Method for inferring silkworm chrysalis stage development process based on trimethyl lysine hydroxylase
By screening a combined model of the trimethyllysine hydroxylase gene and actin gene 3, the problem of accurately predicting the development time of the silkworm pupal stage was solved, and the accurate measurement of the pupal development process was achieved, thus improving the accuracy and efficiency of seed production.
Patent Information
- Application Number
- CN202511500215.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-21
- Publication Date
- 2025-12-05
AI Technical Summary
Existing technologies cannot accurately predict the development time of silkworm pupae. Traditional methods rely on observation of pupa color and are prone to error. The expression level of the cocoon-dissolving enzyme gene is low in the early pupal stage, making it difficult to accurately determine.
By using second-generation Illumina sequencing, the trimethyllysine hydroxylase gene and actin gene 3 were screened together. A model was established to show the relationship between the relative expression level and the pupal development time. Detection primers were optimized to accurately infer the pupal development process.
This provides a precise and quantifiable method that avoids the judgment errors of traditional observation methods, enables accurate measurement of the pupal development time, and improves the accuracy and efficiency of seed production.
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Figure CN121065359A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biological detection technology, and in particular to a method for inferring the developmental process of silkworm pupae based on trimethyllysine hydroxylase. Background Technology
[0002] silkworm( Bombyx mori As an important economic insect, the silkworm has a domestication history of over 5700 years in China. The individual development of the silkworm exhibits a complete metamorphosis, going through four developmental stages: egg, larva, pupa, and adult. The metamorphosis from the pupal stage to the adult stage is called eclosion, lasting approximately 10-15 days. During this crucial stage from pupal to adult, the silkworm does not need to obtain any nutrients from the external environment, but it undergoes an extremely intense and complex physiological remodeling process, characterized by the simultaneous deconstruction of larval tissues and organs and the construction of adult tissues and organs.
[0003] In current production practices, accurately predicting the development time of silkworm pupae is of great significance for carrying out seed production work quickly and effectively. It can provide key time nodes for the orderly development of seed production processes and the efficient utilization of germplasm resources, thereby improving the accuracy and timeliness of seed production work and ensuring the economic benefits and sustainable development of the sericulture industry.
[0004] Traditional methods for predicting the developmental progress of silkworm pupae rely primarily on observing changes in pupal color at specific stages of development, combined with the accumulated production experience of practitioners. However, due to natural variations in pupal color among different silkworm varieties and batches, this traditional method has significant limitations in practical application. This approach, relying solely on visual observation and subjective experience, often fails to ensure high accuracy in assessing the pupal development process. Consequently, errors in judgment can easily lead to unreasonable subsequent production processes, affecting seed production efficiency and the stability of cocoon quality.
[0005] Previous studies have constructed models relating the relative transcriptional expression level of the cocoon-lysin gene to the pupal development time. By measuring the relative expression level of this gene in the target silkworm pupa and combining it with the model, the pupal development time can be calculated. This method can accurately determine the pupal development time of one or more silkworm pupae. However, the synthesis of cocoon-lysin prozymase occurs at an accelerated rate between days 9 and 14 of the pupal to adult stage, while the expression level of the cocoon-lysin gene remains low during the first 9 days of the pupal to adult stage. Therefore, this method may not be able to accurately determine the early pupal development time. Thus, there is an urgent need to find a method that can accurately predict the entire pupal development process of silkworm pupae. Summary of the Invention
[0006] To solve the above technical problems, the present application carries out second-generation Illumina sequencing and screens out a combination of trimethyl lysine hydroxylase and actin gene 3 that can be used for pupal development time inference of the silkworm, and further optimizes the detection primers to obtain a set of primers with higher detection accuracy, thereby providing a brand-new way for accurate determination of pupation time.
[0007] The first object of the present application is to provide a method for inferring the pupal development time of silkworm pupae, comprising the following steps: S1, taking silkworm larvae, detecting the expression amount of trimethyl lysine hydroxylase gene and reference gene at certain time intervals after pupation to obtain the relative expression amount of trimethyl lysine hydroxylase gene relative to the reference gene, and establishing a relationship model of the relative expression amount and the pupal development time; S2, taking the silkworm pupae to be tested, obtaining the relative expression amount of trimethyl lysine hydroxylase gene relative to the reference gene in the sample of the silkworm pupae to be tested by the method of S1, and bringing the relative expression amount into the relationship model of S1 to calculate the pupal development time of the silkworm pupae to be tested.
