WO2022087857A1 - 一种判断家蚕化蛾时间的方法 - Google Patents

一种判断家蚕化蛾时间的方法 Download PDF

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WO2022087857A1
WO2022087857A1 PCT/CN2020/124174 CN2020124174W WO2022087857A1 WO 2022087857 A1 WO2022087857 A1 WO 2022087857A1 CN 2020124174 W CN2020124174 W CN 2020124174W WO 2022087857 A1 WO2022087857 A1 WO 2022087857A1
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silkworm
time
moth
gene
cocoon
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李兵
顾芝亚
叶文涛
李凡池
孙海娜
卫静
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苏州大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms

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  • the invention relates to a method for judging the time of silkworm morphing into moths, in particular to a method for accurately detecting the development time of silkworm pupa based on the relative transcription and expression level of genes.
  • the silkworm ( Bombyx mori ) is an important economic insect and has been domesticated in China for more than 5,700 years.
  • the silkworm goes through four different developmental stages in its life, namely egg, larva, pupa and adult.
  • the metamorphosis development process from the pupal stage to the adult stage is called eclosion. This process generally takes 10 to 15 days, and the cocoon is usually harvested about 7 days after the cocooning.
  • Silkworms do not need to take in nutrients from the external environment during the pupal stage, but the destruction of larval tissues and organs is vigorously carried out in the body, and the formation of adult tissues and organs occurs.
  • the traditional method is to observe the color change from the development of the pupae to the later stage (the color becomes darker before pupation) under specific temperature and humidity conditions (such as temperature 25 ⁇ 1°C, humidity 75 ⁇ 80%), and combine the production Experience to determine the developmental process of the pupal stage, and then infer the time of moth. Due to the differences in the color of pupae of different varieties or batches, it is easy to cause errors by relying only on traditional visual observation combined with production experience. In addition, silkworm germplasm resources are the valuable wealth of sericulture production. There are various types of germplasm resources in my country, covering cocoon color limit, coarse denier, fine denier, etc. The preservation of germplasm resources is of great strategic significance to my country's sericulture industry.
  • the purpose of the present invention is to provide a new method for judging the time of moth transformation of silkworm, so as to accurately infer the time of moth transformation of silkworm.
  • the technical scheme adopted in the present invention is: a method for judging the time of silkworm transformation into moths, comprising the following steps: (1) respectively constructing the relative transcription and expression levels of the cocoon lysase genes of silkworm male pupae and female pupae and An association model of developmental timing in the pupal stage.
  • step (2) Determination of the relative expression level of the silkworm pupa cocolysolytic enzyme gene of the silkworm to be detected, according to the model obtained in step (1) of the sex control, to determine the development time of the pupal stage, and then to complete the judgment of the time of the silkworm to transform into a moth.
  • step (1) and step (2) are the same, and the elapsed time of the pupal stage of each variety is determined.
  • step (1) specifically includes the following steps: take the silkworm chrysalis of the same variety as the silkworm to be detected, and divide them into two groups, male and female, and the first time is the blood of the pupae within 2 to 3 hours of pupation; then every 48 Blood was taken once every hour until moths were killed; then total RNA was extracted, treated with DNase, and cDNA was prepared by reverse transcription; Actin gene 3 ( Actin3 ) and lysase gene were used as templates, respectively, by Real-time PCR method Amplification was performed; the relative transcriptional expression of lysin gene was the transcriptional expression of lysinase gene/the transcriptional expression of Actin3 gene. Then, according to the sex of the pupa, the curve equation of the relative transcriptional expression of cocoon lysin and the development time of the pupal stage was established as the correlation model.
  • step (2) specifically includes the following steps: firstly identifying the sex of the silkworm pupae to be tested, then extracting total RNA from the blood of the silkworm pupae to be tested, after DNase treatment, reverse transcription to prepare cDNA, and the actin gene 3
  • the lysin gene and the cocoon lysin gene were respectively used as templates, and were amplified by the Real-time PCR reaction to calculate the relative transcription and expression level of the cocoon lysin gene of the sample to be tested, and then the development time of the silkworm pupae to be tested was determined according to the model obtained in step (1). This infers moth time.
  • step (1) the starting point of the development time of the pupa stage is 2 to 3 hours of pupation, and the end point is the moth, and under normal environmental conditions, the silkworm pupae are protected from pupation to moth; For 48 hours; for example, in step (1), the first sampling is within 2 to 3 hours of pupation, and the mathematical model will be affected if it is too early or too late. , which are respectively recorded as P3, P5, P7, P9, P11... .
  • step (1) takes the blood of the silkworm chrysalis of different pupal stage development times, each time more than 6 silkworm chrysalis are selected for sampling, so as to avoid errors caused by individual differences.
