CN102808033B - Method for identifying silkworm silk color in larval stage - Google Patents

Abstract

The invention belongs to the field of biotechnology, and discloses a method for identifying a silkworm silk color in a larval stage. The method provided by the invention comprises the following steps of: extracting the ribonucleic acid (RNA) of a silkworm silk gland of a 5th instar stage to be detected by utilizing a genetic engineering technology; performing reverse transcription to synthesize complementary deoxyribonucleic acid (cDNA); taking the obtained cDNA as a template, performing reverse transcription-polymerase chain reaction (RT-PCR) amplification reaction by using a specific primer; judging whether a silkworm spins yellow silk or green silk or white silk by detecting the type and the length of a PCR amplification product by agarose electrophoresis; and further judging whether the silkworm which spins the yellow silk is a homozygous yellow silk spinning silkworm or a heterozygous yellow silk spinning silkworm, and other silkworms spin green silk or white silk by analyzing the content of the RT-PCR amplification product relative to an electrophoresis gel product. By the method provided by the invention, the pedigree with consistent internal cocoon color and external cocoon color and high color fastness also can be identified and separated from a mixed group with different cocoon layer colors and chromaticities. The identification method provided by the invention is simple, convenient and rapid to operate; and the accuracy for identifying the color of the silkworms is high.

Description

A method for identifying the silk color of silkworm silkworm in the larval stage

technical field

The invention belongs to the field of biotechnology, and in particular relates to a method for identifying the silk color of silkworm silkworm, specifically using the specific sequence of mRNA transcribed from the Cbp gene and the Cbp-like gene of silkworm, to specifically identify the silk silk color of silkworm, and at the same time distinguish the silkworm silk The silkworm with yellow silk and the silkworm with part of yellow silk, the silkworm with homozygous yellow silk and the silkworm with heterozygous yellow silk, and the difference in chromaticity and color fastness of yellow silk.

Background technique

Silkworm (Bombyx mori), also known as silkworm, is an insect that is industrially used to produce silk protein fibers in large quantities. Silk is a continuous long fiber that is solidified by secreting silk liquid when cooked silkworms make cocoons, also known as cocoon silk. The modern cocoon and silk industry mainly uses the white cocoon silk of silkworms to produce colored silk products through chemical dyeing and other production processes. A considerable part of the dyes and other chemicals used in the dyeing and finishing process contain substances that are harmful to the human body or even carcinogenic. Printing and dyeing Wastewater has become an important remediation target of industrial sewage.

Silkworm natural colored cocoon is a resource that humans have discovered long ago. The natural color of silkworm cocoon mainly includes three types: yellow-red cocoon, yellow-green cocoon and white cocoon. The yellow-red cocoon series includes various cocoon colors such as light yellow, golden yellow, flesh-colored, red, wormwood, and rust; the yellow-green cocoon series includes bamboo green (light green) and green. Silkworm natural colored cocoon fiber has strong moisture absorption and moisture conduction performance, excellent antibacterial performance, good skin care ability such as absorbing ultraviolet rays and anti-oxidation, and has outstanding properties such as natural color and elegant color. The fiber products produced are soft in color, non-toxic and harmless, and colorful Natural, is a high-grade natural green textile material. Silkworm natural colored cocoon silk fibers do not need to be dyed during the production process, can effectively reduce the pollution of silk printing and dyeing, are friendly to the environment and human body, and have great economic value and important social development significance. However, in the past hundred years, the production of colored cocoon raw materials has not been able to solve the major defects of cocoon silk fiber characteristics such as mixed cocoon color, easy fading, poor silk quality, low silk volume, and irregular cocoon shape. Without qualified raw material cocoons, it is impossible to carry out technical research and breakthroughs, and there is an industry technical bottleneck that cannot spin fine high-grade natural colored silk threads and cannot produce high-grade fabrics. Therefore there is an urgent need to innovate the breeding method of natural colored cocoon varieties, improve the accuracy of selection, and speed up the breeding speed of natural colored cocoon varieties.