[0008] Further, in step S1, the first detection time is within 0-3 hours after the silkworm larvae pupate.
[0009] Further, in step S1, the detection interval is 1-3 days.
[0010] Further, the calculation formula of the relative expression amount of trimethyl lysine hydroxylase gene relative to the reference gene is: relative expression amount of trimethyl lysine hydroxylase gene relative to the reference gene = expression amount of trimethyl lysine hydroxylase gene / expression amount of the reference gene.
[0011] Further, the expression amount of trimethyl lysine hydroxylase gene and the reference gene can be obtained by any feasible quantitative method, such as fluorescence quantitative PCR.
[0012] Further, the reference gene can be selected from ribosomal protein L32 gene or actin 3 gene, and most preferably is actin 3 gene.
[0013] Further, the nucleotide sequence of trimethyl lysine hydroxylase gene is shown in SEQ ID NO. 1, and the nucleotide sequence of actin gene 3 is shown in SEQ ID NO. 2.
[0014] Further, the steps for detecting gene expression amount include dissecting the silkworm pupae to obtain fat body tissue, extracting total RNA, removing DNA interference, reverse transcribing cDNA, and amplifying trimethyl lysine hydroxylase gene and reference gene using primers.
[0015] Further, the primers for amplifying the trimethyl lysine hydroxylase gene comprise SEQ ID NO. 3-4 (most preferred) or SEQ ID NO. 7-8.
[0016] Further, when the internal reference gene is actin gene 3, the primers for amplifying the internal reference gene comprise SEQ ID NO. 5-6.
[0017] A second object of the present application provides an inference model for the pupal development time of Bombyx mori, which comprises a relative expression curve of the trimethyl lysine hydroxylase gene relative to the actin gene 3 in the silkworm pupa and the time corresponding to the detected expression.
[0018] Further, the time corresponding to the detected expression is taken as the starting time of 0-3 hours of the pupation of the Bombyx mori larva.
[0019] Further, the primers for detecting the expression of the trimethyl lysine hydroxylase gene are SEQ ID NO. 3-4, and the primers for detecting the expression of the actin gene 3 are SEQ ID NO. 5-6.
[0020] A third object of the present application is to provide a primer combination for inferring the pupal development time of Bombyx mori, the nucleotide sequence of which is shown in SEQ ID NO. 3-6.
[0021] A fourth object of the present application is to provide a kit for inferring the pupal development time of Bombyx mori, which is used for (quantitatively) detecting the expression of the trimethyl lysine hydroxylase gene and the actin gene 3.
[0022] Further, the kit contains primers for detecting the trimethyl lysine hydroxylase gene and the actin gene 3, which comprise the above-mentioned primer combination.
[0023] A fifth object of the present application is to provide the use of the inference model, primer combination or kit in inferring the pupal development time of Bombyx mori, such as preparing detection products, detection instruments, detection equipment, etc.
[0024] By the above-mentioned scheme, the present application has at least the following advantages: 1. The present application obtains a group of differentially expressed genes closely related to the pupal development time of Bombyx mori by second-generation Illumina sequencing screening, and further screens an internal reference gene, constructs a correlation model based on the differential genes and the internal reference gene, and provides a method for accurately inferring the whole development process of the pupal stage of Bombyx mori, which can realize accurate determination of the pupal development time by using a small amount of fat body sample, and is more representative than the traditional observation method and the weighing method.
[0025] 2、The determination method of the present application can avoid the judgment error of the traditional observation method and the weighing method, can accurately determine the whole development process of the silkworm pupa, and has the characteristics of quantifiable data, accurate results, strong practicality and other significant advantages, and has wide popularization and application value.
[0026] The above description is only a summary of the technical solutions of the present application, in order to more clearly understand the technical means of the present application, and can be implemented according to the content of the specification, the following is the preferred embodiment of the present application and the detailed description of the drawings as follows. BRIEF DESCRIPTION OF DRAWINGS
[0027] In order to make the content of the present application more easily understood, the following is a further detailed description of the present application according to the specific embodiments of the present application and in combination with the drawings.
[0028] Figure 1 For time series analysis results.
[0029] Figure 2 For the transcriptional expression change trend of different molecular markers.
