CN107484722A - Silkworm variety breeding method for secreting silk fibroin fibrils containing sericin protein - Google Patents

Abstract

The invention provides a method for breeding silkworm varieties containing sericin protein in the secreted silk fibrils, which comprises the following steps: using the female moth of the transgenic mutant of the Japanese system variety that synthesizes and secretes sericin protein in the rear silk gland as the female In this paper, the male moths of the silkworm species of the Japanese system were mated with dimorphic hybrids, and further methods such as self-crossing, backcrossing, and marker gene tracking were used to breed the original species of the Japanese system with high-efficiency expression of sericin in the posterior silk gland; The original species of the Japanese system directly crossed with the original species of the Chinese system in the above-mentioned dichroic hybrid combination, and the silkworm hybrid combination variety containing sericin protein in the secreted silk fibrils was bred. The method of the present invention can efficiently and quickly select and breed silkworm hybrids that produce common silkworm cocoons into silkworms that contain sericin protein in the secreted silk fibrils, and then obtain new silkworm silk fibers with changed fiber properties, which are suitable for commercial breeding need.

Description

Silkworm Breeding Method Containing Sericin Protein in Secreted Silk Fibrils

technical field

The invention relates to the field of silkworm variety breeding for special purposes, in particular to a silkworm variety breeding method capable of efficiently and quickly breeding common cocoon-producing silkworm hybrid varieties into mutant silkworm varieties containing sericin protein in the produced silk fibroin fibers.

Background technique

The silkworm is a silk insect that has been used for thousands of years. The silk fiber produced by the cocoon silk produced by the mature larvae of the silkworm is known as the queen of fibers. The silk gland of silkworm is a specialized organ for synthesizing and secreting silk protein, and it is the main target of human domestication, selection and transformation of silkworm. The protein synthesis and secretion function of the silk gland of silkworm determines the yield and quality of silkworm cocoons, and fundamentally affects the main yield and quality level of the whole silk industry. Improving the protein fiber composition synthesized by the silk gland is a major topic of world attention in the sericulture industry.

The silk glands of the silkworm are divided into three functional regions: anterior silk glands, middle silk glands and posterior silk glands. The anterior silk gland is the channel through which silk protein is exported from the body, and the middle and posterior silk glands are the synthesis and secretion sites of sericin protein and silk fibroin protein, respectively. The silk fibroin protein synthesized by the posterior silk gland is a kind of β-fold crystal structure, tough and elastic, water-insoluble high-molecular-weight fiber protein, which accounts for 70-80% of the total cocoon silk, and is the basic raw material for making silk and other textiles. A complex composed of silk fibroin light chain (Fib-L), heavy chain (Fib-H) and P25 protein at a molar ratio of 6:6:1 is used as the basic structural unit of silk fibroin (Inoue, 2000), P25 protein is also called fibrohexamerin (Fhx/P25) (Yamaguchi et al., 1989; Sprague 1975; Couble et al., 1983). The silk fibroin protein synthesized in the posterior silk gland cells in the silkworm larval stage 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. During silkworm larva spinning and cocooning after maturation, silk fibroin protein molecules continuously aggregate into fibrils with a diameter of about 0.1 μm and an irregular circular section. Silk fibroin "core". Fibrils are composed of crystalline fibers and non-crystalline fiber molecular aggregates alternately connected in series along the uranium direction. The fibrils are close to each other, firmly connected by the side chains of peripheral molecules, and there is no interstitial filling. This is the molecular basis for the special luster of silk fibroin and the ability to produce ultrafine protein fibers.

During the process of forming silk fibrils and moving to the front, the silk fibrils in the silk gland of the silkworm are wrapped with 4 layers of sericin in the middle silk gland, and the innermost layer is the 4 layers encoded by the sericin gene Ser1. Four kinds of sericin proteins translated by Ser1A (2.8kb), Ser1B (4kb), Ser1C (10kb) and Ser1D (9kb) (Michaille et al., 1986), the outer layer is the 3 sericin proteins encoded by Ser2 and Ser3 genes Ser2A (5.4kb), Ser2B (3.1kb) (Kludkiewicz et al., 2009) and Ser3 (9kb) are translated into three sericin proteins (Takasu et al., 2007). Silkworm sericin protein accounts for 20-30% of the total cocoon silk, contains β structure, but has no α helical structure, the molecular conformation is mainly random coil, and the molecular space structure is loose and disordered. The contents of glycine, alanine and tyrosine in sericin were significantly lower than that of silk fibroin, and the content of free amino acids was significantly higher than that of silk fibroin. There are many long side chain amino acids such as arginine, lysine, glutamic acid, methionine, tryptophan and tyrosine on the sericin chain, and the surface of the polypeptide chain also contains -OH, -COOH, -NH ₂ and other polar hydrophilic groups. Therefore, sericin has excellent functions of moisture absorption, moisturizing and odor absorption. Among them, the water absorption and fluidity of the sericin protein SER3 were significantly higher than those of the four sericin proteins translated by the Ser1 gene. Studies have shown that silkworm sericin protein also has good antioxidant, antibacterial and anti-ultraviolet functions. The content of serine in sericin protein is as high as 30% or more (Zhu Liangjun et al., 1997), and its role in cosmetics is similar to the natural moisturizing factor in the stratum corneum of human skin.

