Bombyx mori BmOBP6 gene and application of bombyx mori BmOBP6 gene in screening artificial feed high-intake variety

Abstract

The invention discloses a bombyx mori BmOBP6 gene and application of the bombyx mori BmOBP6 gene in screening of artificial feed high-intake varieties. The nucleotide sequence of the BmOBP6 gene is shown as SEQ ID NO.2. The invention further discloses a preparation method of the bombyx mori BmOBP6 gene. Compared with the prior art, the method has the advantages that when a large number of breeding materials are screened, the artificial feed intake of the breeding materials can be effectively judged according to the expression level of the BmOBP6 gene in the newly-hatched silkworm period, feed screening is not needed, the test period is shortened, and the workload is reduced; and the transcription level of the screened bombyx mori BmOBP6 gene is matched with the 24-hour hair thinning rate detected by an artificial feed containing 30% of mulberry leaf powder. And (2) when the BmOBP6 gene is high in expression quantity and the artificial feed is fed for 24h, the hair thinning rate of the medium line is greater than 85%, the hair thinning rate of the Japanese line is greater than 90%, and the assembled hybrid combination meets the standard requirement that the 24h hair thinning rate of the silkworm breed bred by the artificial feed is greater than 95%.

Description

Technical Field

[0001] The present invention belongs to the technical field of silkworm molecular breeding, and relates to the selection of silkworm varieties with high artificial feed intake by genetic engineering means, specifically the silkworm BmOBP6 gene and its application in screening varieties with high artificial feed intake. Background Art

[0002] Silkworms, or Bombyx mori, are oligophagous insects, and mulberry leaves are their preferred food source, a result of their co-evolution with the mulberry tree. However, since the freshness of mulberry leaves is significantly affected by factors such as region and season, pesticide contamination is also common, severely restricting the timely availability of sericulture. Artificial diet sericulture has become an important method for achieving high-quality silkworm rearing year-round. High artificial diet intake is a prerequisite and foundation for factory-based artificial diet sericulture. Artificial diet intake is controlled by multiple genes and varies significantly between strains and individuals. Currently, the selection of silkworm strains adapted to artificial diets is primarily conducted through multi-generational screening, which is a large-scale and time-consuming process. Different feed formulations significantly affect feeding efficiency and lack stability, hindering accurate assessment of a strain's feeding efficiency.

[0003] Insect feeding habits are closely linked to their olfactory system. Odorant-binding proteins (OBPs) are crucial for the proper functioning of the olfactory system, enhancing the intensity and sensitivity of the olfactory system's response to odorants. BmOBP6 is the most highly expressed member of the OBP family and serves as a receptor for β-sitosterol, a volatile compound found in mulberry leaves. However, the function of the BmOBP6 gene and its expressed protein in silkworms has not been previously reported. Summary of the Invention

[0004] Technical Problem Solved: To overcome the shortcomings of the existing technology, the present invention analyzes the correlation at the gene level based on the principle that odor-binding proteins in the olfactory system are closely related to the feeding habits of insects. The BmOBP6 gene, which has the highest expression in silkworms, is used as a detection indicator. Furthermore, based on the characteristic that the expression of the BmOBP6 gene decreases with the growth and development of silkworms, newly hatched silkworms are selected as detection targets. The artificial feed feeding habits of different varieties are preliminarily determined based on the transcription levels of the BmOBP6 gene, thus shortening the breeding material screening time and improving breeding efficiency. In view of this, the present invention provides the silkworm BmOBP6 gene and its application in screening varieties with high artificial feed feeding habits.

[0005] Technical solution: Bombyx mori BmOBP6 gene, the amino acid sequence of the BmOBP6 gene is shown in SEQ ID NO.1.

[0006] SEQ ID NO.1:

[0007] MSIKWRHIERVGSFCYLGSIVDDRGGTEADIAARINKARAAFSQLRPVWSSSTLTRRTKALT

[0008] EEQKAEITKSSLPLIAECSKEFSVNQGDIDAAKKLGDPSGLNSCFVGCFMKKAGIINASGLF

[0009] DVAATIEKSKKYLTSEEDLKAFEKLTETCAPENDKPVSDSDKGCERAKLLLDCFVANKGSNTRIVALSKDLDEEAEDRAQWLAIGEAYVQQ。

[0010] Preferably, the nucleotide sequence of the BmOBP6 gene is as shown in SEQ ID NO.2.