[0030] Figure 3 For the transcriptional expression change trend of trimethyl lysine hydroxylase gene under different internal reference genes.
[0031] Figure 4 For the relative relationship curve between the relative transcriptional expression amount of Bombyx mori trimethyl lysine hydroxylase gene and the pupal development time. DETAILED DESCRIPTION
[0032] The present application will be further described below in combination with the drawings and specific embodiments, so that those skilled in the art can better understand the present application and can be implemented, but the embodiments are not as a limitation of the present application.
[0033] The principle and data analysis method of quantitative PCR (Real-time PCR reaction) in the present application have been disclosed by J.H.Schefe (J.H.Schefe, K.E.Lehmann, I.R.Bushchmann, T.Unger, H.Funke-Kaiser, Quantitative real-time RT-PCR data analysis: current concepts and the novel “gene expression’s CT difference” formula, Journal of molecular medicine 2006 (84): 901-910), those skilled in the art can refer to it.
[0034] The present application aims at the problem of the prior art that the accuracy is insufficient and the time range is limited in predicting the development time of the silkworm pupa, and discloses a scientific and accurate judgment method for the first time, that is, screening a marker gene related to the development specificity of the silkworm pupa, and establishing a correlation model according to the expression of the marker gene and the development process of the pupa stage, and then verifying the development process of the pupa stage of the same variety of silkworm pupa, and the result is accurate. Except for the primer, the substances involved in the present application are conventional substances in the art, and the specific operation method is a conventional method in the art.
[0035] A method for accurately predicting the development time of the silkworm pupa in the pupa stage, comprising the following preferred steps: (1) Constructing a correlation model of the relative transcription expression of trimethyl lysine hydroxylase gene and the development time of the pupa stage according to the known silkworm pupa sample; (2) Determining the relative expression of trimethyl lysine hydroxylase gene of the silkworm pupa to be detected, and calculating the development time of the pupa stage according to the correlation model obtained in step (1).
[0036] In the present application, the silkworm variety in step (1) and step (2) is preferably the same variety, and the pupa stage duration of each variety is determined.
[0037] In the present application, step (1) specifically comprises the following steps: raising silkworms by conventional method to upper cocoon, taking down the cocoon after 2-4 days of upper cocoon, and cutting open the cocoon to take out the hair foot silkworm without pupation; the first material is the pupa of the hair foot silkworm (referring to the state that the silkworm has not completely pupated after spinning silk and cocooning, and the silkworm body shape is still retained, and the foot can still be seen) within 0-3 hours of pupation; then take the female pupa every 24 hours until the adult is hatched; dissect the fat body tissue to extract total RNA, and prepare cDNA after DNA enzyme treatment; use actin gene 3 and trimethyl lysine hydroxylase gene as templates, and amplify by Real-time PCR method; the relative transcription expression of trimethyl lysine hydroxylase gene is the expression of trimethyl lysine hydroxylase gene / the expression of internal reference gene (in the examples of the present application, it refers to the expression of actin gene 3). Then a curve equation of the relative transcription expression of trimethyl lysine hydroxylase and the development time of the pupa stage is established, which is a correlation model.
[0038] In the above technical solution, in step (1), the starting point of the pupa stage development time is 0-3 hours of pupation, and the end point is hatching into adult, and the silkworm is raised by conventional method from pupation to hatching into adult; the interval of adjacent pupa stage development time is 24 hours; for example, the first material in step (1) is within 0-3 hours of pupation, and this pupa stage development time is recorded as P1, and then the material is taken every 12-36 hours (preferably every 24 hours), which is recorded as P2, P3, P4, P5, P6, …, respectively.
[0039] In the technical scheme, in the step (1), more than 3 silkworm pupae are selected as a group each time, and the material is repeated for three groups, so as to avoid errors caused by different individuals.
[0040] In the technical scheme, the method for accurately inferring the pupal development time of the domestic silkworm is suitable for different varieties of the domestic silkworm species.
[0041] In the preferred technical scheme, when the correlation model between the relative transcription expression of the silkworm pupa trimethyl lysine hydroxylase gene and the pupal development time is established, the pupal development time is taken as the horizontal coordinate, and the relative transcription expression of the trimethyl lysine hydroxylase gene is taken as the vertical coordinate, so as to obtain an exponential trend curve.