  • the detection method for inferring the time of silkworm transformation is applicable to all silkworm varieties.
  • the development time of pupa stage is taken as the abscissa
  • the relative transcription expression level of cocoon lysase gene is taken as ordinate to get a polynomial trend curve.
  • cocoon lysin gene changes, until before moth, Coc proenzyme is activated and secreted to play a role, hydrolyzing sericin and silk fibroin, which is convenient for silkworm moth to break out of cocoon. Therefore, it is feasible to judge the time of Bombyx moth by measuring the relative expression of lysase gene.
  • the present invention discloses primers for judging the time of silkworm moth transformation, as follows.
  • the sample (blood of silkworm chrysalis) is stored in a -80°C ultra-low temperature refrigerator to avoid RNA degradation, and the storage time should not exceed 30 days.
  • the present invention has the following advantages compared with the prior art.
  • the present invention provides a kind of method for accurately judging the time of silkworm moth transformation, it only needs to adopt a small amount of silkworm pupa blood samples to realize accurate measurement of the pupal stage development time, without damaging the pupa, more representative than traditional methods. .
  • the determination method of the present invention can avoid the judgment error of the traditional method, has the characteristics of accurate results, convenient operation and strong practicability, and is suitable for popularization and application.
  • Fig. 1 is a graph showing the relative relationship between the relative transcriptional expression level of Suxiu ⁇ Chunfeng male pupa cocoon lysing enzyme gene and the development time of the pupal stage in the embodiment.
  • Fig. 2 is a graph showing the relative relationship between the relative transcriptional expression level of the Suxiu ⁇ Chunfeng female cocoon lysing enzyme gene and the development time of the pupal stage in the embodiment.
  • the present invention discloses a scientific and accurate judgment method for the first time, and uses the designed primers and the operation method to obtain a correlation model, and then judges the moth removal of the same variety of silkworm, and the result is accurate .
  • the substances involved in the present invention are all conventional substances in the field, and the specific operation methods are all conventional methods in the field; the present invention is further described below with reference to the examples and accompanying drawings.
  • Embodiment 1 a method for inferring the time of silkworm moths.
  • the silkworm chrysalis were bred from Soochow University (Suxiu ⁇ Chunfeng); the incubator was a 250D artificial climate chamber produced by Shaoguan Xinteng Science Instrument Co., Ltd., Guangdong province; and the quantitative PCR instrument was ABI ViiA7.
  • Sample Take more than 6 male or female pupae for model construction every 48 hours.
  • primers were sent and synthesized according to conventional methods: 5'-TCGCCGGATCGAAACTGAAA-3'; 5'-GCACCGTTACTTTGGAAATACCTT-3'; 5'-CGGCTACTCGTTCACTACC-3'; 5'-CCGTCGGGAAGTTCGTAAG-3'.
  • RNA extraction, DNase treatment, and reverse transcription synthesis of cDNA The extraction of total RNA, DNase treatment and reverse transcription synthesis of cDNA were performed according to the Molecular Cloning Guidelines (Third Edition) and were routine techniques.
  • Quantitative PCR detection using the fluorescent dye SYBR GreenI on a ViiA7 instrument.
  • Real-time PCR reaction system 2 ⁇ SYBR Ex Taq (2 ⁇ ) 10 ⁇ l; ROX Reference Dye (50 ⁇ ) 0.4 ⁇ l; upstream primer (10 ⁇ mol/L) 0.4 ⁇ l; downstream primer (10 ⁇ mol/L) 0.4 ⁇ l; cDNA 2.0 ⁇ l; dH 2 O 6.8 ⁇ l; total system 20 ⁇ l.
  • the reaction conditions were as follows: 95 °C for 1 min; 45 cycles of 95 °C for 5 s, 55 °C for 10 s, and 72 °C for 10 s.
  • upstream primers were 5'-TCGCCGGATCGAAACTGAAA-3', 5'-CGGCTACTCGTTCACTACC-3', and downstream primers were 5'-GCACCGTTACTTTGGAAATACCTT-3', 5'-CCGTCGGGAAGTTCGTAAG-3'.
  • Table 1 data is carried out statistical drawing, accompanying drawing 1 is above-mentioned Suxiu ⁇ Chunfeng breed male pupa cocoon lysing enzyme gene relative transcriptional expression and the relative relationship curve of the pupal stage development time, from which it can be seen that the cocoon lysing enzyme gene is relatively
  • Table 2 data is carried out statistical drawing, accompanying drawing 2 is above-mentioned Suxiu ⁇ Chunfeng variety female pupa cocoon lysing enzyme gene relative transcriptional expression and the relative relationship curve of the pupal stage development time, from which it can be seen that the cocoon lysing enzyme gene is relatively
  • Application example 1 Take a female pupae that needs to accurately judge the time of moth transformation and carry out quantitative PCR detection.
  • the average Ct value of the cocoon lysing enzyme gene is 19.085
  • the average Ct value of the Actin3 gene is 25.796.
  • the corresponding log values are 3.169 and 1.103 respectively
  • the corresponding lysin gene transcription and expression level is 1474.1937
  • the Actin3 gene transcription and expression level is 12.6862.
  • the relative transcript expression of the gene was 116.2049. It can be seen from Fig. 2 that the development time of silkworm chrysalis is between the 12th and 13th days.
  • Comparative application Change the 2 hours after the pupation of the hairy silkworm in (2) to 1 hour after the pupation of the hairy foot silkworm, and the rest remain unchanged to obtain the development time of the male pupa and the relative transcriptional expression of the cocoon lysing enzyme gene. Quantitative relationship model, the application example 2 is brought into the relationship model, and x ⁇ 7.38 is calculated.