[0011] SEQ ID NO.2:

[0012] ATGTCGATCAAATGGAGGCACATCGAGCGGGTGGGAAGCTTCTGTTACCTGGGCAGCA

[0013] TTGTGGACGATAGGGGAGGCACCGAGGCAGACATTGCGGCTCGTATAAATAAAGCCA

[0014] GAGCCCTTTTAGTCAACTTAGACCAGTGTGGAGTTCCTCAACCTTAACAAGACGAACA

[0015] AAGGCGCTAACAGAAGAACAAAAGGCCGAGATAACGAAATCATCATTGCCTCTAATA

[0016] GCGGAATGCTCCAAAGAGTTCAGCGTGAATCAGGGCGACATTGACGCGGCTAAGAAA

[0017] CTTGGTGACCCGAGCGGTTTGAACTCTTGCTTTGTAGGATGTTTCATGAAGAAAGCTGG

[0018] AATCATCAACGCAAGTGGTCTTTTCGATGTAGCCGCTACTATTGAGAAGAGCAAGAAA

[0019] TATCTTACCAGCGAGGAGGACTTGAAAGCCTTCGAGAAACTAACGGAGACGTGCGCTC

[0020] CAGAAAACGACAAACCAGTCAGTGACAGTGACAAGGGCTGCGAAAGAGCAAAACTTC

[0021] TCCTCGACTGTTTCGTTGCAAACAAAGGAAGTAATACACGGATAGTAGCACTCAGCAA

[0022] GGATCTGGATGAGGAAGCCGAGGATCGTGCTCAATGGCTTGCAATTGGAGAGGCTTATGTCCAACAGTAG.

[0023] The application of the silkworm BmOBP6 gene described above in screening silkworm varieties with high feeding ability on artificial feed.

[0024] Preferably, the screening method comprises the following steps:

[0025] S1. Select Chinese and Japanese silkworm varieties, raise them on mulberry leaves until they form cocoons, and then self-pollinate to produce seeds after moths emerge;

[0026] S2, taking the silkworm eggs prepared in S1, relieving them from diapause and accelerating them to hatch, and using the silkworms as test materials;

[0027] S3. Total RNA was extracted from S2 silkworms and reverse transcribed into cDNA. Primers were designed based on the full-length sequence of the BmOBP6 gene. The transcription level of the BmOBP6 gene in each silkworm variety was detected by qRT-PCR using the cDNA as a template. The BmGAPDH gene was used as an internal reference gene.

[0028] S4. Screening silkworm varieties with a relative expression level of 2^(-ΔCt)≧1 in S3, i.e., silkworm varieties with high artificial diet ingestion.

[0029] The method for relieving silkworm egg diapause adopts a refrigeration method or a pickling method, and the incubation is a simplified two-stage incubation method.

[0030] Preferably, in S1, the selected Chinese silkworm varieties include: Xiafang, Ganxia, ​​Qun S, 8bai S, Ying S, Jing S, and Lu S; the Japanese silkworm varieties include: Hao S, Chun S, and Fu S.

[0031] Preferably, in S3, the upstream primer sequence for qRT-PCR of the silkworm BmOBP6 gene is shown in SEQ ID NO.3, and the downstream primer sequence is shown in SEQ ID NO.4; the upstream primer sequence for the internal reference gene BmGAPDH is shown in SEQ ID NO.5, and the downstream primer sequence is shown in SEQ ID NO.6. Specifically:

[0032] SEQ ID NO.3: 5'-CATCAACGCAAGTGGTCTA-3';

[0033] SEQ ID NO.4: 5'-CGTCTCCGTTAGTTTCTCG-3';

[0034] SEQ ID NO.5: 5'-TTCATGCCACAACTGCTACA-3';

[0035] SEQ ID NO. 6: 5'-AGTCAGCTTGCCATTAAGAG-3'.

[0036] Preferably, the silkworm varieties with high artificial feed intake obtained by screening in S4 are used as parents, and artificial feed is continuously screened and subcultured to improve their adaptability and stability in artificial feed intake.

[0037] Preferably, the silkworm varieties with high artificial feed intake obtained by screening in S4 are used as parents, and hybrid combinations are formed in the Chinese·Chinese×Japanese·Japanese manner, and the combinations with a 24h hair thinning rate greater than 95% are used as the varieties with high artificial feed intake for production applications.

[0038] Preferably, in S4, the verification standard is that the hair thinning rate of Chinese varieties after consuming artificial feed for 24 hours is greater than 85% and that of Japanese varieties is greater than 90%, and the selected varieties are consistent with the verification results. The artificial feed contains 30% mulberry leaf powder and is purchased from Chongqing Zhengjia Feed Co., Ltd. or Hai'an Xinyuan Agricultural Development Co., Ltd. for young silkworms.

