CN107484722B - Silkworm variety breeding method for secreting silk fibroin fibrils containing sericin protein - Google Patents
Silkworm variety breeding method for secreting silk fibroin fibrils containing sericin protein Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention provides a breeding method of silkworm varieties with secreted silk fibroin fibers containing sericin protein, which comprises the following steps: mating with male moth of Japanese system silkworm variety in bivoltine hybridization combination with female moth of Japanese system variety transgenic mutant capable of synthesizing and secreting sericin from posterior silk gland as female parent, and further utilizing methods of selfing, backcrossing, marker gene tracing and the like to breed Japanese system stock with sericin efficiently expressed from posterior silk gland; directly hybridizing the bred Japanese system stock with the Chinese system stock in the bivoltine hybridization combination to breed a silkworm hybridization combination variety containing sericin proteins in secreted silk fibroin fibrils. The method can efficiently and quickly breed the silkworm hybrid variety for producing the common silkworm cocoon into the silkworm with the secreted silk fibroin fibrils containing sericin protein, thereby obtaining the novel silkworm silk fiber with changed fiber performance, and being suitable for the commercial breeding requirement.
Description
Technical Field
The invention relates to the field of breeding of silkworm varieties with special purposes, in particular to a breeding method of a mutant silkworm variety containing sericin protein in produced silk fibroin fiber, which can efficiently and quickly breed a silkworm hybrid variety for producing common silkworm cocoons.
Background
Silkworm is a sericin insect which is applied for thousands of years, and silk fiber produced by cocoon silk spitted by mature silkworm larvae is known as fiber queen. The silk gland of silkworm is a specialized organ for synthesizing and secreting silk protein, and is a main object for domesticating, selecting and modifying silkworm. The protein synthesis and secretion function of the silkworm silk gland determines the yield and quality of silkworm cocoons, and fundamentally influences the main yield and quality level of the whole silk industry. Protein fiber components that improve silk gland synthesis are a significant issue of interest in the silkworm industry world.
The silkworm silk gland comprises three functional areas, namely a front silk gland, a middle silk gland and a rear silk gland. The anterior silk gland is a pipeline for outputting silk protein out of a body, and the middle silk gland and the posterior silk gland are synthesis and secretion parts of sericin protein and silk fibroin protein respectively. The fibroin protein synthesized by the posterior silk gland is a tough and elastic fiber protein with high molecular weight and insoluble in water, has beta-folded crystal structure, accounts for 70-80% of the total weight of cocoon silk, is a basic raw material for preparing textiles such as silk and the like, and is prepared from fibroin light chain (light chain, Fib-L), heavy chain (heavy chain, Fib-H) and P25 protein in a weight ratio of 6: 6: a molar ratio of 1 constitutes a complex, and as a basic structural unit of fibroin (Inoue,2000), P25 protein is also called fibrohexamer (Fhx/P25) (Yamaguchi et al, 1989; Sprague 1975; Couble et al, 1983). The silk fibroin synthesized by posterior silk gland cells in the larva stage of silkworms is continuously secreted to the gland cavity and gradually moves from the gland cavity of the posterior silk gland to the gland cavity of the middle silk gland. In the process of spinning and cocooning after silkworm larvae are mature, silk fibroin molecules are continuously polymerized into fibrils with the diameter of about 0.1 mu m and irregular round sections, and the fibrils which are also called fibrils are further tightly bundled to form a silk fibroin core of silk. The fibrils are formed by alternately connecting crystalline fibers and non-crystalline fiber molecular polymers in series along the uranium direction. The fibrils are closely connected through the side chains of the molecules at the periphery, and have no filling of the interstitial substance, which is the molecular basis of the special luster of the silk fibroin fiber and the production of the superfine protein fiber.
In the process of silk fiber formation and forward movement of silk fiber in silk gland of silkworm, the silk gland is coated with 4 layers of sericin, wherein the innermost layer is4 mRNAs encoded by sericin gene Ser1, namely Ser1A (2.8kb), Ser1B (4kb), Ser1C (10kb) and Ser1D (9kb) translated 4 sericin proteins (Michaille et al, 1986), and the outer layers are 3 mRNAs encoded by Ser2 and Ser3 genes, namely Ser2A (5.4kb), Ser2B (3.1kb) (Kludkiewicz et al, 2009) and Ser3(9kb) translated 3 sericin proteins (Takasu et al, 2007), the sericin protein accounts for 20-30% of the total cocoon silk, contains a β polypeptide chain structure, but does not have a α helix structure, the main conformation of the molecule is random coil, the spatial structure of the molecule is loose, disordered, the glycine, alanine and tyrosine content in sericin is significantly lower than that of sericin, and the free amino acid content is significantly higher than that of the sericin protein which has many methionine, lysine, tyrosine, amino acid2An isopolar hydrophilic group. Therefore, sericin has excellent effects of moisture absorption, moisture retention, odor absorption and the like. Wherein, the hydroscopicity and the fluidity of the sericin SER3 are obviously higher than those of 4 sericin proteins translated by a Ser1 gene. The prior research shows that the silkworm silk collagen also has good antioxidant, antibacterial and ultraviolet resistant functions. The serine content in sericin is up to 30% or more (Zhuliang equi, 1997), and its effect in cosmetics is similar to natural moisturizing factor in human skin stratum corneum.
In the operation processes of silk reeling, scouring and the like for producing the silk textile, sericin on the surface of silk fiber is almost completely removed, which is also a technological requirement for improving the gloss of silk and ensuring the dyeing uniformity. The silk fabric without removing the sericin on the outer layer has the problems of poor gloss and uneven dyeing, and poor softness and poor wearing comfort of the fabric. Although the water absorption of degummed silk fiber molecules is still about 30% higher than that of cotton fibers, the silk feel is not absorbed by cotton cloth when the silk is taken. This is because silk is expensive and is almost a thin fabric. Therefore, sericin protein is doped into the fibroin protein, the fiber structure and the physical and chemical properties of fibroin are expected to be changed, the purpose of changing the fiber function is realized, and particularly, the dual characteristics of fibroin and sericin are used simultaneously, so that sericin is remained on the surface of the fibroin fiber, or sericin coating is carried out on the surface of silk fiber or fabric, and the function utilization can not be realized.