In the process of reeling and scouring in the production of silkworm silk textiles, the sericin on the surface of the silk fiber is almost completely removed, which is also a technological requirement to improve the luster of silk and ensure the uniformity of dyeing. The silk fabric without removing the outer layer sericin not only has the problems of poor luster and uneven dyeing, but also has the problems of poor fabric softness and poor wearing comfort. Although the water absorption of silk fiber molecules after degumming is still nearly 30% higher than that of cotton fibers, silk is not as absorbent as cotton cloth when taken. This is due to the high price of silkworm silk, which is almost all thin fabrics. Therefore, adding sericin protein into silk fibroin protein is expected to change the fiber structure and physical and chemical properties of silk fibroin, and realize the purpose of changing fiber function, especially the dual characteristics of silk fibroin and sericin are used at the same time. Sericin remains on the fiber surface, or a functional utilization that cannot be achieved by sericin coating on the surface of silk fibers or fabrics.

Based on the current understanding of silk protein structural genes in silkworms, if the synthesis efficiency and molecular ratio of different structural components of silk protein in the silk gland can be induced, it may subvert the known properties of silk protein fibers. Sericulture researchers have also been trying to transfer exogenous high-yield, high-quality or special performance genes to silk glands of silkworms for expression, the purpose of which is to greatly change the fiber properties of silk proteins, such as transgenic spider silk genes (Wen et al., 2010; Teulé et al., 2012; Zhang Yuansong et al., 2012), transgenic silk gene (Li Zhengang & Fan Jiuge, 1997), etc. At present, relevant researches are all focused on introducing exogenous protein fiber coding genes or developmental regulatory genes into the silkworm genome, so as to achieve the purpose of changing silk protein synthesis efficiency and silk protein fiber properties. However, silkworms transfected with exogenous genes, without exception, have a series of problems such as a significant decrease in the synthesis and secretion efficiency of silk protein, and a series of problems such as the fiber clarity or strength of silk protein fiber is not significantly improved, and even the performance is reduced. The degree hinders the further industrialization research and application of related research work (Wang et al., 2015). The inventors connected amino acids with nanometer quantum dots, and found that the absorption efficiency of alanine by silk gland cells was significantly higher than that of glycine, although the content of glycine in sericin and silk protein was 150% and 280% of that of alanine ( Xing et al., 2016). The present inventors conducted a transgene-induced posterior silk gland degenerate system and normal silk gland transcriptomic investigation and found that there were 891 significantly differentially expressed genes involved in protein synthesis, processing, secretion and transportation of cells, as well as energy supply of cells, etc. aspect (Wang et al., 2015). Therefore, the present inventors believe that the above-mentioned problem of significantly decreased silk protein synthesis and secretion efficiency of the transgenic silkworm is related to the amino acid utilization preference of the silk gland cells of the silkworm. Therefore, it is very necessary to invent a transgenic method that adapts to the amino acid utilization preference of silk gland cells of silkworm.

The present inventors selected sericin specifically expressed in the middle silk gland of silkworm with tissue expression specificity, realized high-efficiency expression in silkworm posterior silk gland, and prepared a transgenic mutant silkworm that synthesizes and secretes sericin in the posterior silk gland ( Invention name: Preparation method of silkworm whose posterior silk gland can synthesize and secrete hydrophilic sericin, Chinese patent application number: 201610348866.0). The transgenic mutant with highly efficient expression of sericin in the rear silk gland obtained by using the above patented technology, although it is a mutant homozygous silkworm, has various excellent cocoon silk production such as silk quantity, fineness, clarity, cocoon color, etc. The potential of traits, but it does not meet the production practical needs of modern sericulture. It is a genetic resource called material in the field of silkworm breeding. The mutant retains the non-diapause and short growth period of the wild-type original variety suitable for transgenic operations. , Low amount of cocoon silk, poor development uniformity and other problems, cocoon silk quality such as cocoon silk fineness, clarity and strength also need directional transformation. On the other hand, the silkworm species used in sericulture production at home and abroad are preferably the first-generation hybrids of the dimorphic Chinese system and the Japanese system, using the Chinese patent application "Posterior silk glands can synthesize and secrete hydrophilic sericin The preparation method of the silkworm "(Application No.: 201610348866.0) The transgenic mutant silkworm of the Japanese systemic variety lacks the high combining ability of the Chinese systemic inbreeding variety, and cannot efficiently produce a generation of hybrids for silkworm production.

Therefore, the development of silkworm mutants that synthesize and secrete sericin in the posterior silk gland is very necessary and has great commercial prospects.

Contents of the invention

In order to solve the above-mentioned technical problems, the present invention provides a method for breeding silkworm varieties containing sericin protein in the secreted silk fibrils, which can rapidly and directionally transform the cocoon fineness, clarity and strength of transgenic mutants.

To achieve the above object, the present invention adopts the following technical solutions:

A silkworm variety breeding method containing sericin protein in secreted silk fibrils, comprising the following steps:

(1) The female moth of the transgenic mutant of the Japanese lineage adiapause variety that synthesizes and secretes sericin in the rear silk gland is used as the female parent, and mated with the male moth of the Japanese lineage silkworm variety in the duality-optimized cross combination, and the single moth Lay eggs and obtain the first generation of silkworm eggs; Among them, the transgenic mutant of the Japanese systemic non-diapause variety that synthesizes and secretes sericin in the posterior silk gland is prepared from the Japanese systemic non-diapause variety according to the method of Chinese patent application 201610348866.0 .

