Application of BmPriL in Improving Economic Traits of Silkworm

Abstract

This invention discloses the application of BmPriL in improving the economic traits of silkworms. Using the piggyBac transposition system, silkworms were constructed in which BmPriL was overexpressed specifically in the middle silk gland, mediated by the Ser1 promoter, and in the posterior silk gland, mediated by the FibH promoter. Results showed that the sericin content in cocoons of silkworms overexpressing BmPriL specifically in the middle silk gland was significantly higher than that in controls; and the cocoons overexpressing BmPriL specifically in the posterior silk gland were significantly higher in cocoons with significantly higher cocoon layer volume, cocoon layer rate, and fibroin content than in controls. These results demonstrate that BmPriL can improve the economic traits of silkworms.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to application of silkworm BmPriL in improving the economic traits of silkworms. Background Art

[0002] The silkworm (Bombyx mori) is an important economic insect, primarily due to the significant economic value of its silk, which can be processed and manufactured in various industries. Silk protein is primarily composed of an inner layer of fibroin and an outer layer of sericin, with fibroin accounting for 70%-80% and sericin 20%-30%. These two types of silk protein have different applications due to their different structural properties. Fibroin, an insoluble fibrous protein, is primarily used in the textile industry to produce raw silk and other silk products. Sericin, on the other hand, is a soluble globular protein with excellent moisturizing, antioxidant, and UV protection properties. It is primarily used in cosmetics, pharmaceuticals, food, and fiber modification. Increasing silk protein content is crucial for improving the economic benefits of the industry and promoting the development of silk production. The silk gland of the Bombyx mori is the only organ capable of synthesizing and secreting silk protein. The efficient endoreduplication of silk gland cells is a prerequisite for the synthesis and expression of large amounts of silk protein. Silk gland cells in the Bombyx mori exhibit typical endoreduplication characteristics. During the embryonic stage, silk gland cells undergo normal mitosis until around the eighth day after egg laying. Mitosis ceases once the silk gland is fully developed, and the number of silk gland cells becomes fixed and does not change. Subsequently, during the larval stage, silk gland cells undergo endoreduplication, focusing solely on DNA replication and cell enlargement, without further cell division. Through approximately 190,000 to 200,000 DNA replications, they accumulate a large amount of genetic material for the synthesis and secretion of silk proteins. Current research suggests that improving the endoreduplication efficiency of silk gland cells can promote silk gland development and silk protein synthesis, thereby increasing cocoon yield. DNA primase large subunit (PriL), a gene that plays a key role in the initiation of DNA replication, has been shown to have a significant impact on individual development. Ser1 is specifically expressed in the mid-posterior region of the silk gland, while FibH is specifically expressed in the posterior region of the silk gland. Summary of the Invention

[0003] In view of this, one of the objects of the present invention is to provide an application of BmPriL, a silkworm's middle silk gland-specific overexpression method, in improving the economic traits of silkworms; a second object of the present invention is to provide an application of BmPriL, a silkworm's posterior silk gland-specific overexpression method, in improving the economic traits of silkworms; a third object of the present invention is to provide a method for improving the economic traits of silkworms.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] 1. Application of BmPriL specifically overexpressed in the middle silk gland of silkworm in improving the economic traits of silkworm, wherein the nucleotide sequence of BmPriL is shown in SEQ ID NO.1.

[0006] Preferably, the economic trait of the present invention is an increased content of sericin in silk.

[0007] 2. Application of BmPriL specifically overexpressed in the posterior silk gland of silkworm in improving the economic traits of silkworm. The nucleotide sequence of BmPriL is shown in SEQ ID NO.1.

[0008] Preferably, the economic trait of the present invention is to increase the cocoon layer rate, cocoon layer weight or cocoon fibroin content.

[0009] 3. A method for improving the economic traits of silkworms, wherein BmPriL is specifically overexpressed in the middle silk gland or the posterior silk gland of silkworms, and the transgenic silkworm strain obtained is a silkworm with improved economic traits.

[0010] Preferably, in the method for specifically overexpressing BmPriL in the middle silk gland of the silkworm, the middle silk gland-specific promoter Ser1P is used to mediate the expression of BmPriL.

[0011] In a preferred embodiment of the present invention, the method for specifically overexpressing BmPriL in the posterior silk gland of the silkworm is to utilize the FibHP promoter to mediate the expression of BmPriL.