Based on the structural gene of the silkworm silk protein which is known at present, if the structural gene can induce the synthesis efficiency and the molecular ratio change of different structural components of silk protein in silk glands, the fiber characteristics of the known silk protein can be overturned. Silkworm researchers have also attempted to transfer exogenous genes of high yield, quality or special properties to the silkworm silk gland expression with the aim of substantially altering the fiber properties of silk proteins, such as spidroin genes (Wen et al, 2010; Teul et al, 2012; yananyong et al, 2012), tussian genes (lisang & fanjiogo, 1997), etc. At present, related researches are focused on introducing exogenous protein fiber coding genes or development regulation genes into the genome of silkworms, so that the aims of changing the synthesis efficiency of silkworm silk proteins and the performance of silk protein fibers are fulfilled. However, silkworm transformed with exogenous gene has a series of problems such as obvious decrease of silk protein synthesis and secretion efficiency, insignificant improvement of fiber administration performances such as fiber purity and strength of silk protein, and even performance decrease, thereby largely hindering further industrial research and application of related research works (Wang et al 2015). The present inventors connected amino acids with nano quantum dots, and found that the absorption efficiency of serine gland cells to alanine was significantly higher than that of glycine, although the content of glycine in sericin and silk fibroin was 150% and 280% of alanine, respectively (Xing et al, 2016). The inventor finds that 891 genes with obvious difference in expression are involved in protein synthesis, processing, secretion and transportation of cells, energy supply of cells and the like through the research of silk gland transcriptomics of a rear silk gland degeneration system and a normal system induced by transgenes (Wang et al, 2015). Therefore, the present inventors considered that the above-mentioned problem of a significant decrease in the efficiency of silk protein synthesis and secretion in transgenic silkworms is related to the preference of amino acid utilization in silk gland cells of silkworms. Therefore, the invention is necessary to provide a preferential transgenic method for amino acid utilization suitable for silk gland cells of silkworms.
The inventor selects the sericin which has tissue expression specificity and is specifically expressed by the middle silk gland of the silkworm, realizes high-efficiency expression of the silk gland at the rear part of the silkworm, and prepares the transgenic mutant silkworm which synthesizes and secretes the sericin by the rear silk gland (the invention name is a preparation method of the silkworm which can synthesize and secrete the hydrophilic sericin by the rear silk gland, and the Chinese patent application number is 201610348866.0). The transgenic mutant of the sericin protein efficiently expressed by the rear silk gland obtained by the patent technology is a mutant homozygous silkworm, has the potential of cultivating various excellent cocoon silk production characters such as silk quantity, fineness, purity, cocoon color and the like, but does not meet the production practicability requirement of the modern silkworm breeding industry, is a genetic resource called as a material in the field of silkworm breeding, reserves the problems of non-delayed fertility, short growth period, low cocoon silk quantity, poor development uniformity and the like of a wild type original variety suitable for transgenic operation, and has the cocoon silk quality such as cocoon silk fineness, purity, strength and the like which needs to be directionally transformed. On the other hand, silkworm varieties used in domestic and foreign silkworm breeding industry, preferably first generation hybrids of the bivoltine chinese system and the japanese system, are relatively poor in compatibility with the chinese phylogenetic silkworm strain, which is a japanese system transgenic mutant prepared by the method for preparing silkworms capable of synthesizing and secreting hydrophilic sericin in the posterior silk gland of the chinese patent application (application No. 201610348866.0).
Therefore, the development of silkworm mutants for synthesizing and secreting sericin from the posterior silk gland is necessary, and the method has great commercial prospect.
Disclosure of Invention
In order to solve the technical problems, the invention provides a breeding method of silkworm varieties with secreted silk fibroin fibers containing sericin protein, which can rapidly and directionally transform cocoon silk fineness, purity and strength of a transgenic mutant.
In order to achieve the purpose, the invention adopts the following technical scheme:
a breeding method of silkworm breed containing sericin protein in secreted silk fibroin fibril comprises the following steps:
(1) mating a female moth of a transgenic mutant of a non-diapause variety of a Japanese system, which synthesizes and secretes sericin from a posterior silk gland, serving as a female parent with a male moth of a variety of a Japanese system in a bivoltine optimal hybridization combination, and allowing a single moth to lay eggs to obtain a first generation of silkworm eggs; among them, a transgenic mutant of a Japanese systematic non-diapauzing variety, which synthesizes and secretes sericin from the posterior silk gland, is prepared from the Japanese systematic non-diapauzing variety according to the method of Chinese patent application 201610348866.0.