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Abstract

一种判断家蚕化蛾时间的方法，具体包括以下步骤：(1)分别构建待检测品种雄蛹、雌蛹的溶茧酶(Cocoonase，Coc)基因的相对转录表达量与蛹期发育时间的关联模型；(2)测定待检测蚕蛹溶茧酶基因相对表达量，根据性别雌雄对照步骤(1)所得关联模型，计算得出蛹期发育时间，进而判断化蛾时间。该方法可通过对一个或多个蚕蛹的蛹期发育时间进行精确测定而精确判断家蚕化蛾时间，可减少传统肉眼观察造成的误差，为家蚕制种工作中判定化蛾时间提供新的检测方法。

Description

一种判断家蚕化蛾时间的方法 技术领域
本发明涉及一种判断家蚕化蛾时间的方法，具体涉及一种基于基因相对转录表达量的家蚕蛹期发育时间的精确检测方法。
 背景技术
家蚕( Bombyx mori)是一种重要的经济昆虫，在中国已经有5700多年的驯化历史。家蚕的一生经历4个不同的发育阶段，即卵、幼虫、蛹和成虫。由蛹期到成虫期的变态发育过程称为羽化，这个过程一般经历10~15天，一般上蔟后7天左右采茧。家蚕在蛹期不需要从外界环境摄取营养物质，但体内剧烈地进行着幼虫组织器官的破坏，成虫组织器官的发生形成。
目前生产技术中，传统方法是在特定的温湿度条件下（如温度25±1℃，湿度75~80%）通过观察蛹发育到后期颜色的变化（化蛹前颜色变深），并结合生产经验来确定蛹期发育进程，进而推断化蛾时间。由于不同品种或不同批次蛹的颜色存在差异，仅依靠传统的肉眼观察结合生产经验很容易造成误差。另外，家蚕种质资源是蚕业生产的宝贵财富，我国种质资源类型多样，涵盖茧色限性、粗纤度、细纤度等，保存种质资源对我国蚕桑产业具有重要的战略意义，而其中能够准确推断化蛾时间进而快速有效开展制种工作，将稀缺的种质资源保留继代下去显得尤为重要。因此，需要寻找一种能够对家蚕化蛾时间精确推断的方法。
 技术问题
本发明的目的是提供一种新的判断家蚕化蛾时间的方法，以精确推断家蚕的化蛾时间。
 技术解决方案
为达到上述发明目的，本发明采用的技术方案是：一种判断家蚕化蛾时间的方法，包括以下步骤：（1）分别构建家蚕雄蛹、雌蛹的溶茧酶基因的相对转录表达量与蛹期发育时间的关联模型。
（2）测定待检测家蚕的蚕蛹溶茧酶基因相对表达量，根据性别雌雄对照步骤（1）所得模型，从而确定蛹期发育时间，进而完成家蚕化蛾时间的判断。
本发明中，步骤（1）、步骤（2）的家蚕品种一致，每个品种蛹期经历时间确定。
本发明中，步骤（1）具体包括如下步骤：取与待检测家蚕同品种的蚕蛹，分为雌雄两组，第1次取材为化蛹2~3小时内的蛹的血液；然后每隔48小时取一次血液，直至化蛾；然后提取总RNA，经DNA酶处理后，反转录制备cDNA；以肌动蛋白基因3( Actin3)、溶茧酶基因分别为模板，通过Real-time PCR法进行扩增；溶茧酶基因相对转录表达量即溶茧酶基因转录表达量/ Actin3基因转录表达量。然后依据蛹的性别分别建立溶茧酶相对转录表达量与蛹期发育时间的曲线方程，为关联模型。