(2) Raise the hatched larvae of the first-generation silkworm eggs with the method of single moth breeding, take the individual with cocoon silk as a positive indicator, and select the moth area where positive individuals account for more than 90%, and further optimize the selected moth area The positive individuals with inner cocoon-shaped long ellipse and shallow girdle are mated in the moth area to obtain the second generation of silkworm eggs, and the cocoon silk quantity and cocoon silk quality of the positive individuals are respectively ranked within the top 20% of the selected moth area ;

(3) Raise the hatched larvae of the second-generation silkworm eggs with the method of single moth breeding, take the individual with cocoon silk as a positive indicator, and select the moth area where positive individuals account for more than 90%, and further optimize the selected moth area The positive individuals with inner cocoon-shaped oblong and shallow girdle are mated in the moth area to obtain the third generation of silkworm eggs, and the cocoon silk quantity and cocoon silk quality of the positive individuals are respectively ranked within the top 20% of the selected moth area ;

(4) The larvae hatched from the third-generation silkworm eggs are reared by ants in the moth area, and the individual with cocoon silk showing green fluorescence is a positive indicator, and the moth area where the positive individuals account for more than 90% is selected, and the selected moth is further selected A female positive individual with a cocoon-shaped oblong and shallow girdle in the area, the female positive individual is mated with the male moth of the Japanese silkworm variety used in step (1), that is, backcrossing treatment, a single moth lays eggs, and the fourth generation silkworm is obtained Eggs, wherein the cocoon silk quantity and cocoon silk quality of the female positive individual are respectively ranked within the top 20% in the selected moth area;

(5) Raise the hatched larvae of the fourth-generation silkworm eggs with the method of single moth breeding, take the individual with cocoon silk as green fluorescence as the positive index, select the moth area where positive individuals account for more than 90%, and further optimize the selected moth area The positive individuals with inner cocoon-shaped long ellipse and shallow girdle are mated in the moth area to obtain the fifth generation of silkworm eggs, and the cocoon silk quantity and cocoon silk quality of the positive individuals are respectively ranked within the top 20% of the selected moth area ;

(6) The hatched larvae of the fifth generation silkworm eggs are reared by ants in the moth area, and the individual with cocoon silk showing green fluorescence is a positive indicator, and the moth area where the positive individuals account for more than 90% is selected, and the selected one is further preferably selected. The positive individuals with cocoon-shaped oblong and shallow girdle in the moth area were mated in the moth area to obtain the sixth generation of silkworm eggs, and the cocoon silk quantity and cocoon silk quality of the positive individuals ranked top 20 in the selected moth area respectively within %;

(7) Raise the hatched larvae of the sixth-generation silkworm eggs with ants in the moth area, take the cocoon silk as a positive indicator, and select the moth area where the positive individuals account for more than 90%, and further optimize the selected moth The cocoon-shaped oblong and shallow girdle positive individuals in the area were mated in the moth area to obtain the seventh-generation silkworm eggs, and the cocoon silk quantity and cocoon silk quality of the positive individuals were respectively ranked in the top 20% of the selected moth area within;

(8) Raise the hatched larvae of the seventh-generation silkworm eggs with a single moth breeding method, take the individual with cocoon silk showing green fluorescence as a positive indicator, and select the moth area where positive individuals account for more than 90%, and further optimize the selected moth area Positive individuals with inner cocoon-shaped long ellipse and shallow girdle are mated in the moth area to obtain the eighth generation of silkworm eggs, wherein the cocoon silk quantity and cocoon silk quality of the positive individuals are respectively ranked within the top 20% of the selected moth area ;

(9) Raise the hatched larvae of the eighth generation of silkworm eggs with the method of single moth breeding, take the individual with cocoon silk showing green fluorescence as a positive indicator, select the moth area where the positive individual accounts for more than 90%, and further optimize the selected moth The cocoon-shaped oblong and shallow girdle positive individuals in the area were mated in the moth area to obtain the ninth generation of silkworm eggs, and the cocoon silk quantity and cocoon silk quality of the positive individuals were respectively ranked in the top 20% of the selected moth area within;

(10) Raise the hatched larvae of the ninth generation of silkworm eggs with the method of single moth breeding, take the individual with cocoon silk as green fluorescence as the positive index, choose the moth area with more than 98% positive individuals as the pre-selected moth area, and select In the pre-selected moth area, the cocoon shape is oblong, the shallow waist, and the cocoon color are basically the same, and the positive individuals of the silkworm cocoon that meet the breeding target (cocoon silk quantity and cocoon silk quality are respectively ranked in the top 20% or less in the selected moth area) , then carry out living pupa silk reeling to the seed cocoons in the pre-selected moth area, and select individuals with high cocoon unwinding rate, high cocoon silk clarity and strength, and cocoon silk fineness in line with the breeding target to perform moth interval mating (cocoon unwinding) Individuals ranked within the top 20% in rate, cocoon silk clarity and strength respectively), obtained the tenth generation of silkworm eggs, that is, the practical Japanese system-specific original species of the mutant homozygous highly expressed sericin protein in the posterior silk gland;

(11) Hybridize the practicable original species of the Japanese system that is homozygous for the mutation that highly expresses sericin in the posterior silk gland, and the Chinese silkworm variety that is used in step (1) to obtain the secreted silk. Silkworm hybrids containing sericin protein in the fibrils.

Preferably, in each step, the mated male and female moths are separated, and the female moths are placed on the oviposition paper to lay eggs.

Preferably, the female moths are separated from each other with iron or plastic moth rings.

Preferably, the eggs laid by the female moths are placed in an environment of 25°C-27°C, 75%-80% relative humidity and natural light, and the non-diapause silkworm eggs of the N4 variety left behind are eliminated, and the eggs with good diapause are left. The moth area keeps seeds.

In the present invention, the diapause eggs gradually change from light yellow to yellow in 20 to 30 hours after laying eggs, light brown in 30 to 50 hours, and dark brown in 50 hours.

In the present invention, non-diapause silkworm eggs remain light yellow and do not change color within one week after laying eggs.