(2) Breeding larvae hatched by the first generation silkworm eggs by a single moth breeding method, selecting and reserving a moth area with more than 90% of positive individuals by taking individuals with green fluorescence cocoon silks as positive indexes, and further preferably performing moth in-area mating on the positive individuals with a cocoon type ellipse and a shallow girdling waist in the selected moth area to obtain second generation silkworm eggs, wherein the cocoon silk amount and the cocoon silk quality of the positive individuals are respectively ranked within the top 20% in the selected moth area;
(3) breeding larvae hatched by the second generation silkworm eggs by a single moth breeding method, selecting and reserving a moth area with more than 90% of positive individuals by taking individuals with green fluorescence cocoon silks as positive indexes, and further preferably performing moth in-area mating on the positive individuals with a cocoon type ellipse and a shallow girdling waist in the selected moth area to obtain third generation silkworm eggs, wherein the cocoon silk amount and the cocoon silk quality of the positive individuals are respectively ranked within the top 20% of the selected moth area;
(4) breeding larvae hatched by the third generation silkworm eggs by using the quantity of ants in a moth area, selecting and reserving the moth area with more than 90% of positive individuals by using individuals with green fluorescence cocoon silks as positive indexes, further preferably selecting female positive individuals with a cocoon type ellipse length and a shallow girdling waist in the moth area, and mating the female positive individuals with male moths of the Japanese system silkworm variety used in the step (1), namely backcrossing treatment and single moth oviposition to obtain fourth generation silkworm eggs, wherein the cocoon silk quantity and the cocoon silk quality of the female positive individuals are respectively ranked within the top 20% in the selected moth area;
(5) breeding larvae hatched by the fourth generation of silkworm eggs by a single-moth breeding method, selecting and reserving a moth area with more than 90% of positive individuals by taking green fluorescence individuals with cocoon silks as positive indexes, and preferably performing moth in-area mating on the positive individuals with a cocoon type ellipse and a shallow girdling waist in the selected moth area to obtain fifth generation silkworm eggs, wherein the cocoon silk amount and the cocoon silk quality of the positive individuals are respectively ranked within 20% of the selected moth area;
(6) breeding larvae hatched by the fifth generation of silkworm eggs by using the quantity of ants in a moth area, selecting and reserving the moth area with more than 90% of positive individuals by using green fluorescence individuals of cocoon filaments as positive indexes, and further preferably performing moth in-area mating on the positive individuals with a cocoon type prolate ellipsoid and a shallow girdling waist in the selected moth area to obtain the sixth generation of silkworm eggs, wherein the cocoon filament quantity and the cocoon filament quality of the positive individuals are respectively ranked within the top 20% of the selected moth area;
(7) breeding larvae hatched by the sixth generation of silkworm eggs by using the quantity of ants in a moth area, selecting and reserving the moth area with more than 90% of positive individuals by using green fluorescence individuals of cocoon filaments as positive indexes, and further preferably performing moth in-area mating on the positive individuals with a cocoon type ellipse and a shallow girdling waist in the selected moth area to obtain seventh generation silkworm eggs, wherein the cocoon filament quantity and the cocoon filament quality of the positive individuals are respectively ranked within the top 20% of the selected moth area;
(8) breeding larvae hatched by the seventh generation of silkworm eggs by a single moth breeding method, selecting and reserving a moth area with more than 90% of positive individuals by taking green fluorescence individuals with cocoon silks as positive indexes, and preferably performing moth in-area mating on the positive individuals with a cocoon type ellipse and a shallow girdling waist in the selected moth area to obtain eighth generation silkworm eggs, wherein the cocoon silk amount and the cocoon silk quality of the positive individuals are respectively ranked within 20% of the selected moth area;
(9) breeding larvae hatched from the eighth generation of silkworm eggs by a single-moth breeding method, selecting and remaining moth areas with more than 90% of positive individuals by taking green fluorescence individuals with cocoon silks as positive indexes, and further preferably performing moth in-area mating on the positive individuals with a cocoon type prolate ellipsoid and a shallow girdling waist in the selected moth areas to obtain ninth generation silkworm eggs, wherein the cocoon silk amount and the cocoon silk quality of the positive individuals are respectively ranked within the top 20% of the selected moth areas;
(10) breeding the larva hatched by the ninth generation of silkworm eggs by a single moth breeding method, selecting an individual with green fluorescence cocoon silks as a positive index, selecting a moth area of more than 98% of the positive individuals as a pre-selected moth area, selecting the cocoons in the preselected moth area with the shape of long ellipse, the shape of shallow girdling and the color of cocoons basically consistent, and the cocoons conform to the positive individuals of the breeding target silkworm cocoons (the cocoon silk quantity and the cocoon silk quality are respectively ranked within the top 20 percent in the selected moth area), then carrying out live pupa reeling on the cocoons in the preselected moth area, selecting the individuals with the cocoon reelability rate high, the cocoon silk cleanliness and the strength high, and the cocoon silk fineness conforming to the breeding target to carry out interval moth mating subculture (the individuals with the cocoon reelability rate, the cocoon silk cleanliness and the strength respectively ranked within the top 20 percent), obtaining tenth generation silkworm eggs, namely, the practical Japanese system special stock of the mutant homozygotic type of the posterior silk gland high-efficiency expression sericin;
(11) hybridizing the mutation homozygous practical Japanese system stock for efficiently expressing sericin in the posterior silk gland with the Chinese system silkworm variety of the bivoltine optimized hybridization combination used in the step (1) to obtain the silkworm hybridization combination variety containing sericin in the secreted silk fibroin fibrils.
Preferably, in each step, the mated males and females are separated and the females are placed on a oviposition paper to lay eggs.
Preferably, the female moths are isolated from each other by a moth ring made of iron or plastic.
Preferably, eggs laid by female moths are placed in an environment with the temperature of 25-27 ℃, the relative humidity of 75-80% and natural illumination, the silkworm eggs which leave the N4 variety and are not diapause are eliminated, and the moth areas with good diapause are left for seed reservation.
In the invention, the diapause eggs gradually change from light yellow to yellow 20-30 hours after spawning, change to light brown 30-50 hours after spawning, and change to dark brown 50 hours after spawning.
In the present invention, the non-diapause silkworm eggs remain pale yellow and do not change color within one week after the egg laying.
By means of the scheme, compared with the prior art, the invention at least has the following advantages:
the present invention provides a breeding method of silkworm variety containing sericin protein in secreted silk fibroin, and said method can utilize optimum hybridization combination, such as high combining power of hybridization combination for producing practical variety in sericulture industry and excellent cocoon silk production property, and can quickly breed hybrid combination variety by introducing mutant gene of rear silk gland high-effective expression sericin protein into Japanese system, and can high-effectively produce first generation hybrid seed for sericulture production, and has no need of synchronously cultivating practical Chinese system special-purpose stock of rear silk gland high-effective expression sericin protein mutation homozygote, and can obviously reduce breeding workload.