The material that forms the cocoon color of the silkworm comes from the mulberry leaves (feed) that the larvae feed on. The natural pigments in the mulberry leaves can be absorbed by the silkworm's digestive tract after being combined with the pigment transporter in the body, and then transferred into the silk gland through the blood. Eventually a colored cocoon is formed. Silkworms also feed on mulberry leaves and can produce cocoons of different colors, even white cocoons. The fundamental reason is the difference in the ability to protect and process pigments in the process of pigment absorption and transport in silkworms. Classical genetic analysis has identified nearly 20 genes that control the cocoon color of silkworms. Therefore, the inheritance of cocoon color is quite complicated. Not only are some types of yellow cocoons difficult to distinguish from green cocoons, there are also many genetic types of yellow cocoons, and even There are also differences between the outer yellow cocoon and the inner yellow cocoon. The homozygosity of the genetic gene cannot be guaranteed only by the cocoon color, and it is also difficult to isolate and purify through hybridization. This directly affects the breeding of practical varieties of colored cocoons and the utilization of hybrid F1 generations. .

Carotenoids, lutein and flavonoid pigments are also pigments that exist in large quantities in the blood of Asian humans. They have functions such as anti-oxidation, affecting reproductive performance, and participating in the body's immune response in animals. Birds have the habit of depositing carotenoids in their eggs, which deposit pigments that promote embryonic development and hatching. So far, the absorption and transport mechanism of carotenoid-related pigments in animals, including humans, is still unclear, and the research on its regulatory genes only refers to the experimental animal data of silkworm. Therefore, the discovery and application of methods for identifying genes and molecular markers at the molecular level has practical significance in biomedicine.

In 1996, Jouni and Wells reported a lutein binding protein (LBP) isolated from the silkworm midgut, but there is no in-depth report on its sequence and function. Tabunoki H et al. (2002), Tsuchida et al. (2004), Sakudoh et al. (2005, 2007), Niu Yanshan et al. (2010) reported a silkworm carotenoid binding protein (CBP), which interacts with carotenoids, especially leaves The absorption and transport of flavin (Lutin) are closely related, and the gene was cloned and functionally identified. CBP, as a carotenoid carrier in the cytosol, cooperates with the CAMEO2 protein encoded by the C gene as a transmembrane receptor on the cell surface to jointly mediate the transport of carotenoids (Sakudoh et al., 2010). The Cbp gene of silkworm is located at the BmStart1 gene locus whose function is still unclear. It consists of 7 exons and 6 introns. The integrity of the second exon sequence is related to the formation of yellow cocoon. The reason may be a retrotransposon CATS (CBP-associated TRAS-like sequence), the insertion of the second intron of Cbp leads to gene rearrangement, resulting in the 3′ end sequence of the second exon and the second The deletion of the intron and CATS 5′ end sequence resulted in more than 4 gene structures in the Cbp gene, and the rearrangement process in this gene affected the formation of yellow cocoon and white cocoon varieties.

On the basis of the comparative analysis of the Cbp gene of multiple silkworm cocoon color systems (variety) and three kinds of Cbp-like gene structures, combined with the analysis of their silk gland pigments and cocoon color, it was found that: the translation protein product CBP of the silkworm Cbp gene is the silkworm The key factors of pigment transfer in the process of yellow cocoon formation only exist in silk glands and other tissues of the 4th and 5th instar larvae of silkworm yellow cocoon varieties, but the silkworm Cbp gene and Cbp-like gene and their transcription RNA products exist in various cocoon color inside the silkworm. The Cbp gene and Cbp-like gene of silkworm yellow cocoon variety can simultaneously transcribe the complete mRNA with CBP function and the mRNA lacking the second exon; the Cbp gene and Cbp-like gene of green cocoon variety and white cocoon silkworm variety are only Can transcribe mRNA lacking exon 2 (Niu Yanshan, 2010).

At present, domestic and foreign silkworm colored cocoon breeding methods are based on cocoon color evaluation and selection of families and individuals. However, due to the aforementioned yellow and red cocoon colors, homozygous and heterozygous families cannot be judged. Consistency and high color fastness The selection of families and individuals can only be achieved through cocoon color measurement and cocoon silk color fastness testing, resulting in a lack of accuracy in the selection of silkworm varieties with yellow and red cocoons, and it is difficult to breed strains Stable cocoon color, long breeding cycle, high cost and poor effect. Although theoretically by examining the mRNA structure of the Cbp gene and Cbp-like gene transcription, or detecting the CBP protein product, the carotenoid-binding protein genotype of the individual can be confirmed, but the Cbp gene and Cbp-like gene of the silkworm are mainly found in the 5th instar larvae. Silk gland and digestive tract transcription and expression, identification of Cbp gene and Cbp-like gene transcription and expression product types need to kill larvae to take out silk gland or digestive tract tissue, it is impossible to achieve both identification of gene type and seed retention, and it is also impossible to extract from silkworm cocoons The identification and separation of the mixed populations of yellow-red cocoon systems with different cocoon layer colors and different chromaticity and separation of families with consistent cocoon layer color inside and outside and high color fastness cannot meet the needs of natural color cocoon variety breeding.