By means of the above solution, compared with the prior art, the present invention has at least the following advantages:

The present invention provides a silkworm variety breeding method that contains sericin protein in the secreted silk fibrils, the method can utilize the preferred hybrid combination, such as the hybrid combination of sericulture to produce practical varieties with high combining ability and excellent cocoon Silk production performance, by introducing the mutant gene that highly expresses sericin protein in the posterior silk gland into the Japanese system, hybrid varieties can be bred quickly, and a generation of hybrids for silkworm production can be efficiently produced without the need for simultaneous cultivation of posterior silk glands The practical Chinese system-specific original species of the mutant homozygous high-efficiency expression sericin protein can significantly reduce the breeding workload.

The method of the present invention can change the non-diapause property of the original variety suitable for transgenic use reserved by the transgenic mutant into the diapause property of stable inheritance, and overcome the non-diapause property of silkworm eggs that are not resistant to preservation and require continuous feeding and conservation all year round question.

The method of the invention can improve the problems of short growth period, low amount of cocoon silk, poor development uniformity and the like of the original variety retained by the mutant.

The method of the invention can rapidly and directionally transform cocoon silk properties such as cocoon silk fineness, clarity and strength of mutants, so as to meet the requirements of practical varieties.

detailed description

The present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following examples are only used to illustrate the present invention, rather than limit the protection scope of the present invention.

Example 1

Breeding of yellow cocoon hybrids with high expression of sericin in the posterior silk gland

Present embodiment 1 comprises the following steps:

(1) The Japanese system N4 variety that synthesizes and secretes sericin in the posterior silk gland is prepared according to the method of Chinese patent application 201610348866.0 to obtain a transgenic mutant. The female moth of the mutant is used as the female parent, and the yellow cocoon-bearing moth is used as the female parent. The male moths of the Japanese system M in the dimorphic practical hybrid combination Mzhong×Mri (name of silkworm variety, Caicoon No. 1) mate, and a single moth lays eggs to obtain the first generation of silkworm eggs;

(2) Raise the hatched larvae of the first generation of silkworm eggs with the method of single moth breeding, take the individual with cocoon silk as green fluorescence as the positive indicator, select the moth area with more than 90% positive individuals, and further optimize the positive individual in the moth area , select yellow cocoons with oblong cocoon shape and shallow waist, and then further select individuals with high cocoon silk content within the top 20% of the selected moth area for mating in the moth area to obtain the second generation Silkworm eggs;

(3) Raise the hatched larvae of the second-generation silkworm eggs with the method of raising single moths, take the individuals whose cocoon silk is green fluorescent as positive indicators, and select the moth areas where positive individuals account for more than 90% of the moths, and further optimize the moths in the selected moths. Yellow cocoons with oblong cocoon shape and shallow waist, and then further select individuals with high cocoon silk content within the top 10% of the selected moth area for mating in the moth area to obtain the third generation of silkworm eggs;

(4) The larvae hatched by the third generation of silkworm eggs are reared by ants in the moth area. The individual with cocoon silk showing green fluorescence is a positive indicator, and the moth area where the positive individuals account for more than 90% is selected to further select the selected moth area. Cocoon-shaped long ellipse, yellow cocoon with shallow waist, and then further select the female positive individuals whose high cocoon silk content ranks within the top 10% in the selected moth area. Female positive individuals and steps (1 ) The male moth of the Japanese system M in M × M day used in M is backcrossed, and a single moth lays eggs to obtain the fourth generation of silkworm eggs;

(5) Raise the larvae hatched by the fourth generation of silkworm eggs with the method of single moth breeding, take the individual with cocoon silk as green fluorescence as the positive indicator, select the moth area where positive individuals account for more than 90%, and further optimize the selected moth area The yellow cocoon with long oval cocoon shape and shallow waist, and then further select individuals with high cocoon silk content within the top 20% of the selected moth area for mating in the moth area to obtain the fifth generation of silkworm eggs;

(6) The larvae hatched by the fifth generation of silkworm eggs are reared by ants in the moth area, and the individual with cocoon silk showing green fluorescence is a positive indicator, and the moth area where positive individuals account for more than 90% is selected to stay, and the selected moth area is further optimized The yellow cocoons with oblong inner cocoon shape and shallow waist, and then further select individuals with high cocoon silk content within the top 10% of the selected moth area for mating in the moth area to obtain the sixth generation of silkworm eggs ;

(7) The larvae hatched by the sixth generation of silkworm eggs are reared by ants in the moth area, and the individual with cocoon silk showing green fluorescence is a positive indicator, and the moth area where positive individuals account for more than 90% is selected to stay, and the selected moth area is further optimized The yellow cocoons with oblong inner cocoon shape and shallow girdle, further select individuals with high cocoon silk content within the top 10% of the selected moth area for mating in the moth area to obtain the seventh generation of silkworm eggs ;;

(8) Raise the hatched larvae of the seventh-generation silkworm eggs with the method of single-moth breeding, and take the individuals whose cocoon silk is green fluorescent as positive indicators, and select the moth areas where positive individuals account for more than 90%, and further optimize the selected moth-retaining areas The yellow cocoons with oblong cocoon shape and shallow waist, and further select individuals with high cocoon silk content within the top 5% of the selected moth area for mating in the moth area to obtain the eighth generation of silkworm eggs ;

(9) Raise the hatched larvae of the eighth generation silkworm eggs with the method of single moth breeding, take the individual with cocoon silk showing green fluorescence as a positive indicator, select the moth area where positive individuals account for more than 90%, and further optimize the selected moth area The yellow cocoons with oblong cocoon shape and shallow waist were further selected to retain individuals with high cocoon silk content within the top 5% of the selected moth area for mating in the moth area to obtain the ninth generation of silkworm eggs ;