[0042] In the present application, the extraction of total RNA, the DNA enzyme treatment and the reverse transcription synthesis of cDNA are performed according to the molecular cloning experiment guide (the third edition).
[0043] In the present application, the sample (silkworm pupa fat body tissue) is stored in a-80℃ ultra-low temperature refrigerator, so as to avoid RNA degradation, and the storage time should not be more than 30 days.
[0044] In the present application, the sequences of the trimethyl lysine hydroxylase gene (Tmlh) and the actin gene 3 (Actin3) are as follows: Tmlh (Accession Number: XM_004927907.5): Actin3 (Accession Number: U49854):
[0045] (1) Materials and equipment The silkworm pupae were of the variety (Jing Song x Hao Yue) bred at Suzhou University. The incubator was a 250D artificial climate box produced by Shaoquan Xinteng Science and Education Instrument Co., Ltd. in Guangdong Province. The gene amplification instrument was Biosafer 9703. The real-time fluorescent quantitative PCR instrument was LightCycler® 96.
[0046] Samples: Every 24 hours, more than 9 female pupae were taken for model construction.
[0047] (2) Sample collection The silkworms were raised by the conventional method until cocooning. After 3 days of cocooning, the cocoon was cut open to take out the non-pupating hair foot silkworms. The pre-pupa condition of the larvae was observed every 6 hours. After 2 hours of pupation of the hair foot silkworms, 3 female pupae were collected for dissection of the fat body tissue as a group, with three biological replicates. This pupal development stage was recorded as P1. The pupae were protected under the conditions of temperature 25℃ and humidity 75% until the moths emerged. Every 24 hours, 9 female pupae were taken, and the pupal development stages were recorded as P2, P3, P4, P5, P6, P7, P8, P9, P10, P11, and P12, respectively.
[0048] (3) Determination of target genes We used the second-generation Illumina sequencing technology to study the transcriptional expression profiles of the silkworm at different development time points (P1, P3, P5, P7, P9, and P11) from the pupal stage to the adult stage. The Mfuzz package was used to analyze the gene expression data. First, genes with low expression or low variation among time points were removed, and genes with insignificant differences among samples were further removed based on the standard deviation. In the clustering process, the Euclidean distance was used to measure the similarity of gene expression patterns, the optimal fuzzifier was determined to be 1.715, and the number of clusters was set to 10. Subsequently, the acore function was used to evaluate the membership score (MEM.SHIP) of the genes in each cluster, and the top 10 core genes in each cluster were selected as the representative genes of the expression pattern. As shown in Table 1, the genes in cluster 6 showed a trend of continuous increase in expression over time, and the annotations of the top 10 genes in this cluster are shown in Table 1. Figure 1
[0049] Table 1 Information of the top 10 genes in cluster 6
[0050] According to the GO, KEGG function enrichment information of the genes in Table 1, four representative molecular markers related to development were screened, which were leucine-rich repeat-containing G protein-coupled receptor (LOC101745125), dual specificity protein phosphatase 22 (LOC101735761), trimethyl lysine hydroxylase (LOC101743938), and fibrillin-like protein 1 (LOC101738553). The transcriptional level changes of these four molecular markers every 24 hours were detected by fluorescent quantitative PCR. The transcriptional expression change trend of the four molecular markers and their correlation model are shown in Figure 2 The expression of the four molecular markers was normalized by the expression of the internal reference gene.
[0051] As shown in Figure 2 , with the development process of the silkworm pupal stage, the transcriptional level of trimethyl lysine hydroxylase gene (LOC101743938) gradually increased, and the correlation model with the pupal development time was optimal, suggesting that this gene played an important role in the metamorphosis of the silkworm.
[0052] Therefore, it is feasible to accurately infer the whole development process of the silkworm pupa by determining the relative expression amount of trimethyl lysine hydroxylase gene and applying it to the inference of the chrysalis time.
[0053] In the selection of internal reference genes, by combining the transcriptional expression profile data at different development time points from pupal stage to adult stage, we selected two commonly used candidate internal reference genes, which were ribosomal protein L32 gene and actin 3 gene. The two internal reference genes were detected and evaluated by fluorescent quantitative PCR technology. The results showed that when actin 3 gene was selected as the internal reference gene, the correlation model performed better, so actin 3 gene was determined as the optimal internal reference gene (see Figure 3 , A represents that ribosomal protein L32 is used as the internal reference gene, and B represents that actin 3 is used as the internal reference gene).