[0012] The present invention discloses the use of BmPriL in increasing cocoon yield. Using the piggyBac transposition system, strains of silkworms expressing BmPriL specifically overexpressing the middle silk gland (BmPriL-OE-MSG) mediated by the Ser1 promoter and the posterior silk gland (BmPriL-OE-PSG) mediated by the FibH promoter were constructed. Test results showed that the area and weight of the middle and posterior regions (MSG-(M+P)) of the middle silk gland in BmPriL-OE-MSG strains were significantly greater than those in the wild-type (WT) strain. The length and weight of the posterior silk gland (PSG) in BmPriL-OE-PSG strains were also significantly greater than those in the WT strain. EdU staining, DNA content analysis, and qRT-PCR analysis revealed that the silk glands of the overexpressing strains exhibited enhanced DNA replication activity and higher DNA content. Phalloidin staining and measurement of silk gland cells revealed that the cell area of ​​the middle silk gland of BmPriL-OE-MSG and the posterior silk gland of BmPriL-OE-PSG was larger than that of the WT group. Economic trait statistics revealed that the sericin content in BmPriL-OE-MSG cocoons was significantly higher than that of the control, while the cocoon layer volume, cocoon layer rate, and fibroin content of BmPriL-OE-PSG cocoons were significantly higher than those of the control. These results suggest that overexpressing BmPriL in the silk gland promotes endoreduplication, improves the transcription and translation efficiency of silk proteins, and increases silk protein synthesis, thereby increasing cocoon yield. Therefore, by specifically overexpressing BmPriL in the middle and posterior silk glands of B. mori, it is possible to promote endoreduplication and silk protein synthesis in silk gland cells, thereby creating transgenic lines with significantly improved economic traits. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to make the purpose, technical solutions and beneficial effects of the present invention more clear, the present invention provides the following drawings for illustration:

[0014] Figure 1 To prepare transgenic silkworms that specifically overexpress BmPriL in the middle silk gland (A: Schematic diagram of the transgenic vector for specifically overexpressing BmPriL in the middle silk gland; B: Screening of individuals positive for specifically overexpressing BmPriL in the middle silk gland; C: qPCR detection of silkworms that specifically overexpress BmPriL in the middle silk gland; D: Western blot detection of silkworms that specifically overexpress BmPriL in the middle silk gland);

[0015] Figure 2 Observation results of the middle silk gland of the silkworm (A: phenotypic observation of the silk gland of the silkworm on the fifth day of the fifth instar; B: length measurement of MSG-(M+P) on the fifth day of the fifth instar; C: area measurement of MSG-(M+P) on the fifth day of the fifth instar; D: weight measurement of MSG-(M+P) on the fifth day of the fifth instar);

[0016] Figure 3DNA activity detection of the middle silk gland cells (A: EDU staining of silk glands on the second day of the fourth instar to observe cell DNA replication activity; B: Statistics of the number of EDU fluorescent labels in MSG-(M+P) on the second day of the fourth instar; C: Total DNA content per unit of MSG-(M+P) on the fifth day of the fifth instar);

[0017] Figure 4 Observation of silk gland cell area (A. Phalloidin staining to observe the morphology and structure of silk gland cells on the second day of the fourth instar; B. Calculation of the size of MSG-(M+P) cells on the second day of the fourth instar);

[0018] Figure 5 Phenotypes and sericin content of transgenic silk cocoons and pupae (A. Phenotypic observation of cocoons and pupae; B. Detection of sericin content in cocoons);

[0019] Figure 6 qPCR test results for the middle silk gland;

[0020] Figure 7 The construction process of transgenic silkworms that specifically overexpress BmPriL in the posterior silk gland (A. Schematic diagram of the transgenic vector for specifically overexpressing BmPriL in the posterior silk gland; B. Screening of positive individuals for specifically overexpressing BmPriL in the posterior silk gland; C. qPCR detection of silkworms that specifically overexpress BmPriL in the posterior silk gland; D. Western blot detection of silkworms that specifically overexpress BmPriL in the posterior silk gland).

[0021] Figure 8 To observe the silk gland phenotype (A. Observation of silk gland phenotype of silkworm on the fifth day of the fifth instar; B. Measurement of PSG length on the fifth day of the fifth instar; C. Measurement of PSG weight on the fifth day of the fifth instar).