The method of the invention can change the non-diapause property of the original variety suitable for transgene retained by the transgenic mutant into the stable hereditary diapause property, and overcome the problems that the silkworm eggs of the non-diapause variety are not tolerant to be preserved and need to be continuously raised and preserved all the year round.
The method can solve the problems of short growth period, low cocoon silk content, poor development uniformity and the like of the original variety reserved by the mutant.
The method can rapidly and directionally transform cocoon silk properties of the mutant, such as cocoon silk fineness, purity, strength and the like, and meet the requirements of practical varieties.
Detailed Description
The present invention will be described in further detail with reference to specific embodiments. It should be understood that the following examples are only illustrative of the present invention and are not intended to limit the scope of the present invention.
Example 1
Breeding yellow cocoon hybrid variety with high-efficiency expression of sericin protein in rear silk gland
This embodiment 1 includes the following steps:
(1) preparing a transgenic mutant from a Japanese systematic N4 variety which synthesizes and secretes sericin from a rear silk gland according to the method of Chinese patent application 201610348866.0, mating a female moth of the mutant with a Japanese systematic M male moth in multiplied by M days (silkworm variety name, colored cocoon I) in a bivoltine practical hybridization combination M of a yellow cocoon by taking the female moth as a female parent, and allowing a single moth to lay eggs to obtain a first generation silkworm egg;
(2) breeding larvae hatched from first generation silkworm eggs by a single moth breeding method, selecting and reserving a moth area with more than 90% of positive individuals by taking individuals with green fluorescence cocoon silks as positive indexes, further preferably selecting the positive individuals in the moth area, selecting and reserving yellow cocoons with cocoon type oblong and shallow girdling, further selecting and reserving individuals with high cocoon silk content within 20% of the selected moth area before ranking, and performing moth in-area mating to obtain second generation silkworm eggs;
(3) breeding larvae hatched by second generation silkworm eggs by a single moth breeding method, selecting and reserving a moth area with more than 90% of positive individuals by taking individuals with green fluorescence cocoon silks as positive indexes, further preferably selecting yellow cocoons with a cocoon type of long ellipse and shallow girdling in the selected moth area, and further selecting individuals with a high cocoon silk content within 10% of the selected moth area before ranking to perform moth in-area mating to obtain third generation silkworm eggs;
(4) breeding third-generation silkworm egg hatched larvae by using a moth area ant amount, selecting and reserving a moth area with more than 90% of positive individuals by using individuals with green fluorescence cocoon silks as positive indexes, further selecting yellow cocoons with long oval cocoon types and shallow girdling cocoons in the selected moth area, further selecting and reserving female positive individuals with high cocoon silk content within 10% of the individuals with high cocoon silk content in the selected moth area before ranking, backcrossing the female positive individuals with Japanese system M day male moths of multiplied by M days in M used in the step (1), and performing single-egg laying on the moths to obtain fourth-generation silkworm eggs;
(5) breeding larvae hatched by fourth generation of silkworm eggs by a single moth breeding method, selecting and reserving a moth area with more than 90% of positive individuals by taking individuals with green fluorescence cocoon silks as positive indexes, further preferably selecting yellow cocoons with a cocoon type oblong and a shallow girdling shape in the selected moth area, and further selecting individuals with a high cocoon silk content within 20% of the selected moth area before ranking to perform moth in-area mating to obtain fifth generation silkworm eggs;
(6) breeding larvae hatched by fifth-generation silkworm eggs by using the quantity of ants in the moth area, selecting and reserving the moth area with more than 90% of positive individuals by using individuals with green fluorescence cocoon silks as positive indexes, further preferably selecting yellow cocoons with a cocoon type of long ellipse and shallow girdling in the selected moth area, and further selecting individuals with a high cocoon silk quantity within 10% of the quantity of the high cocoon silks arranged in the selected moth area for moth in-area mating to obtain sixth-generation silkworm eggs;
(7) breeding larvae hatched by sixth generation silkworm eggs by using the quantity of ants in a moth area, selecting and reserving the moth area with more than 90% of positive individuals by using individuals with green fluorescence cocoon silks as positive indexes, further preferably selecting yellow cocoons with a cocoon type oblong and a shallow girdling shape in the selected moth area, and further selecting individuals with a high cocoon silk quantity within 10% of the selected moth area before ranking to perform moth in-area mating to obtain seventh generation silkworm eggs; (ii) a
(8) Breeding larvae hatched by seventh generation silkworm eggs by a single moth breeding method, selecting and reserving a moth area with more than 90% of positive individuals by taking individuals with green fluorescence cocoon silks as positive indexes, further preferably selecting and reserving yellow cocoons with a cocoon type oblong and a shallow girdling shape in the selected moth area, and further selecting and reserving individuals with high cocoon silk content within 5% of the high cocoon silk content before ranking in the selected moth area to perform moth in-area mating to obtain eighth generation silkworm eggs;
(9) breeding larvae hatched by the eighth generation of silkworm eggs by a single moth breeding method, selecting and reserving a moth area with more than 90% of positive individuals by taking individuals with green fluorescence cocoon silks as positive indexes, further preferably selecting yellow cocoons with a long oval cocoon shape and a shallow girth cocoon shape in the selected moth area, and further selecting individuals with a high cocoon silk content within 5% of the high cocoon silk content before ranking in the selected moth area to perform moth in-area mating to obtain ninth generation silkworm eggs;
(10) breeding larvae hatched by ninth generation silkworm eggs by a single moth breeding method, selecting and reserving a moth area with over 98% of positive individuals as a preselected moth area by taking individuals with green fluorescence cocoon silks as positive indexes, further preferably selecting the positive individuals in the moth area, selecting and reserving yellow cocoons with uniform colors of cocoon type ellipse and light corset, further performing live pupa reeling on seed cocoons in the preselected moth area, preferably performing interval mating subculture on the moths by using the individuals with the silkworm cocoon reelability, cocoon silk cleanliness and strength respectively ranked within the first 15% to the tenth generation, and obtaining a practical Japanese system special stock MS day of the yellow cocoons which are homozygotic and highly express sericin by silk glands at the rear part;
(11) the main characters of the MS day of the practical Japanese system stock for the mutant homozygous yellow cocoons of which the rear silk glands efficiently express sericin and the Chinese system M for the white cocoons of the XM day in the bivoltine practical hybridization combination silkworm variety M used in the step (1) are shown in the table 1.