(10) Raise the hatched larvae of the ninth generation of silkworm eggs with the method of single moth breeding, take the individual with cocoon silk as green fluorescence as the positive indicator, select the moth area with more than 98% positive individuals as the pre-selected moth area, and further optimize the moth area For the positive individuals in the cocoon, choose cocoon-shaped oblong cocoons with uniform yellow cocoons, and then carry out live pupa silk reeling on the seed cocoons in the pre-selected moth area, and optimize the cocoon unwinding rate, cocoon silk clarity and strength Individuals ranked within the top 15% perform interval mating of moths, until the tenth generation, that is, to obtain the practicable original species of the yellow cocoon with the mutation homozygous highly expressing sericin in the posterior silk gland MS day;

(11) The practical Japanese system special original seed MS day with yellow cocoon-bearing mutation homozygous for highly expressing sericin in the posterior silk gland and the duality practical hybridization combination silkworm variety M middle × used in step (1) M day’s white cocoon-bearing Chinese system M species hybridization, producing a special type of silkworm hybrid that efficiently synthesizes and secretes pure natural sericin in the posterior silk gland with yellow cocoon M x MS day, 2015 mid-autumn silkworm breeding The main characters of the first-generation hybrids are listed in Table 1.

In the above-mentioned steps (1) to (10), after 30 hours after laying eggs, eliminate the non-diapause light yellow silkworm eggs of the N4 variety, and select light brown or dark brown diapause egg moth areas to reserve seeds.

Using the method of Example 1 of the present invention, the high combining ability and excellent cocoon silk production performance of the M middle × M day hybrid combination of the yellow cocoon production practical variety in sericulture can be used to efficiently express silk in the rear silk gland of the N4 mutant. The mutant gene of gelatin was introduced into the Japanese system Mday, and the yellow cocoon hybrid combination variety was rapidly bred, and the first-generation hybrid variety used for silkworm production was produced efficiently.

Using the method of Example 1 of the present invention, the non-diapause property of the N4 original variety retained by the N4 mutant can be changed into a stable genetic diapause property, and the silkworm eggs of the non-diapause species are not resistant to preservation, and need to be continuously fed and maintained all year round. kind of problem;

Using the method of Example 1 of the present invention, the problems of short growth period, low amount of cocoon silk, and poor development uniformity of the N4 original variety retained by the N4 mutant can be improved;

Using the method of Example 1 of the present invention, the cocoon silk quality, such as cocoon silk fineness, clarity and strength, of the N4 mutant can be rapidly and directionally modified to meet the requirements of practical varieties.

The following table 1 shows the main cocoon silk traits of the first-generation hybrids that highly express sericin in the posterior silk glands of Example 1.

Table 1

Example 2

Breeding of white cocoon hybrids with high expression of sericin in the posterior silk gland

Present embodiment 2 comprises the following steps:

(1) The female moth of the transgenic mutant of the Japanese system N4 variety (prepared by the method of Chinese patent application 201610348866.0 by using the Japanese system N4 variety) that synthesizes and secretes sericin from the posterior silk glands is used as the female parent, and forms a white cocoon with the female moth The Japanese system 9206 male moths of the dimorphic practical hybrid combination 9205×9206 mate, and a single moth lays eggs to obtain the first generation of silkworm eggs;

(2) Raise the hatched larvae of the first generation of silkworm eggs with the method of single moth breeding, take the individual with cocoon silk as green fluorescence as the positive indicator, select the moth area with more than 90% positive individuals, and further optimize the positive individual in the moth area , select white cocoons with long oval cocoon shape and shallow waist, and then further select individuals with high cocoon silk content within the top 20% of the selected moth area for mating in the moth area to obtain the second generation Silkworm eggs;

(3) Raise the hatched larvae of the second-generation silkworm eggs with the method of single-moth breeding, take the individuals whose cocoon silk is green fluorescent as positive indicators, select and retain the moth areas where positive individuals account for more than 90%, and further optimize the positive individuals in the moth areas , select white cocoons with long oval cocoon shape and shallow waist, and then further select individuals with high cocoon silk content within the top 10% of the selected moth area to mate in the moth area to obtain the third generation Silkworm eggs;

(4) The larvae hatched by the third generation of silkworm eggs are reared by ants in the moth area, and the individuals with cocoon silk showing green fluorescence are positive indicators, and the moth areas where the positive individuals account for more than 90% are selected to further select the selected moth area For the positive individuals within the cocoon, the cocoon-shaped long ellipse and white cocoon with a shallow waist were selected, and then the female positive individuals with high cocoon silk content within the top 10% of the selected moth area were selected, and the female positive individuals were compared with The male moth of the Japanese system 9206 variety used in step (1) is backcrossed, and a single moth lays eggs to obtain the fourth generation of silkworm eggs;

(5) Raise the hatched larvae of the fourth-generation silkworm eggs with the method of single moth breeding, take the individual with green fluorescence as the positive indicator, select the moth area with more than 90% positive individuals, and further optimize the positive individuals in the moth area , select white cocoons with long oval cocoon shape and shallow waist, and then further select individuals with high cocoon silk content within the top 20% of the selected moth area for mating in the moth area to obtain the fifth generation Silkworm eggs;

(6) The larvae hatched by the fifth generation of silkworm eggs are reared by ants in the moth area, and the individual with cocoon silk showing green fluorescence is a positive indicator, and the moth area with more than 90% positive individuals is selected to further optimize the positive in the moth area Individuals, choose cocoon-shaped long oval, white cocoons with shallow waist, and then further select individuals with high cocoon silk content within the top 10% of the selected moth area for mating in the moth area, and get the sixth substitute silkworm eggs;