Contents of the invention

In view of this, the purpose of the present invention is to provide a method for quickly, accurately and specifically identifying the silk color of silkworm silkworm in the larval stage by using the specific sequence of the mRNA transcribed by the Cbp gene and the Cbp-like gene of the silkworm.

In order to realize the purpose of the present invention, the present invention adopts following technical scheme:

A method for identifying the silking color of silkworm in the larvae stage. In order to extract the silk gland RNA of the silkworm to be tested, cDNA is synthesized by reverse transcription, and the obtained cDNA is used as a template with the upstream of the nucleotide sequence shown in SEQ ID NO: 1. The primer and the downstream primer having the nucleotide sequence shown in SEQ ID NO: 2 carry out RT-PCR amplification, agarose electrophoresis detects the PCR amplification product, and judges silkworm spinning color;

If there is only one nucleotide sequence shown in SEQ ID NO: 4 or its complementary sequence or both the nucleotide sequence shown in SEQ ID NO: 3 or its complementary sequence and the nucleotide sequence shown in SEQ ID NO: 4 If it only has the nucleotide sequence shown in SEQ ID NO: 3 or its complementary sequence, the silkworm spins green cocoon silk or white cocoon silk.

Although the transcribed mRNA and translated protein sequence of the silkworm gene depend on the exon sequence of the gene, other sequences such as its intron sequence and cis-regulator sometimes also affect the transcribed mRNA and translated protein sequence of the gene. The BmStart1 gene locus of silkworm is a complex multi-gene overlapping locus, and the Cbp gene is one of the overlapping genes. The DNA within the overlapping range of the Cbp gene of the silkworm has four different sequences. The differences in DNA sequences include both the sequence differences of the exons of the Cbp gene and the differences of the intron sequences that affect the mRNA transcripts of the Cbp gene. The mRNA transcripts of Cbp gene and Cbp-like gene in the pigment absorption and storage tissues of silkworm cocoons with different colors have 4 different sequences, and the translated protein sequences and functions are also different. Although the DNA sequence in the overlapping range of Cbp gene varied a lot, there was a specific sequence in it that could distinguish the silkworm with yellow cocoons from those with other colored cocoons. The invention utilizes the specific sequence of the mRNA transcribed by the Cbp gene and the Cbp-like gene of the silkworm to quickly, accurately and specifically identify the silk color of the silkworm in the larval stage.

In a specific embodiment, the present invention extracts the silk gland RNA of the 5th instar silkworm, and synthesizes cDNA by reverse transcription, so that the cDNA obtained is used as a template, and the upstream primer with the nucleotide sequence shown in SEQ ID NO: 1 and the primer with SEQ ID NO: 1 are used. The downstream primer of the nucleotide sequence shown in ID NO: 2 was amplified by RT-PCR to amplify the characteristic sequence of silkworm, and the RT-PCR amplified product was detected by agarose electrophoresis. The result showed that the RT-PCR amplified product had only one SEQ The nucleotide sequence shown in ID NO: 4 or the nucleotide sequence shown in SEQ ID NO: 3 and the nucleotide sequence shown in SEQ ID NO: 4 at the same time, the silkworm spins yellow cocoon silk; RT-PCR amplification The product only has the nucleotide sequence shown in SEQ ID NO: 3, and the silkworm spins green cocoon silk or white cocoon silk.

Actin gene 3 (Actin3) belongs to the Actin subfamily of the ARP family. The expression of Actin3 gene is basically stable in different tissues and developmental stages of silkworm. Therefore, Actin3 gene is one of the commonly used internal reference genes in silkworm molecular biology research. In the present invention, the Actin 3 gene is used as an internal reference gene, and the relative content of the electrophoretic gel product of the RT-PCR amplification product and the Actin 3 gene is analyzed by using a gel automatic imaging system to judge whether the silkworm spins green cocoon silk or white cocoon silk, and the results show that The ratio of the optical density value of the electrophoresis band of the RT-PCR amplification product having the nucleotide sequence shown in SEQ ID NO: 3 to the optical density value of the internal reference gene Actin 3 electrophoresis band is > 0.8, and the silkworm spins green cocoon silk; The ratio of the optical density value of the electrophoresis band of the RT-PCR amplification product with the nucleotide sequence shown in SEQ ID NO: 3 to the optical density value of the internal reference gene Actin 3 electrophoresis band is ≤0.4, and the silkworm spins white cocoon silk.