[0022] Figure 9 DNA activity of the posterior silk gland cells of the silkworm (A. Observation of cell DNA replication activity by EdU staining of silk glands on the second day of the fourth instar; B. Statistics of the EdU fluorescent labeling ratio of PSGs on the second day of the fourth instar; C. Total DNA content per unit of PSGs on the fifth day of the fifth instar).

[0023] Figure 10 The area of ​​silk gland cells in the posterior part of the silkworm (A. Phalloidin staining to observe the morphology and structure of silk gland cells on the second day of the fourth instar; B. Calculation of PSG cell size on the second day of the fourth instar).

[0024] Figure 11 Economic traits of the BmPriL-OE-PSG strain (A. Phenotypic observation of cocoons and pupae; B. Cocoon layer rate statistics; C. Cocoon weight statistics; D. Fibroin content detection of cocoons).

[0025] Figure 12 This is the expression of silk fibroin in the posterior silk gland. DETAILED DESCRIPTION

[0026] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0027] Example 1: Preparation of transgenic silkworms with BmPriL overexpression in the middle silk gland

[0028] First, BmPriL (SEQ ID NO. 1) was cloned from Bombyx mori using primers to attach a Flag tag to the N-terminus and a His tag to the C-terminus. The specific primers are as follows:

[0029] PriLOE F:

[0030] 5'-GGATCCATGGATTACAAGGATGACGATGACAAGGATTTCAAAGTTAAAAG-3' (SEQ ID NO. 2);

[0031] PriLOE R: 5'-GCGGCCGCTTAATGATGGTGATGGTGGTGTTCCAGTGGCTCATC-3' (SEQ ID NO. 3).

[0032] The pb-HEAG vector, which already contains the Hr3 enhancer, the Bombyx mori actin 4 promoter (A4P), and the SV40 termination signal sequence, was digested with BamH I and Not I. The hycu-ep32 gene sequence in the pb-HEAG vector was replaced with BmPriL (for the pb-HEAG vector, see Jiang Liang's doctoral dissertation, "Innovation of Bombyx mori Nuclear Polyhedrosis Virus (BmNPV)-Resistant Materials and Research on the Resistance Mechanism Based on Transgenic Engineering," 2013). The fragment was identified by agarose gel electrophoresis and recovered to yield the 1180-hr3-A4P-SV40 fragment. The BmPriL fragment and the 1180-hr3-A4P-SV40 fragment were ligated and transformed, and positive clones were screened to obtain the 1180-hr3-A4P-BmPriL-SV40 vector.

[0033] The middle silk gland-specific promoter Ser1P (SEQ ID NO. 4) was cloned from Bombyx mori using primers. The specific primers are as follows:

[0034] FHP F: 5'-GTCGACTAATTAGGTAGTGTTTAAGCTT-3' (SEQ ID NO. 5);

[0035] FHP R: 5'-GGATCCGAGAGTTGGAACCGAACT-3' (SEQ ID NO. 6);

[0036] The 1180-hr3-A4P-BmPriL-SV40 and Ser1P vectors were double-digested with SalI and BamH I, replacing A4P with Ser1P to generate the 1180-hr3-Ser1P-BmPriL-SV40 vector. The piggyBac[3×p3 DsRed afm] vector and 1180-hr3-Ser1P-BmPriL-SV40 vector were each digested with Asc I. The target bands were recovered and ligated for transformation. Positive clones were screened to generate the piggyBac[hr3-Ser1P-BmPriL-SV40-3×p3 DsRed afm] vector. DsRed expression was driven by the eye- and nerve-specific promoter 3×P3 as a fluorescent selection marker, while BmPriL expression was mediated by the mid-silk gland-specific promoter Ser1P. Figure 1 , A). The piggyBac [hr3-Ser1P-BmPriL-SV40-3×p3 DsRed afm] transgenic expression vector and the auxiliary plasmid A3H were mixed at a ratio of 1:1 and microinjected into D9L silkworm eggs. The hatched larvae were the G0 generation. After self-crossing or backcrossing, the G1 generation was obtained. Silkworms with red fluorescent eyes were screened under a fluorescence microscope and were identified as positive individuals. The transgenic silkworms were named BmPriL-OE-MSG ( Figure 1 , B). Inverse PCR results showed that the exogenous fragment was inserted into the intergenic region of the transgenic silkworm genome. qPCR results showed that the expression level of BmPriL in BmPriL-OE-MSG was significantly higher than that in the control. The increased expression of BmPriL carried a His tag, and Western blot detected the target band in the transgenic individuals ( Figure 1 , C and D). The above results indicate that we have successfully created transgenic silkworms that overexpress BmPriL specifically in the middle silk gland.