[0039] The principle behind the method described in this invention is that insect feeding habits are inextricably linked to their olfactory systems. Odor-binding proteins (OBPs) are crucial for the proper functioning of the olfactory system, enhancing the intensity and sensitivity of the olfactory system's response to odorants. Previous research by our research group has shown that BmOBP6 is the most highly expressed member of the OBP family and acts as a receptor for the volatile compound β-sitosterol from mulberry leaves. This suggests that BmOBP6 is closely linked to silkworm feeding habits. β-sitosterol is an essential food attractant in silkworm artificial diets. The method described in this invention exploits the fact that different silkworm species exhibit varying levels of BmOBP6 gene expression in response to artificial diets. Furthermore, as silkworms grow and develop, their dependence on these attractants decreases, and BmOBP6 gene expression also decreases. Therefore, newly hatched silkworms were selected for testing, as BmOBP6 gene expression is not induced by food. The transcriptional levels of BmOBP6 in different species accurately reflect their artificial diet intake. The existing method for determining a variety's feeding ability by measuring the 24-hour hair thinning rate after artificial diet feeding confirmed that the transcription level of the BmOBP6 gene in silkworm varieties correlated with the 24-hour hair thinning rate measured on artificial diets containing 30% mulberry leaf powder. Specifically, varieties with high BmOBP6 gene expression had a 24-hour hair thinning rate greater than 85% for medium silkworms and greater than 90% for Japanese silkworms. Therefore, the method described in the present invention can be used as an effective method for the targeted breeding of silkworm varieties with high artificial diet feeding ability.

[0040] Beneficial Effects: (1) When screening a large number of breeding materials, the method of the present invention can effectively determine the artificial feed intake of breeding materials by using the expression level of the BmOBP6 gene during the larval stage, eliminating the need for feed screening, shortening the test period, and reducing the workload. The transcription level of the BmOBP6 gene in the selected silkworm varieties is consistent with the 24-hour hair thinning rate detected by artificial feed containing 30% mulberry leaf powder. (2) Varieties with high BmOBP6 gene expression have a 24-hour hair thinning rate of greater than 85% for medium-sized varieties and greater than 90% for Japanese-sized varieties. The resulting hybrid combinations meet the standard requirement of a 24-hour hair thinning rate of greater than 95% for silkworm varieties fed artificial feed. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is the relative expression level of BmOBP6 gene in different species of ant silkworm stages;

[0042] Figure 2 It is the result of 24-hour hair thinning rate of different varieties of artificial feed. DETAILED DESCRIPTION

[0043] The following examples further illustrate the present invention but are not to be construed as limiting the present invention. Modifications and substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance of the present invention are intended to fall within the scope of the present invention. Unless otherwise specified, the techniques used in the examples are conventional means well known to those skilled in the art.

[0044] Example 1

[0045] qRT-PCR was used to detect the differences in the transcription levels of the BmOBP6 gene in various silkworm varieties, which was used as an indicator of the silkworm's feeding habits on artificial diets. The following steps were involved:

[0046] Total RNA was extracted from various silkworm varieties and reverse transcribed to obtain cDNA. The reverse transcribed cDNA was used as a template and specific primers were designed based on the Bombyx mori BmOBP6 gene sequence published in the NCBI database. The forward primer F: 5'-CATCAACGCAAGTGGTCTA-3' and the reverse primer R: 5'-CGTCTCCGTTAGTTTCTCG-3' were used to amplify the CDS sequence of the BmOBP6 gene, as shown in SEQ ID NO. 2. The Bombyx mori BmGAPDH gene was used as an internal reference gene. The upstream primer sequence of the internal reference gene was F: 5'-TTCATGCCACAACTGCTACA-3' and the downstream primer sequence was R: 5'-AGTCAGCTTGCCATTAAGAG-3'. The transcription levels of the BmOBP6 gene were different in the Bombyx mori varieties Xiafang, Ganxia, ​​Qun S, 8bai S, Ying S, Jing S, and Lu S and the Japanese varieties Hao S, Chun S, and Fu S (see attached). Figure 1 ), among which the relative expression of BmOBP6 gene of Chinese silkworm varieties Green S, Qun S and Japanese silkworm varieties Spring S, Fu S, Hao S was higher, i.e. 2^(-ΔCt)≧1, and they can be used as alternative materials for breeding silkworms with high feeding efficiency. At the same time, they were fed with artificial diet containing 30% mulberry leaf powder for 24 hours, and their hair sparseness rate was calculated (see attached). Figure 2 ), Fu S was 97%, Chun S was 92%, Hao S was 92%, Lu S was 98%, Ying S was 80%, Jing S was 80%, Ba Bai S was 90%, Qun S was 85%, Gan Xia was 60%, and Xia Fang was 5%. The positive judgment results of the detection method described in this example on the feeding habit of silkworms on artificial diets were verified by the production standard of 24-hour hair thinning rate, and all of them were high-feeding silkworm varieties on artificial diets. Therefore, the difference in the transcription level of the BmOBP6 gene in various silkworm varieties detected by qRT-PCR can be used as an indicator of the feeding habit of silkworms on artificial diets.