In the steps (1) to (10), the light yellow silkworm eggs which leave the N4 variety and are not diapause are eliminated after 30 hours after the eggs are laid, and light brown or dark brown diapause egg moth areas are selected for reserving the eggs.
By using the method of example 1 of the present invention, the mutant gene of sericin efficiently expressed by silk glands behind the N4 mutant can be introduced into the japanese system M days by utilizing the high combining ability of M-day hybrid combinations of practical varieties produced by yellow cocoons in the silkworm industry and the excellent cocoon silk production performance, and the yellow cocoon hybrid combination variety can be rapidly bred, thereby efficiently producing first-generation hybrids for the silkworm production.
By using the method of the embodiment 1, the non-diapause property of the original N4 variety retained by the N4 mutant can be changed into the stable hereditary fertility, and the problems that the silkworm eggs of the non-diapause variety are not resistant to storage and need to be continuously raised and preserved all the year round are solved;
by using the method of the embodiment 1, the problems of short growth period, low cocoon silk content, poor development uniformity and the like of the original N4 variety reserved by the N4 mutant can be improved;
by using the method of the embodiment 1, cocoon silk properties such as fineness, cleanliness, strength and the like of the cocoon silk of the N4 mutant can be rapidly and directionally transformed, and the requirement of practical varieties is met.
The main cocoon silk characteristics of the first generation hybrid of example 1, in which the posterior silk gland efficiently expresses sericin, are shown in table 1 below.
TABLE 1
Example 2
White cocoon hybrid variety for breeding high-efficiency expression sericin of posterior silk gland
This embodiment 2 includes the following steps:
(1) mating female moth of transgenic mutant of Japanese System N4 variety (prepared by the method of Chinese patent application 201610348866.0 using Japanese System N4 variety) as female parent, which synthesizes and secretes sericin from the posterior silk gland, with Japanese System 9206 male moth of combined practical hybridization of two chemical properties of whitish cocoon 9205X 9206, and single moth oviposits to obtain first generation silkworm egg;
(2) breeding larvae hatched from first generation silkworm eggs by a single moth breeding method, selecting and reserving a moth area with more than 90% of positive individuals by taking individuals with green fluorescence cocoon silks as positive indexes, further preferably selecting the positive individuals in the moth area, selecting and reserving white cocoons with cocoon type oblong and shallow girdling shapes, further selecting and reserving individuals with high cocoon silk content within 20% of the selected moth area before ranking, and performing moth in-area mating to obtain second generation silkworm eggs;
(3) breeding larvae hatched by second generation silkworm eggs by a single moth breeding method, selecting and reserving a moth area with more than 90% of positive individuals by taking individuals with green fluorescence cocoon silks as positive indexes, further preferably selecting the positive individuals in the moth area, selecting and reserving white cocoons with a cocoon type oblong and a shallow girdling shape, further selecting and reserving individuals with high cocoon silk content within 10% of the selected moth area before ranking, and performing moth in-area mating to obtain third generation silkworm eggs;
(4) breeding third-generation silkworm egg hatched larvae by using a moth area ant amount, selecting and reserving a moth area with more than 90% of positive individuals by using green fluorescence individuals with cocoon silks as positive indexes, further selecting the positive individuals in the selected moth area, selecting and reserving white cocoons with cocoon type oblong and shallow girdling shapes, further selecting and reserving a female positive individual with high cocoon silk amount within 10% of the selected moth area before ranking, backcrossing the female positive individual with male moths of the Japanese system 9206 variety used in the step (1), and allowing the female moths to lay eggs to obtain fourth-generation silkworm eggs;
(5) breeding larvae hatched by fourth generation of silkworm eggs by a single moth breeding method, selecting and reserving a moth area with more than 90% of positive individuals by taking individuals with green fluorescence cocoon silks as positive indexes, further preferably selecting the positive individuals in the moth area, selecting and reserving white cocoons with cocoon type oblong and shallow girdling shapes, further selecting and reserving individuals with high cocoon silk content within 20% of the selected moth area before ranking, and performing moth in-area mating to obtain fifth generation silkworm eggs;
(6) breeding larvae hatched by fifth-generation silkworm eggs by using the quantity of ants in a moth area, selecting and reserving the moth area with more than 90% of positive individuals by using individuals with green fluorescence cocoon silks as positive indexes, further preferably selecting the positive individuals in the moth area, selecting and reserving white cocoons with cocoon type oblong and shallow girdling shapes, and further selecting and reserving individuals with high cocoon silk quantity within 10% of the selected moth area before ranking to perform moth in-area mating to obtain sixth-generation silkworm eggs;
(7) breeding larvae hatched by sixth generation silkworm eggs by using the quantity of ants in a moth area, selecting and reserving the moth area with more than 90% of positive individuals by using individuals with green fluorescence cocoon silks as positive indexes, further preferably selecting the positive individuals in the moth area, selecting and reserving white cocoons with cocoon type oblong and shallow girdling shapes, and further selecting and reserving individuals with high cocoon silk quantity within 10% of the selected moth area before ranking to perform moth in-area mating to obtain seventh generation silkworm eggs;
(8) breeding larvae hatched from seventh generation silkworm eggs by a single moth breeding method, selecting and reserving a moth area with more than 90% of positive individuals by taking individuals with green fluorescence cocoon silks as positive indexes, further preferably selecting the positive individuals in the moth area, selecting and reserving white cocoons with cocoon type oblong and shallow girdling shapes, further selecting and reserving individuals with high cocoon silk content within 5% of the selected moth area before ranking, and performing moth in-area mating to obtain eighth generation silkworm eggs;