[0037] Example 2: The middle silk gland of BmPriL-OE-MSG silkworm is larger and heavier

[0038] The silkworms of the fifth instar and fifth day were selected for dissection, and the silk gland phenotype was observed and photographed. Figure 2 The area of ​​the middle posterior portion of the middle silk gland (MSG-(M+P)) was calculated using PS and ImageJ software. The statistical results show that the MSG-(M+P) in BmPriL-OE-MSG is longer and larger than that in the control. Weighing the MSG-(M+P) area revealed that BmPriL-OE-MSG weighed significantly more than the WT.

[0039] Example 3: DNA activity of the middle silk gland cells of BmPriL-OE-MSG silkworms is stronger

[0040] The silk glands of the fourth instar were selected for EdU staining. The results are as follows: Figure 3 The results showed that compared with the control, the green fluorescence signal of BmPriL-OE-MSG silk gland cells was significantly enhanced, and the number of cells emitting fluorescent signals increased significantly, indicating stronger DNA replication activity.

[0041] Example 4: BmPriL-OE-MSG silkworms have a larger silk gland cell area in the middle

[0042] The silk glands of the fourth instar were stained with phalloidin to observe the cell morphology. Figure 4 The results showed no significant difference in the morphology of the central silk gland cells between BmPriL-OE-MSG and WT cultures; both exhibited neat arrangement, distinct cell membranes, and branched nuclei. Using ImageJ software to calculate the size of each cell, the cell area of ​​BmPriL-OE-MSG cells was larger than that of WT cells. These results suggest that the increased silk gland area in BmPriL-OE-MSG cells is due to the enlargement of silk gland cells.

[0043] Example 5: BmPriL-OE-MSG silkworm cocoons have a higher sericin content

[0044] The phenotypes of transgenic silk cocoons and pupae were observed, and the sericin content of the cocoons was detected. Figure 5 The results showed that the cocoon and pupa size of the BmPriL-OE-MSG strain did not change significantly compared to the WT strain. The sericin content of the cocoons in the BmPriL-OE-MSG strain was significantly higher than that in the control strain.

[0045] Example 6: BmPriL-OE-MSG Upregulates Sericin Expression in the Middle Silk Gland of Silkworms

[0046] The middle silk gland of the fifth instar third day larvae was taken for qPCR detection, and the results were as follows Figure 6 The results showed that the expression levels of Ser1 and Ser2 in the overexpression lines were significantly higher than those in the control.

[0047] Example 7: Preparation of transgenic silkworms specifically overexpressing BmPriL in the posterior silk gland

[0048] The posterior silk gland-specific promoter FibHP (SEQ ID NO. 7) was cloned from Bombyx mori using primers as follows:

[0049] Ser1P F: 5'-GTCGACGAAAACAGCACACACACTAC-3' (SEQ ID NO. 8);

[0050] Ser1P R: 5'-GGATCCGTTGGCGGTCTTTGGAT-3' (SEQ ID NO.9)

[0051] The 1180-hr3-A4P-BmPriL-SV40 and FibHP were double-digested with SalI and BamH I, and A4P was replaced with FibHP to obtain the 1180-hr3-FibHP-BmPriL-SV40 vector. The piggyBac[3×p3 DsRed afm] vector and 1180-hr3-FibHP-BmPriL-SV40 were digested with Asc I, respectively. The target bands were recovered and ligated for transformation. Positive clones were screened to obtain the piggyBac[hr3-FibHP-BmPriL-SV40-3×p3 DsRed afm] vector ( Figure 7 , A). DsRed expression was driven by the eye- and nerve-specific promoter 3×P3 as a fluorescent selection marker, while BmPriL expression was mediated by the posterior silk gland-specific promoter FibHP. The piggyBac [hr3-FibHP-BmPriL-SV40-3×p3 DsRed afm] transgenic expression vector was microinjected to screen for G1-generation positive individuals whose eyes emitted red fluorescence ( Figure 7 , B). The transgenic silkworm was named BmPriL-OE-PSG. Reverse PCR results showed that the exogenous fragment was successfully inserted into the genome of the transgenic silkworm. qPCR results showed that the expression level of BmPriL in BmPriL-OE-PSG was significantly higher than that in the control ( Figure 7 , C). The incrementally expressed BmPriL carries a His tag, and the target band was detected in the transgenic individuals by Western blot ( Figure 7 , D). The above results indicate that we have successfully created transgenic silkworms that overexpress BmPriL specifically in the posterior silk gland.

