CN114921469B - Application of silkworm olfactory receptor gene BmOR56 - Google Patents
Application of silkworm olfactory receptor gene BmOR56 Download PDFInfo
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Abstract
The invention discloses application of a silkworm olfactory receptor gene BmOR56, which is used for breeding silkworms and can improve the feeding range of silkworms.
Description
Technical Field
The invention relates to the technical field related to silkworm breeding. More specifically, the invention relates to the use of a silkworm olfactory receptor gene BmOR56.
Background
Silkworm is a typical lepidopteran insect, mainly fed on mulberry leaves, and can also feed on other plants of Moraceae, compositae or Ulmaceae. Studies show that through functional analysis of olfactory receptors expressed by all larvae in xenopus oocytes, one of olfactory receptors BmOR56 is found to have high sensitivity to cis-jasmone, and cis-jasmone is one of main volatile substances of mulberry leaves, so that the mulberry larvae can be attracted specifically. Therefore, the BmOR56 gene is used for breeding silkworms, and thus the feeding range of silkworms can be improved.
Disclosure of Invention
The invention aims to provide the application of the silkworm olfactory receptor gene BmOR56, which can be used for breeding silkworms and improving the feeding range of the silkworms.
To achieve these objects and other advantages and in accordance with one aspect of the present invention, there is provided the use of the silkworm olfactory receptor gene BmOR56 for silkworm breeding.
Further, the silkworm olfactory receptor gene BmOR56 was knocked out, and the silkworm from which BmOR56 was knocked out was used for silkworm breeding.
Further, according to CDS sequence information of BmOR56 genes, a gRNA target site is designed, and 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 sgRNA and cas9 protein into silkworm eggs; after the injected silkworm eggs are hatched, the silkworm strains are selected for breeding the silkworm.
Further, the method is used for cultivating silkworms with different feeding behaviors.
Further, the method is used for cultivating silkworms which eat lettuce.
Further, the method is used for cultivating silkworms which eat cabbage.
Further, the method is used for cultivating 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 breeding silkworms and improving the feeding range of the 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 shows the mutants obtained by CRISPR/cas 9; (A) BmOR56 gene structure and gRNA site, bmOR56 gene is located on chromosome 28, there are 6 exons, black line indicates introns, rectangle indicates exons, met indicates start codon, stop indicates stop codon; the 2 gRNA target sites are all positioned 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; injecting the mixture of sgRNA and cas9 protein into silkworm eggs, identifying the genotype of the silkworm eggs after the silkworm eggs develop into moths and spawn, reserving heterozygote mutants for seed reserving, and finally screening homozygous mutants in G3 generation;
FIG. 2 shows the structural analysis of BmOR56 gene and amino acid structure analysis in the mutant line; (A) Genomic structural changes of both mutants at target number 1; (B) Genomic structural changes of both mutants at target number 2; (C) nonsense mutation of the gene in the mutant;
FIG. 3 shows the analysis of BmOR56 and related gene expression; (a) analysis of expression of BmOR56 at different developmental stages; (B) analysis of BmOrco expression at different developmental stages; (C) expression analysis of the ionic receptor; * Indicating that T-test result P <0.05, < indicating T-test result P <0.01, < indicating T-test result P <0.001;
FIG. 4 is a diagram of a mutant midgut anatomical model; feeding apple (A), pear (B), corn (C), lettuce (D) and cabbage (E) to five-year-old larvae after 12H of starvation respectively, wherein the midgut of silkworms fed for 12H is shown as apple (F), pear (G), corn (H), lettuce (I) and cabbage (J);
FIG. 5 shows the wide feeding performance detection analysis of mutant silkworms; a: weight change; b: a pattern diagram of body shape changes; c: silkworm excrement number; d: a pre-feeding state; e: a post-feeding state; and (5) statistically analyzing average data of each silkworm.
Detailed Description
The present invention is described in further detail below with reference to the drawings to enable those skilled in the art to practice the invention by referring to the description.
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 BmOR56 gene is positioned on chromosome 28, after BmOR56 is knocked out, the olfactory sensitivity of silkworms is reduced, the feeding range is enlarged to a certain extent, and the mutant can better feed foods such as apples, corns, lettuce and the like compared with the wild type, and can be applied to the breeding of wide-feeding silkworms.
In other embodiments, the knockout of the silkworm olfactory receptor gene BmOR56 is performed, the silkworm from which BmOR56 was knocked out is used for silkworm breeding, and cis-jasmone receptor BmOR56 is knocked out in the silkworm using CRISPR/cas9 gene editing technology.
In other embodiments, the gRNA target site is designed according to CDS sequence information of BmOR56 genes, and the sequence of the target site is shown in 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 sgRNA and cas9 protein into silkworm eggs; after the injected silkworm eggs are hatched, the silkworm strains are selected to be used for silkworm breeding, specifically, (1) 2 gRNA target sites are designed on BmOR56 by utilizing a gRNA design website, and the sgRNA is synthesized and purified by utilizing an sgRNA in vitro synthesis kit; (2) Mixing sgRNA with cas9 protein, and injecting silkworm eggs by using a silkworm microinjection system; (3) Normal incubation of silkworm eggs after injection and molecular identification are carried out, and positive individuals are screened out for seed production; (4) Obtaining homozygous mutant through continuous hybridization and molecular identification; (5) Homozygous mutants were used for feeding identification, fed with different foods, comparing differences between wild type and mutant; (6) homozygous mutants are used for crossbreeding.