(9) breeding larvae hatched from eighth generation silkworm eggs by a single moth breeding method, selecting a moth area with more than 90% of positive individuals by taking individuals with green fluorescence cocoon silks as positive indexes, further preferably selecting the positive individuals in the moth area, selecting white cocoons with cocoon type oblong and shallow girdling, further selecting individuals with high cocoon silk content within 5% of the selected moth area before ranking, and performing moth in-area mating to obtain ninth generation silkworm eggs;
(10) breeding larvae hatched by ninth generation silkworm eggs by a single moth breeding method, selecting and reserving a moth area with over 98% of positive individuals as a preselected moth area by taking individuals with green fluorescence cocoon silks as positive indexes, further preferably selecting the positive individuals in the moth area, selecting and reserving white cocoons with uniform colors of cocoon type prolate ellipses and light corset waists, further performing live pupa reeling on the seed cocoons in the preselected moth area, preferably performing interval mating subculture on the moths by the individuals with silkworm cocoon reelability, cocoon silk cleanliness and strength respectively ranking within the first 15%, and obtaining a practical Japanese system special stock 9206S of the white cocoons which are homozygotic and have rear silk gland high-efficiency expression sericin;
(11) the practical Japanese system special stock 9206S for highly expressing mutant genotype homozygous white cocoons of sericin protein by the rear silk glands is hybridized with the Chinese system 9205 variety of the bivoltine practical hybridization combination 9205X 9206 used in the step (1), the special silkworm hybrid 9205X 9206S for highly synthesizing and secreting pure natural sericin protein by the rear silk glands of the white cocoons is produced, and the main characters of the first generation hybrid bred in the autumn silkworm stage in 2015 are shown in Table 2.
In the steps (1) to (10), the light white silkworm eggs which leave the non-fertility-retention N4 variety are eliminated after 30 hours of spawning, and light brown or dark brown diapause egg moth areas are selected for reserving the seeds.
By using the method of example 2 of the present invention, the high combining ability and excellent cocoon silk production performance of the optimized dichotomized practical hybrid combination 9205X 9206 of white cocoons can be utilized, the mutant gene of sericin efficiently expressed by the silk gland at the rear part of the N4 mutant is introduced into the Japanese system 9206, white cocoon hybrid combination varieties can be rapidly bred, and first generation hybrid seeds for silkworm breeding production can be efficiently produced.
By using the method of the embodiment 2 of the invention, the non-diapause property of the original N4 variety retained by the N4 mutant can be changed into the stably inherited fertility, and the problem that the silkworm eggs of the non-diapause variety are not tolerant to storage and need to be continuously raised and preserved all the year round is solved.
By using the method of the embodiment 2, the problems of short growth period, low cocoon silk amount, poor development uniformity and the like of the original N4 variety reserved by the N4 mutant can be improved.
By using the method of the embodiment 2, cocoon silk properties such as titer, cleanliness, strength and the like of cocoon silk of the N4 mutant can be rapidly and directionally transformed, and the requirements of practical varieties are met.
By using the method of the embodiment 2 of the invention, yellow cocoon silks of the N4 mutant can be rapidly transformed into white cocoon silks in a directional manner, so that the requirement of practical white cocoon varieties is met.
The main cocoon silk characteristics of the first generation hybrid of example 2 in which the posterior silk gland highly expresses sericin are shown in table 2 below.
TABLE 2
Example 3
Special yellow cocoon high silk yield hybrid variety for breeding high-efficiency expression sericin of posterior silk gland
This embodiment 3 includes the following steps:
(1) mating a female moth of a transgenic mutant of a Japanese System N4 variety (produced by the method of Chinese patent application 201610348866.0 using the Japanese System N4 variety) which synthesizes and secretes sericin from the posterior silk gland as a female parent with a Japanese System M male moth of X M days in a yellow cocoon-forming practical hybrid combination silkworm variety M, and allowing a single moth to lay eggs to obtain a first generation silkworm egg;
(2) breeding larvae hatched by first generation eggs by a single moth breeding method, selecting and reserving a moth area with more than 90% of positive individuals by taking individuals with green fluorescence cocoon silks as positive indexes, further preferably selecting the positive individuals in the moth area, selecting and reserving yellow cocoons with cocoon type long elliptic and shallow girdling, further selecting and reserving individuals with high cocoon silk content within 5% of the selected moth area before ranking, and performing moth in-area mating to obtain second generation silkworm eggs;
(3) breeding larvae hatched by second generation silkworm eggs by a single moth breeding method, selecting and reserving a moth area with more than 90% of positive individuals by taking individuals with green fluorescence cocoon silks as positive indexes, further preferably selecting the positive individuals in the moth area, selecting and reserving yellow cocoons with a cocoon type oblong and a shallow girdling shape, further selecting and reserving individuals with a high cocoon silk content within 10% of the selected moth area before ranking, and performing moth in-area mating to obtain third generation silkworm eggs;
(4) breeding third generation silkworm eggs hatched larvae by using a moth area ant amount, selecting and reserving a moth area with more than 90% of positive individuals by taking green fluorescence individuals with cocoon silks as positive indexes, further selecting the positive individuals in the selected moth area, selecting and reserving yellow cocoons with cocoon type oblong and shallow girdling shapes, further selecting and reserving female positive individuals with high cocoon silk amount within 10% of the selected moth area before ranking, mating the female positive individuals with Japanese system 9206 male moths of a bivoltine practical hybridization combination 9205X 9206 of white cocoons, enabling single moths to lay eggs, and obtaining fourth generation silkworm eggs;