[0054] (4) Quantitative PCR analysis According to the conventional method, the following primers were synthesized by outsourcing: Tmlh-F1: 5'-AAGCGCGGAAGCTACAGAAA-3' (SEQ ID NO. 3) Tmlh-A1: 5'-GCGCAGCCAAAGGTAGATTG-3' (SEQ ID NO. 4) Actin3-F1: 5'-CGGCTACTCGTTCACTACC-3' (SEQ ID NO. 5) Actin3-A1: 5'-CCGTCGGGAAGTTCGTAAG-3' (SEQ ID NO. 6).
[0055] Total RNA extraction, DNase treatment and reverse transcription to synthesize cDNA: Total RNA extraction, DNase treatment and reverse transcription to synthesize cDNA were processed according to the Molecular Cloning Guide (3rd edition).
[0056] Real-time fluorescent quantitative PCR detection: SYBR Green I fluorescent dye was used in a LightCycler® 96 instrument.
[0057] Real-time PCR reaction system: 2x SYBR Ex Taq (2x) 10 μl; ROX Reference Dye (50x) 0.4 μl; upstream primer (10 μmol / L) 0.4 μl; downstream primer (10 μmol / L) 0.4 μl; cDNA 2.0 μl; dH2O 6.8 μl; total system 20 μl. Reaction conditions were as follows: 95 ℃ 1 min; 95 ℃ 5 s, 55 ℃ 10 s, 72 ℃ 10 s, 45 cycles. All samples were set up three independent repeated experiments.
[0058] (5) Data statistics According to the method disclosed by J.H. Schefe, a standard curve was prepared according to the determined Ct value, and the standard curve equation of trimethyl lysine hydroxylase gene (Tmlh) was y = -2.77x + 31.009, R 2 = 0.9964; the standard curve equation of actin 3 gene (Actin3) was y = -3.213x + 29.341, R 2 = 0.9987. According to the Ct value determined by each sample, the corresponding log value was calculated after three groups of averages, and the corresponding expression amount was calculated according to the 2- △△CT method as follows: Table 2 Relationship between pupal development time and relative transcription expression amount of trimethyl lysine hydroxylase gene
[0059] The data in Table 2 were statistically analyzed and plotted, Figure 4 the relative relationship curve between the relative transcription expression amount of trimethyl lysine hydroxylase gene and the pupal development time of the female pupa, from which it can be seen that the relative transcription expression amount of trimethyl lysine hydroxylase gene conforms to an exponential trend curve model, and the equation is y = 0.0001e 0.7559x , R 2 = 0.9675.
[0060] (1) Bombyx mori pupae were adopted as the variety (Jing Song x Hao Yue) bred by Suzhou University, and female pupae were randomly selected in the conventional rearing process to infer the pupal development process.
[0061] A female pupa requiring accurate inference of the pupal development process was randomly taken for quantitative PCR detection, and the same method as in Example 1 was used to measure the Ct average of the trimethyl lysine hydroxylase gene as 24.99 and the Ct average of the actin gene 3 as 20.66. According to the standard curve equation of the two genes, the corresponding log values were 2.1729 and 2.7018, respectively. Then, the corresponding expression amount of the trimethyl lysine hydroxylase gene was calculated as 148.9101 and the expression amount of the actin gene 3 was calculated as 503.3108 according to the formula 2 -△△CT . The relative expression amount of the trimethyl lysine hydroxylase gene of the silkworm pupa was 0.2959. The control Figure 4 It can be seen that the pupal development time of the silkworm pupa is about 10 days. According to the formula y = 0.0001e 0.7559x , x is calculated as about 10.57, and thus it is judged that the development time of the sample female pupa is 10.57 days.
[0062] (2) Verification of the detection result: The silkworm pupae in the same batch in the above step (1) were further protected to be adult insects after hatching, and it was observed that the silkworm pupae hatched into adult insects after 2 days. According to the known pupal development time of 12 days of the Bombyx mori variety Jing Song x Hao Yue from pupal stage to hatching, it can be proved that the detection method of the present application is accurate. At the same time, the detection method is more accurate than the traditional method, and the number of days is accurate to the decimal point.