The present invention further judges whether the silkworm is a homozygous yellow cocoon or a heterozygous yellow cocoon by analyzing the relative content of the electrophoretic gel product of the RT-PCR amplification product with a gel automatic imaging system. The results show that it has SEQ ID The optical density ratio of the RT-PCR amplification product electrophoresis band with the nucleotide sequence shown in NO: 4 to the RT-PCR amplification product electrophoresis band with the nucleotide sequence shown in SEQ ID NO: 3 > 3 , the silkworm is a homozygous type that spins yellow cocoons, that is, all the silkworms spin yellow cocoons; the electrophoresis band of the RT-PCR amplification product with the nucleotide sequence shown in SEQ ID NO: 4 is the same as that shown in SEQ ID NO: 3 The RT-PCR amplification product of the nucleotide sequence has an optical density ratio of ≤3, and the silkworm is a heterozygous type that spits yellow cocoon silk, that is, part of the silkworm spits yellow cocoon silk, the outer layer of the cocoon is yellow, and the inner layer is white .

In another embodiment, the present invention distinguishes and separates cocoon color from mixed populations with differences in cocoon layer color and chromaticity by using a gel automatic imaging system to analyze the relative content of the electrophoretic gel product of the RT-PCR amplification product A family with consistent interior and exterior and high color fastness. The result shows that the RT-PCR amplification product electrophoresis band with the nucleotide sequence shown in SEQ ID NO: 4 is different from the RT-PCR amplification product electrophoresis band with the nucleotide sequence shown in SEQ ID NO: 3 When the optical density ratio is greater than 5, the chromaticity and color fastness of the yellow cocoon silk produced by the silkworm are high. The accuracy rate of the method for identifying silkworm spinning color of the present invention is 100%. The method of the present invention can determine whether the progeny of the moth area is a homozygous yellow-spitting cocoon silk with high chroma and high color fastness at the larval stage, without selfing in the moth area for more than 3 generations, which greatly reduces Feeding workload and feeding costs in the breeding process.

In order to obtain colored cocoon silk with high silk quantity, excellent silk quality and strong resistance, people usually cross the silkworm yellow cocoon pure line Y12 with silkworm silkworm variety JS with high silk quantity, high silk quality and strong resistance pure white cocoon. The cocoons of the F1 generation are all yellow, and there are two kinds of cocoons, yellow and white, in the F2 generation. Among them, there are two kinds of individuals in the yellow cocoon, the cocoon color separation and the cocoon color separation. The existing methods cannot remove the individual cocoon color separation. The method currently adopted is to backcross the yellow cocoon of the above-mentioned F2 generation with the pure line Y12 of the yellow cocoon. For each generation of backcrossing, the number of individuals with cocoon color separation in the yellow cocoon will be reduced by half, but theoretically there will never be individuals with cocoon color separation. After being culled, the number of individuals with cocoon color separation in the yellow cocoon of the third generation of backcrossing is still 1/16, and the silk quantity, silk quality and resistance of the yellow cocoon will drop rapidly at this time. In one embodiment, the present invention adopts the hybridization of the identified homozygous yellow silk-spitting silkworm with high chromaticity and high color fastness and a combination of excellent varieties with white cocoons to obtain a large amount of silk, high silk quality, and strong resistance , The yellow cocoon system with the same color inside and outside.

The present invention also provides a kit for identifying silkworm spinning color at the larval stage, comprising an upstream primer having the nucleotide sequence shown in SEQ ID NO: 1 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 2 primers.

In some embodiments, the kit for identifying silkworm spinning color of the present invention further includes one or more of PCR amplification reaction buffer, dNTP, and Taq enzyme.

The present invention also provides a DNA molecular group for identifying the color of silkworm silk spinning in the larval stage, which is composed of the nucleotide sequence shown in SEQ ID NO: 3 and the nucleotide sequence shown in SEQ ID NO: 4.