In other embodiments, the method is used for cultivating silkworms with different feeding behaviors, and the silkworms can feed foods such as apples, corns, lettuce and cabbages after cultivation, so that dependence on mulberry leaves is reduced.
In other embodiments, the method is used to cultivate silkworms that feed lettuce.
In other embodiments, the method is used to cultivate silkworms feeding on cabbage.
In other embodiments, the method is used for cultivating silkworms with different growth speeds, and after lettuce and cabbage are eaten, the silkworms grow fast in weight and can be matched with other genotypes to cultivate silkworms with higher growth speeds.
Specific examples are described below.
(1) gRNA design and synthesis of sgRNA
Based on the CDS sequence information of the BmOR56 gene (SEQ ID NO. 1), 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 each 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 according to the target sequence, and the sgRNA was synthesized separately with reference to the EnGensgRNA synthesis kit, and diluted to 1000 ng/. Mu.L for use after measuring the concentration.
(2) Silkworm egg microinjection
2 sgRNAs and cas9 proteins are mixed according to the volume ratio of 2:2:1, and 5 mu L of the mixed solution is taken into a glass needle of a microinjection system for standby. Sticking non-diapause eggs within 2 hours after the spawning on a glass slide, pricking a small hole on the silkworm eggs by using a steel needle, penetrating the glass needle filled with the mixed solution into the small hole, injecting 10-15pL of solution, sealing the small hole by using nontoxic biological glue, fumigating and sterilizing by using formalin solution after solidification, and moisturizing and hastening green.
(3) Homozygous line selection
Normal hatching of injected silkworm eggs at 28 ℃, feeding fresh mulberry leaves under standard conditions (temperature 25+/-2 ℃, humidity 75+/-5%, darkness for 12 hours/daytime), carrying out random mating after moth formation, carrying out molecular identification on male and female moths respectively after spawning, designing primers BmOR56_test_F (SEQ ID NO. 7) and BmOR56_test_R (SEQ ID NO. 8) at two ends of a target site, extracting genome DNA by male and female moths, sequencing PCR products after PCR, carrying out positive individuals when overlapping peaks appear, continuously feeding silkworm eggs produced, carrying out random selfing in a offspring region, carrying out PCR identification on the silkworm moths after spawning, selecting hybrid combination seed retaining that male and female moths are positive, obtaining a homozygous mutant strain, and carrying out final confirmation on the mutation type by a clone sequencing mode (figure 1B).
By screening, 2 mutant lines were obtained in which 2 bases in mutant line M1 were replaced by 113 bases at target site 1 (fig. 2A), target site 2 was unchanged (fig. 2B), 4 bases in mutant line M2 were deleted at target site 1 (fig. 2A), 25 bases in target site 2 (fig. 2B), and nonsense mutations were 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, the expression quantity analysis is carried out on BmOR56, bmOrco and partial ion receptor to further confirm that the expression quantity of the gene related to feeding habit after the BmOR56 is knocked out is changed, and the result shows that the expression quantity of the 3-class olfactory receptor is up-regulated and is obviously influenced (figure 3)
(5) Mutant feeding habit identification and analysis
When the silkworms are raised at the age of 5 years, 2 mutants and wild silkworms are respectively fed with apples, pears, corns, lettuce and cabbages, each group of silkworms is 20 silkworms, and the feeding effect, the weight change of the silkworms and the silkworm excrement quantity are counted and analyzed by using the average value of 20 silkworms after 12 hours. The silkworms were dissected, the midgut tissue was checked, and it was confirmed that the silkworms had ingested and had digested the food, and the midgut was filled with the corresponding food residues (fig. 4). By counting the weight of silkworms and silkworm excrement, the silkworm effect of feeding lettuce and cabbage is better, the weight of wild silkworms is increased by 0.111 g/head and 0.043 g/head respectively, M1 is increased by 0.241 g/head and 0.114 g/head respectively, and M2 is increased by 0.226 g/head and 0.047 g/head respectively, and the result shows that the weight increase of the mutant is faster after feeding lettuce and cabbage (figures 5A and 5B). The silkworm excrement amount was counted, and it was found that the mutant was more overall than the wild type, in which feeding lettuce and cabbage groups were most varied, with wild type being 2.47 and 2.45 pieces/head, respectively, and M1 being 11.67 and 9.75 pieces/head, respectively, and M2 being 5.91 and 4.05 pieces/head, respectively (FIG. 5C).
In conclusion, the BmOR56 deletion mutant has obviously better feeding effect on lettuce, cabbage and the like than wild type, which shows that BmOR56 can be used for breeding silkworms, particularly for breeding wide-feeding silkworms, and after the mutant feeds lettuce and cabbage, the weight of the mutant increases more rapidly, which shows that BmOR56 can also be used for breeding silkworms with different growth rates.