[0063] (3) Reproducibility verification of accurate inference of the pupal development time of the silkworm pupa According to the method of step (1), 30 female silkworm pupae with different time intervals were randomly detected, and the pupal development time obtained according to the formula was consistent with the actual pupal development process. Figure 1
[0064] Tmlh-F2: 5'-GGCCCTTCGATTCCCTTTGA-3' (SEQ ID NO. 7) Tmlh-A2: 5'-GCGACAGAAAGTCCGCTTTG-3' (SEQ ID NO. 8) Actin3-F1: 5'-CGGCTACTCGTTCACTACC-3' (SEQ ID NO. 5) Actin3-A1: 5'-CCGTCGGGAAGTTCGTAAG-3' (SEQ ID NO. 6).
[0065] The primer of step (4) in Example 1 is replaced by the above primer to obtain a model of the relationship between the pupal development time of the silkworm and the relative transcriptional expression of the trimethyl lysine hydroxylase gene, and the remaining steps are the same as step (1) of Example 2, and the relationship model is introduced to calculate the pupal development time, and the calculation result is x≈9.78.
[0066] It can be seen that the detection accuracy of the method constructed after replacing the primer is slightly worse than that of the method constructed by the primer used in Example 1, but it can still be used to infer the pupal development time of the silkworm.
[0067] Obviously, the above examples are only examples for the purpose of clarity, and are not a limitation on the embodiments. For those skilled in the art, other different forms of changes or variations can be made on the basis of the above description. It is not necessary and impossible to enumerate all the embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present application.
Claims
1. A method of inferring the developmental progress of a silkworm pupal stage, characterized by, The method comprises the following steps: S1, taking silkworm larvae, detecting the expression amount of trimethyl lysine hydroxylase gene and reference gene in the pupa at a certain time interval after pupation, obtaining the relative expression amount of trimethyl lysine hydroxylase gene relative to the reference gene, and establishing a relationship model of the relative expression amount and pupal development time; S2, taking the silkworm pupa to be tested, obtaining the relative expression amount of trimethyl lysine hydroxylase gene relative to the reference gene in the silkworm pupa to be tested by the method of S1, and bringing the relative expression amount into the relationship model of S1 to calculate the pupal development time of the silkworm pupa to be tested.
2. The method of claim 1, wherein, The nucleotide sequence of the trimethyl lysine hydroxylase gene is shown as SEQ ID NO. 1; And / or, the reference gene is actin gene 3 or ribosomal protein L32 gene; the nucleotide sequence of the actin gene 3 is shown as SEQ ID NO.
2.
3. The method according to claim 1 or 2, characterized in that, The step of detecting the expression amount of trimethyl lysine hydroxylase gene or reference gene comprises an amplification step: The primers for amplifying the trimethyl lysine hydroxylase gene are SEQ ID NO. 3-4 or SEQ ID NO. 7-8; The primers for amplifying the reference gene are SEQ ID NO. 5-6.
4. The method of claim 1, wherein, In step S1, the first detection time is within 0-3 hours after the silkworm pupates.
5. A model for inferring the development time of a silkworm pupa, characterized by, The inference model comprises a relationship curve between the relative expression amount of trimethyl lysine hydroxylase gene relative to actin gene 3 in silkworm pupa and the time corresponding to the detection expression amount; wherein the primers for detecting the expression amount of trimethyl lysine hydroxylase gene are SEQ ID NO. 3-4 or SEQ ID NO. 7-8.
6. The inference model of claim 5, wherein, The primers for detecting the expression amount of the actin gene 3 are SEQ ID NO. 5-6.
7. The inference model of claim 5, wherein, The time corresponding to the detection expression amount is taken as the starting time of 0-3 hours after the silkworm pupates.
8. A primer combination for inferring the pupal developmental time of Bombyx mori, characterized by, The nucleotide sequences of the primer combinations are shown as SEQ ID NO. 3-6.
9. A kit for inferring the pupal developmental time of Bombyx mori, characterized by, The kit is used for detecting trimethyl lysine hydroxylase gene and actin gene 3, and contains primers for detecting trimethyl lysine hydroxylase gene and actin gene 3, wherein the primers for detecting trimethyl lysine hydroxylase gene are SEQ ID NO. 3-4 or SEQ ID NO. 7-8, and the primers for detecting actin gene 3 are SEQ ID NO. 5-6.
10. The inference model of any one of claims 5-7, the primer combination of claim 8, or the kit of claim 9 is used for inferring the pupal development time of silkworm.