(7) The larvae hatched by the sixth generation of silkworm eggs are reared by ants in the moth area, and the individual with cocoon silk showing green fluorescence is a positive indicator, and the moth area with more than 90% positive individuals is selected, and the positive in the moth area is further optimized Individuals, choose white cocoons with cocoon-shaped long ellipse and shallow waist, and then further select individuals with high cocoon silk content within the top 10% of the selected moth area for mating in the moth area, and get the seventh substitute silkworm eggs;

(8) Raise the hatched larvae of the seventh-generation silkworm eggs with the method of single-moth breeding, and take the individuals whose cocoon silk is green fluorescent as positive indicators, and select the moth area with more than 90% positive individuals, and further optimize the positive individuals in the moth area , select white cocoons with cocoon-shaped long ellipse and shallow waist, and then further select individuals with high cocoon silk content within the top 5% of the selected moth area for mating in the moth area, and obtain the eighth generation Silkworm eggs;

(9) Raise the hatched larvae of the eighth generation silkworm eggs with the method of single moth breeding, take the individual with cocoon silk showing green fluorescence as a positive indicator, select the moth area with more than 90% positive individuals, and further optimize the positive individuals in the moth area , select white cocoons with cocoon-shaped long ellipse and shallow waist, and further select individuals with high cocoon silk content within the top 5% of the selected moth area for mating in the moth area, and obtain the ninth generation Silkworm eggs;

(10) Raise the hatched larvae of the ninth generation of silkworm eggs with the method of single moth breeding, take the individual with cocoon silk as green fluorescence as the positive indicator, select the moth area with more than 98% positive individuals as the pre-selected moth area, and further optimize the moth area For the positive individuals in the cocoon, select the white cocoons with long oval cocoon shape and shallow waist with uniform color, and then carry out live pupa silk reeling on the seed cocoons in the pre-selected moth area, and optimize the cocoon unwinding rate, cocoon silk clarity and strength Individuals ranked within the top 15% perform interval mating of moths, until the tenth generation, that is, to obtain the practicable original species of white cocoons with a mutation homozygous for highly expressing sericin in the posterior silk gland 9206S;

(11) Combining 9205×9206, the 9205×9206 practicable Japanese system-specific original seed 9206S of the mutant genotype homozygous for the high-efficiency expression of sericin in the posterior silk gland with the white cocoon The hybridization of the Chinese system 9205 variety, producing a special type silkworm hybrid 9205×9206S with white cocoon-forming posterior silk glands that efficiently synthesizes and secretes pure natural sericin, and the main characteristics of the first generation hybrid raised in the mid-autumn silkworm stage in 2015 are shown in Table 2 .

In the above-mentioned steps (1) to (10), after 30 hours after laying eggs, weed out non-diapause light white silkworm eggs of N4 varieties, and select light brown or dark brown diapause egg moth areas to reserve seeds.

Using the method of Example 2 of the present invention, it is possible to efficiently express sericin in the posterior silk gland of the N4 mutant by utilizing the high combining ability and excellent cocoon silk production performance of the preferred dichroic practical hybrid combination 9205×9206 with white cocoons The mutant gene of the protein was introduced into the Japanese system 9206, and the white cocoon hybrid variety was quickly bred, and the first generation of hybrids for silkworm production was produced efficiently.

Using the method of Example 2 of the present invention, it is possible to change the non-diapause property of the N4 original variety retained by the N4 mutant into a stable genetic diapause property, and overcome the non-diapause variety silkworm eggs that are not resistant to preservation and need to be continuously raised all year round conservation issues.

Using the method of Example 2 of the present invention, the problems of the N4 original variety retained by the N4 mutant, such as short growth period, low amount of cocoon silk, and poor developmental uniformity, can be improved.

Using the method of Example 2 of the present invention, the cocoon silk quality such as cocoon silk fineness, clarity and strength of the N4 mutant can be rapidly and directionally transformed to meet the requirements of practical varieties.

Using the method of Example 2 of the present invention, the yellow cocoon silk of the N4 mutant can be quickly and directionally transformed into white cocoon silk, meeting the requirements of practical white cocoon varieties.

Table 2 below shows the main cocoon silk traits of the first-generation hybrid that highly expresses sericin in the posterior silk gland of Example 2.

Table 2

Example 3

Breeding of special yellow cocoon high-silk hybrid varieties with high-efficiency expression of sericin in the posterior silk gland

Present embodiment 3 comprises the steps:

(1) The female moth of the Japanese system N4 variety transgenic mutant (prepared by the method of Chinese patent application 201610348866.0 by using the Japanese system N4 variety) that synthesizes and secretes sericin from the posterior silk glands is used as the female parent, and has yellow cocoons The dimorphic practical hybridization combination silkworm variety Mzhong×Mday Japanese system Mday male moth mating, single moth lays eggs, and obtains the first generation of silkworm eggs;

(2) Raise the larvae hatched by the first-generation eggs with the method of raising single moths, and the individuals with cocoon silk showing green fluorescence are positive indicators, and select the moth districts with more than 90% positive individuals, and further optimize the positive individuals in the moth districts, Select yellow cocoons with oblong cocoon shape and shallow waist, and then further select individuals with high cocoon silk content within the top 5% of the selected moth area for mating in the moth area to obtain the second generation of silkworm eggs ;

(3) Raise the hatched larvae of the second-generation silkworm eggs with the method of single-moth breeding, take the individuals whose cocoon silk is green fluorescent as positive indicators, select and retain the moth areas where positive individuals account for more than 90%, and further optimize the positive individuals in the moth areas , select yellow cocoons with oblong cocoon shape and shallow waist, and then further select individuals with high cocoon silk content within the top 10% of the selected moth area to mate in the moth area to obtain the third generation of silkworms egg;

