNA, wherein the primer sequence is shown in SEQ ID NO. 4-6; the sgRNA and the cas9 protein are injected into silkworm eggs; after the silkworm eggs are injected and incubated, the required silkworm strain is screened out for silkworm breeding.

4. The use of the silkworm olfactory receptor gene BmOR56 as claimed in claim 1 for breeding silkworms of different feeding behaviors.

5. The use of the silkworm olfactory receptor gene BmOR56 as defined in claim 4 for breeding silkworms fed with lettuce.

6. The use of the silkworm olfactory receptor gene BmOR56 as defined in claim 4 for breeding silkworms that feed on cabbage.

7. The use of the silkworm olfactory receptor gene BmOR56 as defined in claim 1 for breeding silkworms of different growth rates.