[0047] Example 2

[0048] A method for screening silkworm varieties with high artificial feed intake is used to utilize the BmOBP6 gene, and hybrid combinations are formed to cultivate practical varieties with high artificial feed intake.

[0049] According to the results of Example 1, the expression levels of the BmOBP6 gene in the Chinese silkworm varieties Green S, Group S and Japanese silkworm varieties Spring S, Fu S, and Hao S were higher. The artificial feed 24-hour hair thinning rate of the varieties ranked first in BmOBP6 gene expression was greater than 85% for the Chinese silkworms and greater than 90% for the Japanese silkworms, i.e., 98% for the Chinese silkworm variety Green S and 85% for Group S, 97% for the Japanese silkworm variety Fu S, 92% for Spring S, and 92% for Hao S. According to the conventional Chinese·Chinese×Japanese·Japanese hybrid combination, such as the combination Green S·Group S×Fu S·Spring S, the 24-hour hair thinning rate was greater than 95%, meeting the target requirements.

Claims

1. The silkworm BmOBP6 gene, characterized in that The amino acid sequence of the BmOBP6 gene is shown in SEQ ID NO.

1.

2. The silkworm BmOBP6 gene according to claim 1, characterized in that The nucleotide sequence of the BmOBP6 gene is shown in SEQ ID NO.

2.

3. Use of the silkworm BmOBP6 gene according to claim 1 or 2 in screening silkworm varieties with high artificial feed intake.

4. The use according to claim 3, characterized in that: The screening method includes the following steps: S1. Select Chinese and Japanese silkworm varieties, raise them on mulberry leaves until they form cocoons, and then self-pollinate to produce seeds after moths emerge; S2, taking the silkworm eggs obtained in S1, relieving them from diapause and accelerating them to hatch, and using the silkworms as test materials; S3, extracting total RNA from S2 silkworms and reversely transcribing it into cDNA, designing primers based on the full-length sequence of the BmOBP6 gene, using cDNA as a template, detecting the transcription level of the BmOBP6 gene in each silkworm variety by qRT-PCR, and selecting the BmGAPDH gene as an internal reference gene; S4. Screening the silkworm varieties with a relative expression level of 2^(-ΔCt)≧1 in S3, that is, the silkworm varieties with high artificial feed intake.

5. The use according to claim 4, characterized in that: In S1, the selected Chinese silkworm varieties include: Xiafang, Ganxia, ​​Qun S, 8bai S, Ying S, Jing S, and Lu S; the Japanese silkworm varieties include: Hao S, Chun S, and Fu S.

6. The use according to claim 4, characterized in that: In S3, the upstream primer sequence of the silkworm BmOBP6 gene qRT-PCR is shown in SEQ ID NO.3, and the downstream primer sequence is shown in SEQ ID NO.4; the upstream primer sequence of the internal reference gene BmGAPDH is shown in SEQ ID NO.5, and the downstream primer sequence is shown in SEQ ID NO.

6.

7. The use according to claim 4, characterized in that: The silkworm varieties with high artificial diet intake obtained in S4 were used as parents, and artificial diet was used for continuous screening and succession to improve their adaptability and stability in artificial diet intake.

8. The use according to claim 4, characterized in that: The silkworm varieties with high artificial feed intake obtained in S4 were used as parents, and hybrid combinations were formed in the Chinese·Chinese×Japanese·Japanese manner. The combinations with a 24h hair thinning rate greater than 95% were used as the varieties with high artificial feed intake for production applications.

9. The use according to claim 4, characterized in that: In S4, the verification standard was that the hair thinning rate of Chinese breeds was greater than 85% and that of Japanese breeds was greater than 90% after consuming artificial feed for 24 hours, and the breeds screened out were consistent with the verification results.

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