(4) The larvae hatched by the third-generation silkworm eggs are reared by ants in the moth area, and the individuals whose cocoon silk is green fluorescent are used as positive indicators, and the moth areas where the positive individuals account for more than 90% are selected, and the positives in the selected moth area are further selected. Individuals, choose cocoon-shaped long oval, yellow cocoons with shallow waist, and then further select female positive individuals with high cocoon silk content within the top 10% of the selected moth area, female positive individuals and white knots Cocoon dimorphism practical hybrid combination 9205×9206 Japanese system 9206 male moth mating, single moth lays eggs, and obtains the fourth generation of silkworm eggs;

(5) Raise the hatched larvae of the fourth-generation silkworm eggs with the method of single moth breeding, take the individual with green fluorescence as the positive indicator, select the moth area with more than 90% positive individuals, and further optimize the positive individuals in the moth area , select yellow cocoons with oblong cocoon shape and shallow waist, and then further select individuals with high cocoon silk content within the top 10% of the selected moth area to mate in the moth area to obtain the fifth generation silkworm egg;

(6) The larvae hatched by the fifth generation of silkworm eggs are reared by ants in the moth area, and the individual with cocoon silk showing green fluorescence is a positive indicator, and the moth area with more than 90% positive individuals is selected to further optimize the positive in the moth area Individuals, the yellow cocoons with oblong cocoon shape and shallow waist were selected, and then the individuals with high cocoon silk quantity remaining within the top 10% in the selected moth area were selected for mating in the moth area, and the sixth generation was obtained Silkworm eggs;

(7) The larvae hatched by the sixth generation of silkworm eggs are reared by ants in the moth area, and the individual with cocoon silk showing green fluorescence is a positive indicator, and the moth area with more than 90% positive individuals is selected, and the positive in the moth area is further optimized Individuals, the cocoon-shaped long oval, yellow cocoon with a shallow waist were selected, and then the individuals with high cocoon silk content within the top 5% of the selected moth area were selected for mating in the moth area, and the seventh generation was obtained Silkworm eggs;

(8) Raise the hatched larvae of the seventh-generation silkworm eggs with the method of single-moth breeding, and take the individuals whose cocoon silk is green fluorescent as positive indicators, and select the moth area with more than 90% positive individuals, and further optimize the positive individuals in the moth area , select yellow cocoons with oblong cocoon shape and shallow waist, and then further select individuals with high cocoon silk content within the top 5% of the selected moth area for mating in the moth area, and obtain the eighth generation of silkworms egg;

(9) Raise the hatched larvae of the eighth generation silkworm eggs with the method of single moth breeding, take the individual with cocoon silk showing green fluorescence as a positive indicator, select the moth area with more than 90% positive individuals, and further optimize the positive individuals in the moth area , select yellow cocoons with oblong cocoon shape and shallow girdle, and further select individuals with high cocoon silk content within the top 5% of the selected moth area for mating in the moth area to obtain the ninth generation of silkworms egg;

(10) Raise the hatched larvae of the ninth generation of silkworm eggs with the method of single-moth breeding, and use the individual with cocoon silk as green fluorescence as a positive preparation, select the moth area with more than 98% positive individuals as the pre-selected moth area, and further optimize the moth area For the positive individuals in the cocoon, select the white cocoons with long oval cocoon shape and shallow waist with uniform color, and then carry out live pupa silk reeling on the seed cocoons in the pre-selected moth area, and optimize the cocoon unwinding rate, cocoon silk clarity and strength Individuals ranked within the top 15% perform interval mating of moths, until the tenth generation, that is, to obtain the practicable original species of white cocoons with a mutation homozygous for highly expressing sericin in the posterior silk gland 9206MS;

(11) The Chinese system 9205×9206, which combines the practical Japanese system 9206MS of the mutant homozygous white cocoon-bearing species with high expression of sericin in the posterior silk gland and the duality practical hybrid used in step (4) 9205×9206 The 9205 variety was crossed to produce a special type silkworm hybrid 9205×9206MS whose posterior silk glands with white cocoons efficiently synthesized and secreted pure natural sericin.

In the above-mentioned steps (1) to (10), after 30 hours after laying eggs, weed out non-diapause light white silkworm eggs of N4 varieties, and select light brown or dark brown diapause egg moth areas to reserve seeds.

Using the method of Example 3 of the present invention, the mutant gene that highly expresses sericin in the rear silk gland of the N4 mutant is introduced into the Japanese system of the M middle × M day hybrid combination of the yellow cocoon production practical variety in the sericulture industry. N4 mutant posterior silk gland highly expresses the mutant gene of sericin and M day yellow cocoon gene into Japanese system 9206, and utilizes the high combining ability and excellent combination of 9205×9206, which is a practical hybrid combination with optimal white cocoons. Excellent cocoon silk production performance, rapid breeding of yellow cocoon hybrid varieties, efficient production of a generation of hybrids for silkworm production.

The method of Example 3 of the present invention can change the non-diapause property of the N4 original variety retained by the N4 mutant into a stable genetic diapause property, and overcome the non-diapause variety silkworm eggs that are not resistant to preservation and require continuous breeding and conservation throughout the year The problem.

The method of Example 3 of the present invention can utilize the advantages of the cocoon color, robustness (chrysalis rate) and unwinding rate of the Japanese system M day of the M middle × M day hybrid combination of the yellow cocoon to produce practical varieties, and utilize the advantages of the 9206 variety The advantages of high cocoon silk yield (cocoon layer rate and cocoon silk length) and cocoon silk quality (cleanliness and clarity) meet the requirements of excellent practical varieties.

The following table 3 shows the main cocoon silk traits of the first-generation hybrids that highly express sericin in the posterior silk glands of Example 3.

table 3

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention. It should be pointed out that for those of ordinary skill in the art, some improvements can also be made without departing from the technical principle of the present invention. and modifications, these improvements and modifications should also be considered as the protection scope of the present invention.