(5) breeding larvae hatched by fourth generation of silkworm eggs by a single moth breeding method, selecting and reserving a moth area with more than 90% of positive individuals by taking individuals with green fluorescence cocoon silks as positive indexes, further preferably selecting the positive individuals in the moth area, selecting and reserving yellow cocoons with cocoon type oblong and shallow girdling, further selecting and reserving individuals with high cocoon silk content within 10% of the selected moth area before ranking, and performing moth in-area mating to obtain fifth generation silkworm eggs;
(6) breeding larvae hatched by fifth-generation silkworm eggs by using the quantity of ants in a moth area, selecting and reserving the moth area with more than 90% of positive individuals by using individuals with green fluorescence cocoon silks as positive indexes, further preferably selecting the positive individuals in the moth area, selecting and reserving yellow cocoons with cocoon type oblong and shallow girdling shapes, further selecting and reserving individuals with high cocoon silk quantity within 10% of the selected moth area before ranking, and performing moth-area mating to obtain sixth-generation silkworm eggs;
(7) breeding larvae hatched by sixth generation silkworm eggs by using the quantity of ants in a moth area, selecting and reserving the moth area with more than 90% of positive individuals by using individuals with green fluorescence cocoon silks as positive indexes, further preferably selecting the positive individuals in the moth area, selecting and reserving yellow cocoons with cocoon type oblong and shallow girdling shapes, further selecting and reserving individuals with high cocoon silk quantity within 5% of the selected moth area before ranking, and performing moth in-area mating to obtain seventh generation silkworm eggs;
(8) breeding larvae hatched by seventh generation silkworm eggs by a single moth breeding method, selecting and reserving a moth area with more than 90% of positive individuals by taking individuals with green fluorescence cocoon silks as positive indexes, further preferably selecting the positive individuals in the moth area, selecting and reserving yellow cocoons with cocoon type oblong and shallow girdling, further selecting and reserving individuals with high cocoon silk content within 5% of the selected moth area before ranking, and performing moth in-area mating to obtain eighth generation silkworm eggs;
(9) breeding larvae hatched from eighth generation silkworm eggs by a single moth breeding method, selecting and reserving a moth area with more than 90% of positive individuals by taking individuals with green fluorescence cocoon silks as positive indexes, further preferably selecting the positive individuals in the moth area, selecting and reserving yellow cocoons with cocoon type oblong and shallow girdling, further selecting and reserving individuals with high cocoon silk content within 5% of the selected moth area before ranking, and performing moth in-area mating to obtain ninth generation silkworm eggs;
(10) breeding larvae hatched by ninth generation silkworm eggs by a single moth breeding method, preparing by taking individuals with green fluorescence cocoon silks as positive, selecting and reserving a moth area with more than 98% of positive individuals as a preselected moth area, further preferably selecting positive individuals in the moth area, selecting and reserving white cocoons with uniform colors of cocoon type ellipse and light corset, further performing live pupa reeling on seed cocoons in the preselected moth area, preferably performing interval mating subculture on the moths of individuals with silkworm cocoon reelability, cocoon silk cleanliness and strength respectively ranking within the first 15% to the tenth generation, and obtaining the practical Japanese system special stock 9206MS of the white cocoons with mutation homozygous type of sericin efficiently expressed by silk gland at the rear part;
(11) the practical Japanese system special stock 9206MS for highly expressing mutant homozygous white cocoons of sericin protein by the rear silk glands is hybridized with the Chinese system 9205 variety of the bivoltine practical hybridization combination 9205X 9206 used in the step (4), the special silkworm hybrid 9205X 9206MS for highly synthesizing and secreting pure natural sericin protein by the rear silk glands of the white cocoons is produced, and the main characters of the first generation hybrid bred in the autumn silkworm period in 2015 are shown in table 3.
In the steps (1) to (10), the light white silkworm eggs which leave the non-fertility-retention N4 variety are eliminated after 30 hours of spawning, and light brown or dark brown diapause egg moth areas are selected for reserving the seeds.
By using the method of example 3 of the present invention, after M days, the mutant gene of sericin efficiently expressed in the silk gland of the N4 mutant was introduced into Japanese system of crossing combination of M.times.M days for producing a practical variety of yellow cocoons in the silkworm industry, the mutant gene of sericin efficiently expressed in the silk gland of the N4 mutant and the yellow cocoon color gene of M days were further introduced into Japanese system 9206, and by utilizing the high combining ability and excellent cocoon silk production performance of the preferred practical combination of bivoltine hybridization of white cocoons 9205X 9206, a yellow cocoon cross-bred variety was rapidly bred, and a first generation hybrid species for silkworm production was efficiently produced.
The method of the embodiment 3 of the invention can change the non-diapause of the original N4 variety retained by the N4 mutant into the stable hereditary diapause, and overcomes the problem that the silkworm eggs of the non-diapause variety are not tolerant to storage and need to be continuously raised and preserved all the year round.
The method of example 3 of the present invention can achieve the requirements of excellent practical varieties by using the advantages of high cocoon silk yield (cocoon layer rate and cocoon silk length) and high cocoon silk quality (cleanness and cleanliness) of 9206 varieties while using the advantages of cocoon color, robustness (worm pupa rate) and releasing rate of the japanese system M day of hybridization combination of M-M day of M-medium × M day of practical varieties of yellow cocoon production.
The main cocoon silk characteristics of the first generation hybrid of example 3 in which the posterior silk gland highly expresses sericin are shown in table 3 below.