[0052] Example 8: The posterior silk gland of BmPriL-OE-PSG silkworm is longer and heavier

[0053] The silk glands of the fifth instar and fifth day were selected for dissection, the silk gland phenotype was observed, photos were taken, and the length of the posterior silk gland (PSG) was calculated using ImageJ software. Figure 8 Statistical results showed that the PSG of BmPriL-OE-PSG was longer than that of the control. Weighing the PSG using a precision electronic balance also revealed that the PSG weight of BmPriL-OE-PSG was significantly higher than that of the WT.

[0054] Example 9: BmPriL-OE-PSG silkworm posterior silk gland cells have stronger DNA activity

[0055] The silk glands of the fourth instar were selected for EdU staining. The results are as follows: Figure 9 The results showed that compared with the control, the green fluorescence signal in the BmPriL-OE-PSG silk gland cells was significantly enhanced, and the number of cells emitting fluorescent signals increased significantly, indicating stronger DNA replication activity. DNA extracted from PSGs on the fifth day of the fifth instar showed that the DNA content in the posterior silk gland of BmPriL-OE-PSGs was significantly higher than that of the WT.

[0056] Example 10: BmPriL-OE-PSG silkworm posterior silk gland cell area is larger

[0057] The silk glands of the fourth instar were stained with phalloidin to observe the cell morphology. Figure 10 As shown in Figure 3 , the posterior silk gland cells of BmPriL-OE-PSG and WT cultures showed no significant morphological differences; both cells were neatly arranged, had clear cell membranes, and branched nuclei. Using ImageJ software to calculate the size of each cell, BmPriL-OE-PSG cells were larger than WT cells. These results suggest that the increased length and weight of the silk glands in BmPriL-OE-PSG is due to the enlargement of the silk gland cells.

[0058] Example 11: BmPriL-OE-PSG strain has better economic traits

[0059] The sizes of cocoons and pupae of the BmPriL-OE-PSG strain were counted. The results are as follows: Figure 11 The results showed that the cocoon and pupa size of the BmPriL-OE-PSG strain did not change significantly compared to the WT. Statistical analysis of cocoon layer percentage and cocoon weight showed that the cocoon layer percentage and cocoon weight of the BmPriL-OE-PSG strain were significantly higher than those of the control. Fibroin content in the cocoons of the BmPriL-OE-PSG strain was significantly higher than that of the control.

[0060] Example 12: Upregulation of silk fibroin expression in the posterior silk gland of BmPriL-OE-PSG silkworms

[0061] qPCR detection of the posterior silk gland of the fifth instar third day larvae, the results are as follows Figure 12 The results showed that compared with the control, the expression of FibH, FibL and P25 in BmPriL-OE-PSG was significantly upregulated.

[0062] The above embodiments are merely preferred embodiments for the purpose of fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

Claims

1. Application of BmPriL specifically overexpressed in the middle silk gland of silkworm to improve the economic traits of silkworm, characterized by: The nucleotide sequence of BmPriL is shown in SEQ ID NO. 1; the economic trait is increased sericin content in silk.

2. The use according to claim 1, characterized in that: The method for specifically overexpressing BmPriL in the middle silk gland of silkworm is to use the middle silk gland specific promoter Ser1P to mediate the expression of BmPriL.

3. A method for improving the economic traits of silkworms, characterized by: BmPriL is specifically overexpressed in the posterior silk gland of silkworms to obtain a transgenic silkworm strain with improved economic traits; the nucleotide sequence of the BmPriL is shown in SEQ ID NO.1, and the economic traits are increased cocoon layer rate, cocoon layer weight or cocoon fibroin content.

4. The method according to claim 3, wherein: The method of specifically overexpressing BmPriL in the posterior silk gland of silkworm is to use the FibHP promoter to mediate the expression of BmPriL.

Images