The number of equipment and the scale of processing described herein are intended to simplify the description of the present invention. The use, modification and variation of the use of the silkworm olfactory receptor gene BmOR56 of the present invention will be apparent to those skilled in the art.
Although embodiments of the present invention have been disclosed above, it is not limited to the details and embodiments shown and described, it is well suited to various fields of use for which the invention would be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the specific details and illustrations shown and described herein, without departing from the general concepts defined in the claims and their equivalents.
SEQUENCE LISTING
<110> Guangxi Zhuang nationality silkworm industry technical popularization station, china agricultural science college silkworm industry institute
<120> use of silkworm olfactory receptor gene BmOR56
<130> CN21NN13180I
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 1206
<212> DNA
<213> BmOR56 Gene
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gtatctcgag ctgatttaga gcaaagggcc acttcggact cgagaaagtt ggctctgata 360
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gcctggtggt cgagagatgt gaagtaccgc aaaaatttat atatattggt ggcgcagttt 1080
aacaaggtta ttgtcttttc cgcgggcccc ttcactaagc ttacggtcgc cacttttatc 1140
aggatcctca aaggagccta tagttactac acactgctca gtcagtcgca aatgaataaa 1200
acatga 1206
<210> 2
<211> 23
<212> DNA
<213> target site 1
<400> 2
ggtcctagta aaggacgatc ggg 23
<210> 3
<211> 23
<212> DNA
<213> target site 2
<400> 3
ggtcctagta aaggacgatc ggg 23
<210> 4
<211> 55
<212> DNA
<213> sgRNA_OR56_1F
<400> 4
ttctaatacg actcactata gggtcctagt aaaggacgat cgttttagag ctaga 55
<210> 5
<211> 55
<212> DNA
<213> sgRNA_OR56_2F
<400> 5
ttctaatacg actcactata gaatgcctgg cgtgacctca tgttttagag ctaga 55
<210> 6
<211> 80
<212> DNA
<213> sgRNA_R
<400> 6
gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 60
ggcaccgagt cggtgctttt 80
<210> 7
<211> 21
<212> DNA
<213> BmOR56_test_F
<400> 7
atacaatgga gcttgattcg t 21
<210> 8
<211> 23
<212> DNA
<213> BmOR56_test_R
<400> 8
ggtcgtatac atcaagaacc agt 23
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.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016076240A1 (en) * | 2014-11-14 | 2016-05-19 | 国立研究開発法人農業生物資源研究所 | Female silkworm lethal strain of bombyx mori |
| JP2017085958A (en) * | 2015-11-09 | 2017-05-25 | 国立研究開発法人農業・食品産業技術総合研究機構 | Method for producing genetically modified silkworm |
| WO2018062201A1 (en) * | 2016-09-29 | 2018-04-05 | 住友化学株式会社 | Odorant receptor complex and cells expressing same |
| CN108795933A (en) * | 2017-04-27 | 2018-11-13 | 中国科学院上海生命科学研究院 | A kind of method and its application changing silkworm feeding habits |
| CN109804067A (en) * | 2016-09-29 | 2019-05-24 | 住友化学株式会社 | Olfactory receptor coreceptor |
| WO2022024902A1 (en) * | 2020-07-27 | 2022-02-03 | 住友化学株式会社 | Mutant insect olfactory receptor protein |
-
2022
- 2022-04-12 CN CN202210380688.5A patent/CN114921469B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016076240A1 (en) * | 2014-11-14 | 2016-05-19 | 国立研究開発法人農業生物資源研究所 | Female silkworm lethal strain of bombyx mori |
| JP2017085958A (en) * | 2015-11-09 | 2017-05-25 | 国立研究開発法人農業・食品産業技術総合研究機構 | Method for producing genetically modified silkworm |
| WO2018062201A1 (en) * | 2016-09-29 | 2018-04-05 | 住友化学株式会社 | Odorant receptor complex and cells expressing same |
| CN109804067A (en) * | 2016-09-29 | 2019-05-24 | 住友化学株式会社 | Olfactory receptor coreceptor |
| CN108795933A (en) * | 2017-04-27 | 2018-11-13 | 中国科学院上海生命科学研究院 | A kind of method and its application changing silkworm feeding habits |
| WO2022024902A1 (en) * | 2020-07-27 | 2022-02-03 | 住友化学株式会社 | Mutant insect olfactory receptor protein |
Non-Patent Citations (4)
| Title |
|---|
| Song WenTing等.The molecular mechanisms and factors affecting the feeding habits of silkworm (Lepidoptera: Bombyxidae).Journal of Asia-Pacific Entomology.2021,第24卷第955-962页. * |
| 刘群.家蚕嗅觉相关基因的功能研究.博士电子期刊出版库.2019,(第05期),第1-147页. * |
| 家蚕嗅觉相关基因的功能研究;刘群;博士电子期刊出版库(第05期);第1-147页 * |
| 稚蚕摄食人工饲料的家蚕种质资源筛选及实用品种选育;贾晓虎;硕士电子期刊出版库(第05期);1-50 * |
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| CN114921469A (en) | 2022-08-19 |
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