The invention provides a method for identifying the silk color of the silkworm. The method of the present invention utilizes genetic engineering technology to extract the silk gland RNA of silkworm silk glands to be tested at 5 instars, and synthesizes cDNA by reverse transcription. The type and length of the amplified product can be used to determine whether the silkworm spins yellow cocoon silk or green or white cocoon silk, and by analyzing the relative content of the electrophoresis gel product of the RT-PCR amplification product, it is further judged that the silkworm that spins yellow cocoon silk is homozygous silkworm. The yellow cocoon is still the heterozygous type that spins yellow cocoon, and the other silkworms spin green cocoon or white cocoon. The method of the present invention can also identify and separate families with consistent cocoon color inside and outside and high color fastness from mixed populations with differences in cocoon layer color and chroma. The identification method of the invention is simple and quick to operate, and has high accuracy in identifying the silk color of the silkworm. At the same time, the homozygous yellow silk-spitting silkworm with high chromaticity and high color fastness can also be crossed with the combination of excellent varieties of white cocoons to obtain yellow silk with a large amount of silk, excellent silk quality, strong resistance, and consistent internal and external colors. cocoon system.

Detailed ways

The embodiment of the invention discloses a method for identifying the silk color of the silkworm. Those skilled in the art can refer to the content of this article to appropriately improve the process parameters to achieve. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method of the present invention has been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the method described herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention .

In order to further understand the present invention, the method provided by the present invention will be described in detail below in conjunction with the examples.

Example 1:

In order to identify the green cocoon Dazao variety with yellow-green cocoon color, the white cocoon variety C108 with pure white cocoon color, the yellow cocoon system Caicoon No. YO, this embodiment has carried out the following steps:

(1) The total RNA of the silk glands of the above four 5th instar larval samples was extracted with a commercial RNA extraction kit (Qiagen), and cDNA was synthesized by reverse transcription, and stored at -20°C to -40°C until use. For reverse transcription, the reverse transcription kit from TaKaRa Company was used. Reverse transcription reaction system (10 μL): 5×PrimeScript ^TM Buffer 2 μL; PrimeScript ^TM EnzymeMixI 0.5 μL; 50 μmol/L Oligo dT Prime (50 μmol/L) 0.5 μL; Total RNA 1 μL; add RNase FreeH ₂ O to a total volume of 10 μL. The reverse transcription conditions were: 37°C for 15 minutes; 85°C for 5s.

(2) Using the four silkworm cDNAs obtained in step (1) as templates, using an upstream primer having a nucleotide sequence shown in SEQ ID NO: 1 and a downstream primer having a nucleotide sequence shown in SEQ ID NO: 2, Actin 3 gene was used as an internal reference gene, and SYBR PrimeScript ^TM RT-PCR Kit (TaKaRa Company) was used to perform RT-PCR amplification on a Bio-Rad Chromo4 instrument according to the instruction manual. The reaction system was 25 μL, and each sample was tested 3 times. . The upstream primer sequence of the Actin 3 gene is 5'-CCCCATCGAACACGGAATCG-3', and the downstream primer is 5'-CGCTCGGCAGTGGTAGTGAA-3'; the reaction conditions for RT-PCR amplification are: 95°C for 5s; 60°C for 10s; 72°C for 10s; 32 loops.

(3) The types and sequence lengths of the RT-PCR amplification products of the four samples obtained in step (2) were detected by 1.2% agarose electrophoresis added with EB, and the 200bp DNA Ladder was used as the sequence length marker.

(4) Observation step (3) electrophoresis gel under ultraviolet light conditions, in addition to a product band of the internal reference gene Actin 3, there is only one product with a length of 268bp in the electrophoresis lanes of sample 2 and sample 3; samples 1 and 3 In the electrophoresis lane of sample 4, there is a product with a length of 268 bp and a product with a length of 577 bp.

The 268bp product and the 577bp product obtained from the electrophoresis gel of the 4 larval samples were recovered with the PCR Clean-up kit respectively, and the results after cloning and sequencing were as follows:

For the 268bp product from sample 1, 2 of the 3 clones have the nucleotide sequence shown in SEQ ID NO: 3, and the other 1 clone has only 1 base with the nucleotide sequence shown in SEQ ID NO: 3 base difference. For the 577bp product from sample No. 1, one of the three clones has the nucleotide sequence shown in SEQ ID NO: 4, and only one clone has the nucleotide sequence shown in SEQ ID NO: 4 Base difference, the other clone has only 2 base differences with the nucleotide sequence shown in SEQ ID NO:4.