Claims (6)

1. the cultivated silkworm breed variety selection containing sericine in a kind of fibroin fibril of secretion, it is characterised in that Comprise the following steps：

(1) using posterior division of silkgland synthesize and secrete sericin Japanese system kind genetically modified mutant female moth as Female parent, mated with the male moth of the Japanese system Silkworm varieties in bivoltine cross combination, single moth egg, obtain the Generation silkworm seed；

(2) larva of the first generation egg-incubation is raised in one batch rearing method, with silk in green fluorescence Body is positive indication, selects and remain the moth area that positive individuals account for more than 90%, cocoon type in moth area selected by further selection Long oval, shallow positive individuals with a tight waist mated in moth area, obtain second generation silkworm seed；

(3) larva of the second generation egg-incubation is raised in one batch rearing method, with silk in green fluorescence Body is positive indication, selects and remain the moth area that positive individuals account for more than 90%, cocoon type in moth area selected by further selection Long oval, shallow positive individuals with a tight waist mated in moth area, obtain third generation silkworm seed；

(4) larva for raising the third generation egg-incubation is educated with moth area ant amount, the individual for being in green fluorescence with silk For positive indication, the moth area that positive individuals account for more than 90% is selected and remain, further cocoon type is grown in moth area selected by selection Oval, shallow female positive individuals with a tight waist, the female positive individuals and Japanese system silkworm used in step (1) The male moth mating of kind, single moth egg, obtains forth generation silkworm seed；

(5) larva of the forth generation egg-incubation, the individual for being in green fluorescence with silk are raised in one batch rearing method For positive indication, the moth area that positive individuals account for more than 90% is selected and remain, further cocoon type is grown in moth area selected by selection Oval, shallow positive individuals with a tight waist mated in moth area, obtain the 5th generation silkworm seed；

(6) larva for raising the 5th generation egg-incubation is educated with moth area ant amount, the individual for being in green fluorescence with silk For positive indication, the moth area that positive individuals account for more than 90% is selected and remain, further cocoon type is grown in moth area selected by selection Oval, shallow positive individuals with a tight waist mated in moth area, obtain the 6th generation silkworm seed；

(7) larva for raising the 6th generation egg-incubation is educated with moth area ant amount, the individual for being in green fluorescence with silk For positive indication, the moth area that positive individuals account for more than 90% is selected and remain, further cocoon type is grown in moth area selected by selection Oval, shallow positive individuals with a tight waist mated in moth area, obtain the 7th generation silkworm seed；

(8) larva of the 7th generation egg-incubation, the individual for being in green fluorescence with silk are raised in one batch rearing method For positive indication, the moth area that positive individuals account for more than 90% is selected and remain, further cocoon type is grown in moth area selected by selection Oval, shallow positive individuals with a tight waist mated in moth area, obtain eighth generation silkworm seed；

(9) larva of the eighth generation egg-incubation, the individual for being in green fluorescence with silk are raised in one batch rearing method For positive indication, the moth area that positive individuals account for more than 90% is selected and remain, further cocoon type is grown in moth area selected by selection Oval, shallow positive individuals with a tight waist mated in moth area, obtain the 9th generation silkworm seed；

(10) larva of the 9th generation egg-incubation is raised in one batch rearing method, with silk in green fluorescence Body is positive indication, and the moth area for selecting and remain more than 98% positive individuals is used as pre-selection moth area, and selects described pre- Cocoon type grows oval, shallow positive individuals with a tight waist in Xuan E areas, then carries out pupa of living to kind cocoon in the pre-selection moth area Filature, the individual of silkworm cocoon reelability rate, silk cleanliness and intensity difference ranking within preceding 20% is selected to perform moth Section mating subculture, obtain the mutant homozygous type of the tenth generation silkworm seed, i.e. posterior division of silkgland high efficient expression sericin The special original seed of practicality Japanese system；And

(11) by the special original seed of practicality Japanese system of the mutant homozygous type of posterior division of silkgland high efficient expression sericin The Chinese system Silkworm varieties hybridization of the bivoltine cross combination used with step (1), obtains the silk of the secretion Cultivated silkworm breed variety containing sericine in plain fibril.

2. the cultivated silkworm breed variety seed selection side containing sericine in the fibroin fibril of secretion according to claim 1 Method, it is characterised in that：In each step, the male and female moth of post-coitum is separated, female moth is placed on spawning paper and produced Ovum.

3. the cultivated silkworm breed variety seed selection side containing sericine in the fibroin fibril of secretion according to claim 2 Method, it is characterised in that：With the mutually isolated female moth of iron or plastic moth circle.

4. the cultivated silkworm breed variety choosing containing sericine in the fibroin fibril of the secretion according to Claims 2 or 3 Educate method, it is characterised in that：The ovum given birth to is placed on 25 DEG C -27 DEG C, 75%-80% relative humidity and natural light According in the environment of, the silkworm seed for having left the non-Diapause of Japanese system genetically modified mutant kind is eliminated, is left stagnant The moth area of fertility reserves seed for planting.

5. the cultivated silkworm breed variety seed selection side containing sericine in the fibroin fibril of secretion according to claim 4 Method, it is characterised in that select Diapause ovum in such a way：Diapause ovum is 20~30 hours after spawning Yellow is gradually become from light yellow, was changed into filbert at 30~50 hours, until being changed into dark brown after 50 hours.

6. according to the cultivated silkworm breed variety seed selection side containing sericine in the fibroin fibril of the above-mentioned secretion of claim 4 Method, it is characterised in that：Kept after spawning in 1 week light yellow constant for non-Diapause ovum.