TABLE 3
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
Claims (6)
1. A breeding method of silkworm varieties with secreted silk fibroin fibrils containing sericin protein is characterized by comprising the following steps:
(1) mating a female moth of a Japanese systematic variety transgenic mutant which synthesizes and secretes sericin from a posterior silk gland as a female parent with a male moth of a Japanese systematic silkworm variety in a bivoltine hybridization combination, and allowing a single moth to lay eggs to obtain a first generation silkworm egg;
(2) breeding larvae hatched by the first generation silkworm eggs by a single moth breeding method, selecting and reserving a moth area with more than 90% of positive individuals by taking green fluorescence individuals with cocoon silks as positive indexes, and further selecting the positive individuals with a cocoon type ellipse and a shallow girdling waist in the selected moth area to perform moth in-area mating to obtain second generation silkworm eggs;
(3) breeding the larvae incubated by the second generation silkworm eggs by a single moth breeding method, selecting and reserving a moth area with more than 90% of positive individuals by taking green fluorescence individuals of cocoon silks as positive indexes, and further selecting the positive individuals with a cocoon type ellipse and a shallow girdling waist in the selected moth area to perform moth in-area mating to obtain third generation silkworm eggs;
(4) breeding and breeding larvae hatched by third-generation silkworm eggs by using the quantity of ants in a moth area, selecting and reserving the moth area with more than 90% of positive individuals as positive indexes by using individuals with green fluorescence cocoon silks, further selecting female positive individuals with a cocoon type of long ellipse and shallow girdling in the selected moth area, mating the female positive individuals with male moths of Japanese systematic silkworm varieties used in the step (1), and allowing single moths to lay eggs to obtain fourth-generation silkworm eggs;
(5) breeding larvae hatched by the fourth generation of silkworm eggs by a single-moth breeding method, selecting and reserving a moth area with more than 90% of positive individuals by taking green fluorescence individuals with cocoon silks as positive indexes, and further selecting the positive individuals with a cocoon type ellipse and a shallow girdling waist in the selected moth area to perform moth in-area mating to obtain fifth generation silkworm eggs;
(6) breeding larvae hatched by the fifth generation of silkworm eggs by using the quantity of ants in the moth area, selecting and reserving the moth area with more than 90% of positive individuals as positive indexes, and further selecting the positive individuals with a cocoon type prolate ellipsoid and a shallow girdling waist in the selected moth area to perform moth in-area mating to obtain sixth generation of silkworm eggs;
(7) breeding larvae hatched by the sixth generation of silkworm eggs by using the quantity of ants in the moth area, selecting and reserving the moth area with more than 90% of positive individuals as positive indexes by using individuals with green fluorescence of cocoon silks, and further selecting the positive individuals with a cocoon type ellipse and a shallow girdling waist in the selected moth area to perform moth in-area mating to obtain seventh generation silkworm eggs;
(8) breeding larvae hatched by the seventh generation of silkworm eggs by a single moth breeding method, selecting and reserving a moth area with more than 90% of positive individuals by taking green fluorescence individuals with cocoon silks as positive indexes, and further selecting the positive individuals with a cocoon type ellipse and a shallow girdling waist in the selected moth area to perform moth in-area mating to obtain eighth generation silkworm eggs;
(9) breeding larvae hatched by the eighth generation of silkworm eggs by a single moth breeding method, selecting and reserving a moth area with more than 90% of positive individuals by taking green fluorescence individuals with cocoon silks as positive indexes, and further selecting the positive individuals with a cocoon type ellipse and a shallow girdling waist in the selected moth area to perform moth in-area mating to obtain ninth generation silkworm eggs;
(10) breeding larvae incubated by the ninth generation of silkworm eggs by a single-moth breeding method, selecting an individual with green fluorescence cocoon silks as a positive index, selecting a moth area with more than 98% of positive individuals as a preselected moth area, selecting a positive individual with a cocoon type oblong and a shallow girdling shape in the preselected moth area, performing live pupa reeling on seed cocoons in the preselected moth area, selecting individuals with a cocoon reelability rate, cocoon silk cleanliness and strength respectively ranked within the first 20% to perform moth interval mating subculture, and obtaining tenth generation silkworm eggs, namely, a practical Japanese system special stock of mutant homozygoty type with sericin efficiently expressed by a rear silk gland; and
(11) hybridizing the mutant homozygous practical Japanese system stock of the rear silk gland high-efficiently expressing sericin with a Chinese system silkworm variety combined by bivoltine hybridization to obtain the silkworm variety of which the secreted silk fibroin fibrils contain sericin.
2. The method for breeding silkworm varieties containing sericin proteins in secreted silk fibroin according to claim 1, wherein: in each step, mated males and females are separated and placed on a spawning paper to spawn.
3. The method for breeding silkworm varieties containing sericin proteins in secreted silk fibroin according to claim 2, wherein: the female moths are mutually isolated by a moth ring made of iron or plastic.
4. The method for breeding silkworm varieties containing sericin proteins in secreted silk fibrils according to claim 2 or 3, wherein the method comprises the steps of: the eggs laid are placed in the environment with the temperature of 25-27 ℃, the relative humidity of 75-80 percent and natural illumination, the silkworm eggs which leave the non-diapause of the Japanese system transgenic mutant variety are eliminated, and the moth area with the diapause is left for reserving the seeds.
5. The method for breeding silkworm varieties with secreted silk fibroin proteins of claim 4, wherein diapause eggs are selected as follows: the diapause eggs gradually change from light yellow to yellow 20-30 hours after spawning, change to light brown 30-50 hours after spawning, and change to dark brown 50 hours later.
6. The method for breeding silkworm varieties containing sericin proteins in secreted silk fibroin according to claim 4, wherein: the egg remained pale yellow and remained unchanged as a non-diapause egg for 1 week after spawning.
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