For the 268bp product from sample 2, 2 of the 3 clones have the nucleotide sequence shown in SEQ ID NO: 3, and the other 1 clone has only 1 nucleotide sequence shown in SEQ ID NO: 3 base difference.

For the 268bp product from sample 3, 2 of the 3 clones have the nucleotide sequence shown in SEQ ID NO: 3, and only 2 of the other clones have the nucleotide sequence shown in SEQ ID NO: 3 base difference.

For the 268bp product from sample 4, 2 of the 3 clones have the nucleotide sequence shown in SEQ ID NO: 3, and the other clone has only 1 base with the nucleotide sequence shown in SEQ ID NO: 3 base difference. For the 577bp product from sample No. 4, 1 of the 3 clones differs from the nucleotide sequence shown in SEQ ID NO: 4 by only 1 base, and the other 2 clones differ from the nucleotide sequence shown in SEQ ID NO: 4 The nucleotide sequences differ by only 2 bases.

The remaining larvae of the 4 samples were further reared until they spun silk and formed cocoons, and samples 1 and 4 spun yellow cocoons. Therefore judge if only have a nucleotide sequence shown in SEQ ID NO: 4 or have the nucleotide sequence shown in SEQ ID NO: 3 and the nucleotide sequence shown in SEQ ID NO: 4 at the same time, then silkworm spit Yellow cocoon silk; if it only has the nucleotide sequence shown in SEQ ID NO: 3, the silkworm spins green cocoon silk or white cocoon silk.

(5) Use the gel automatic imaging system Tanon 2500, Tanon GIS (Gel Image System) software to analyze the relative content of the electrophoresis gel products of each sample in step (3), the result: the light of the product electrophoresis band with a length of 268bp The ratio of the density value to the optical density value of the internal reference gene Actin 3 product electrophoresis band is 1.2; the ratio of the optical density value of the 268bp product electrophoresis band of sample 3 to the optical density value of the internal reference gene Actin A3 product electrophoresis band is 0.3; the optical density value of the product electrophoresis band with a length of 577bp in sample 1 is 11.2 times higher than that of the product electrophoresis band with a length of 268bp; the optical density value of the product with a length of 577bp in sample 4 is higher than that of The optical density value of the product electrophoresis band with a length of 268bp was 2.3 times.

Observe the cocoons made by the remaining larvae of the 4 samples raised to judge the silkworm species. Sample 1 is the yellow cocoon system Caicoon No. 1 variety that spits out yellow cocoon silk; It is the white cocoon system C108 variety with white cocoon silk; sample 4 is the outer yellow cocoon variety YO with some yellow cocoon silk.

Therefore, it is determined that if the silkworm has the ratio of the optical density value of the RT-PCR amplification product electrophoresis band of the nucleotide sequence shown in SEQ ID NO: 3 to the optical density value of the internal reference gene Actin 3 electrophoresis band > 0.8, the silkworm Spit green cocoon silk; if the ratio of the optical density value of the RT-PCR amplification product electrophoresis band with the nucleotide sequence shown in SEQ ID NO: 3 to the optical density value of the internal reference gene Actin 3 electrophoresis band is ≤0.4, The silkworm spins white cocoons. If silkworm has the RT-PCR amplification product electrophoresis band of the nucleotide sequence shown in SEQ ID NO: 4 and has the light of the RT-PCR amplification product electrophoresis band of the nucleotide sequence shown in SEQ ID NO: 3 If the density ratio is greater than 3, the silkworm is homozygous for producing yellow cocoon silk.

Example 2

From the mixed populations with different cocoon layer colors and different chromaticity, the families with consistent cocoon color inside and outside and high color fastness were isolated.

(1) Raise in a single moth area, take a certain number of larval samples from each of the 100 moth areas at the 5th instar, and extract the total RNA from each larval silk gland according to the method described in Example 1, according to the embodiment cDNA was synthesized by the reverse transcription method described in 1, and stored at -20°C to -40°C for future use.

(2) Using the silk gland cDNA of Bombyx mori in each moth region extracted in step (1) as a template, use the upstream primer with the nucleotide sequence shown in SEQ ID NO: 1 and the primer with the nucleotide sequence shown in SEQ ID NO: 2 For downstream primers, the Actin 3 gene was used as an internal reference gene, and RT-PCR was amplified according to the method described in Example 1.

(3) The RT-PCR amplification products of the larval samples of each moth region obtained in step (2) were detected by 1.2% agarose electrophoresis added with EB, and the 200bp DNA Ladder was used as the sequence length marker.

(4) Observation step under ultraviolet light (3) Electrophoresis gel, in addition to one product band of the internal reference gene Actin 3, only one 577bp in the electrophoresis lane has the nucleoside shown in SEQ ID NO: 4 Samples of products of the acid sequence, such samples are recorded as "+";

(5) In addition to a product band of the internal reference gene Actin 3 in the swimming lane observed under ultraviolet light in step (4), there is also a 268bp nucleotide sequence shown in SEQ ID NO: 3 at the same time The product and a sample of the product with a length of 577bp having the nucleotide sequence shown in SEQ ID NO: 4, using the gel automatic imaging system Tanon 2500, Tanon GIS (Gel Image System) software to analyze the electrophoresis gel products of each sample There are 3 types of results:

Type 1 is that the optical density value of the product of SEQ ID NO.4 of the sample is 3 to 5 times higher than the optical density value of SEQ ID NO.3, and this type of sample is recorded as "+"

Type 2 is that the optical density value of the product of SEQ ID NO.4 of the sample is more than 5 times higher than the optical density value of SEQ ID NO.3, and this type of sample is recorded as "++"

Type 3 is that the optical density value of the product of SEQ ID NO.4 of the sample is less than 3 times higher than that of SEQ ID NO.3, and this type of sample is recorded as "-".

(6) Count the types in step (4) or step (5) of all samples in each moth area, select moth areas without "-" type to reserve seeds, and select No. 3, No. 23, No. 27, and No. 39 No. 61, No. 89 and No. 6 moth areas, among which the proportion of "++" type in No. 3 moth area was higher than 80%. As a result of systematic breeding, no cocoons of other colors appeared in these 6 moth areas, and the inner and outer cocoon layers of the cocoons were all yellow, indicating that the accuracy rate of the method for identifying the silk color of the silkworm of the present invention is 100%. Among them, the silk color of the offspring of the No. 3 moth district was golden yellow, and the color fastness was higher than that of the cocoon silk of the other 5 moth districts. Therefore judge, if have the RT-PCR amplification product electrophoresis strip of the nucleotide sequence shown in SEQ ID NO:4 and have the RT-PCR amplification product electrophoresis strip of the nucleotide sequence shown in SEQ ID NO:3 When the ratio of optical density of the belt is more than 5, the chromaticity and color fastness of the yellow cocoon silk of the silkworm are high.

The method of the present invention can determine whether the offspring of the moth field are homozygous yellow cocoon silks without selfing in the moth field for more than 3 generations, and can reduce 94% of the rearing amount in the late 5th instar.

Example 3

The homozygous yellow silk-spitting silkworm was selected from the mating of the homozygous yellow silk-spitting silkworm and the white cocoon excellent variety.

(1) The pure line Y12 of silkworm yellow cocoon was crossed with JS silkworm variety JS, which has a large amount of silk, high silk quality, and strong resistance. The blood of all larvae is all yellow 100 moth districts to get a certain number of larvae samples, and the total RNA extracted from each larvae silk gland according to the method described in Example 1 is reverse transcribed according to the method described in Example 1. Methods cDNA was synthesized and stored at -20°C to -40°C for future use.

(2) Using the silk gland cDNA of each moth region extracted in step (1) as a template, use the upstream primer with the nucleotide sequence shown in SEQ ID NO: 1 and the downstream primer with the nucleotide sequence shown in SEQ ID NO: 2 Primers, using the Actin 3 gene as an internal reference gene, were amplified by RT-PCR according to the method described in Example 1.

(3) The RT-PCR amplification products of the larval samples of each moth region obtained in step (2) were detected by 1.2% agarose electrophoresis added with EB, and the 200bp DNA Ladder was used as the sequence length marker.

(4) The step of selecting the source of each larva in the moth area (3) The types of RT-PCR amplification products detected by electrophoresis all have a length of 268bp of SEQ ID NO.3 product and a length of 577bp of SEQ ID NO. .3 products, the result was a total of 99 moth areas were selected.

(5) Use the gel automatic imaging system Tanon 2500, Tanon GIS (Gel Image System) software to analyze the relative content of the electrophoresis gel products of each sample, and calculate the electrophoresis band SEQ ID NO.4 product and SEQ ID in the same electrophoresis lane NO.3 The relative optical density value ratio of the product, select the moth area with the ratio greater than 2.0 for each larval sample in the moth area, and as a result, 41 moth areas were selected from the 99 moth areas in step (4).

(6) Continue to raise the larvae of the 41 moth areas selected in step (5), and the systematic breeding method has obtained a stable genetic yellow cocoon variety with significantly higher silk quantity, silk quality and resistance than the silkworm yellow cocoon pure line Y12, Crossbreed with the selected white cocoon variety to obtain a yellow cocoon system with a large amount of silk, excellent silk quality, strong resistance, and consistent internal and external colors.

The method of the present invention does not need to go through repeated backcrossing with Y12, can shorten the breeding time and cost by more than 50%, and the method of the present invention will not cause low production efficiency and variety identification caused by impure cocoon color of bred varieties difficult question.

The descriptions of the above embodiments are only used to help understand the method and core idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principle of the present invention, some improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims (3)

1. a method of differentiating family's silkworms spin silk color in larval stage, is characterized in that,

Extract to be measured 5 the length of time domestic natural silk gland RNA, reverse transcription method synthesizes cDNA, taking obtain cDNA as template use the upstream primer of nucleotide sequence as shown in SEQ ID NO:1 and as shown in SEQ ID NO:2 the downstream primer of nucleotide sequence carry out RT-PCR amplification, agarose electrophoresis detects pcr amplification product, judgement man's silkworms spin silk color;

If only there is nucleotide sequence or its complementary sequence as shown in SEQ ID NO:4, or there is nucleotide sequence or its complementary sequence and the nucleotide sequence as shown in SEQ ID NO:4 or its complementary sequence as shown in SEQ ID NO:3, family's curley waste Yellow Cocoon silk simultaneously; If only there is nucleotide sequence or its complementary sequence as shown in SEQ ID NO:3, the green silk of family's curley waste or white silk;

Also comprise and the RT-PCR amplified production optical density value comparison of the nucleotide sequence reference gene Actin A3 as shown in SEQ ID NO:5, judge the green silk of family's curley waste or white silk;

If there is nucleotide sequence or the optical density value of RT-PCR amplified production electrophoretic band of its complementary sequence and the ratio > 0.8 of the optical density value of reference gene Actin3 electrophoretic band as shown in SEQ ID NO:3, the green silk of family's curley waste; If there is nucleotide sequence or the optical density value of RT-PCR amplified production electrophoretic band of its complementary sequence and ratio≤0.4 of the optical density value of reference gene Actin3 electrophoretic band as shown in SEQ ID NO:3, family's curley waste white silk.

2. discrimination method according to claim 1, it is characterized in that, also comprise the Reinhoit Zahl that utilizes the RT-PCR amplified production electrophoretic band of nucleotide sequence as shown in SEQ ID NO:4 or its complementary sequence and the RT-PCR amplified production electrophoretic band of nucleotide sequence as shown in SEQ ID NO:3 or its complementary sequence, judge that silkworm is homozygously to tell yellow silk or heterozygous is told yellow silk;

If 3 silkworms of Reinhoit Zahl > of the RT-PCR amplified production electrophoretic band of the RT-PCR amplified production electrophoretic band of the nucleotide sequence as shown in SEQ ID NO:4 or its complementary sequence and the nucleotide sequence as shown in SEQ ID NO:3 or its complementary sequence are the homozygous yellow silk of telling.

3. discrimination method according to claim 1, it is characterized in that, utilize the Reinhoit Zahl of the RT-PCR amplified production electrophoretic band of nucleotide sequence as shown in SEQ ID NO:4 or its complementary sequence and the RT-PCR amplified production electrophoretic band of nucleotide sequence as shown in SEQ ID NO:3 or its complementary sequence, colourity and the colour fastness of judgement man curley waste Yellow Cocoon silk;

If the colourity of 5 family's curley waste Yellow Cocoon silks of Reinhoit Zahl > of the RT-PCR amplified production electrophoretic band of the RT-PCR amplified production electrophoretic band of the nucleotide sequence as shown in SEQ ID NO:4 or its complementary sequence and the nucleotide sequence as shown in SEQ ID NO:3 or its complementary sequence is high and colour fastness is high.