本发明中，步骤（2）具体包括如下步骤：首先鉴别待测蚕蛹的性别，然后提取待测定蚕蛹样本血液的总RNA，经DNA酶处理后，反转录制备cDNA，以肌动蛋白基因3、溶茧酶基因分别为模板，通过Real-time PCR反应进行扩增，计算待测样本溶茧酶基因相对转录表达量，然后对照步骤（1）所得模型确定出待测蚕蛹的发育时间，以此推断出化蛾时间。
上述技术方案中，步骤（1）中，蛹期发育时间起点为化蛹2~3小时，终点为化蛾，在常规环境条件下保护蚕蛹由化蛹至化蛾；相邻蛹期发育时间间隔为48小时；比如步骤（1）中第1次取材为化蛹2~3小时内，过早或过晚均会影响数学模型，此蛹期发育时间记为P1，之后每间隔48小时取材一次，分别依次记为P3、P5、P7、P9、P11……。
上述技术方案中，所述步骤（1）取不同蛹期发育时间的蚕蛹血液时，每次选取6个以上的蚕蛹进行取材，避免因个体的不同而导致的误差。
上述技术方案中，所述推断家蚕化蛾时间的检测方法适用于所有家蚕品种。
优选的技术方案中，依据性别雌雄，分别建立家蚕溶茧酶基因相对转录表达量和蛹期发育时间的关联模型时，以蛹期发育时间为横坐标，以溶茧酶基因相对转录表达量为纵坐标，得到一条多项式趋势曲线。
本发明中，总RNA的提取、DNA酶处理和反转录合成cDNA处理根据分子克隆实验指南（第三版）进行。
家蚕从蛹期到成虫的变态发育过程中，溶茧酶基因表达量变化，直至化蛾前，Coc酶原被激活并分泌发挥作用，水解丝胶和丝素，便于蚕蛾破茧而出。因此通过测定溶茧酶基因的相对表达量判断家蚕化蛾时间是可行的。
本发明公开了判断家蚕化蛾时间用引物，如下。
5’-TCGCCGGATCGAAACTGAAA-3’；5’-GCACCGTTACTTTGGAAATACCTT-3；5’-CGGCTACTCGTTCACTACC-3’；5’-CCGTCGGGAAGTTCGTAAG-3’。
本发明中取样（蚕蛹血液）存放于-80℃超低温冰箱保存，避免RNA降解，并且存放时间不宜超过30天。
本发明中定量PCR（Real-time PCR反应）的原理及数据分析方法已经被J.H. Schefe公开(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), 本领域技术人员可以参考。
 有益效果
由于上述技术方案运用，本发明与现有技术相比具有下列优点。
1、本发明提供了一种精确判断家蚕化蛾时间的方法，其只需采用少量蚕蛹血液样本即可实现对蛹期发育时间进行准确的测定，不损伤蛹体，较传统方法更具有代表性。
2、本发明的测定方法能够避免传统方法的判断误差，具有结果准确、操作方便、实用性强的特点，适合推广应用。
 附图说明
图1是实施例中苏秀×春丰品种雄蛹溶茧酶基因相对转录表达量与蛹期发育时间的相对关系曲线图。
图2是实施例中苏秀×春丰品种雌蛹溶茧酶基因相对转录表达量与蛹期发育时间的相对关系曲线图。
 本发明的实施方式
针对现有技术仅能通过人工观测推测化蛾时间的问题，本发明首次公开了科学、准确的判断法，采用设计引物以及操作方法得到关联模型，再对同品种家蚕进行化蛾判断，结果准确。除引物外，本发明涉及的物质都为本领域常规物质，具体操作方法都为本领域常规方法；下面结合实施例及附图对本发明作进一步描述。
实施例一：一种推断家蚕化蛾时间的测定方法。
（1）材料及设备。
家蚕蛹采用苏州大学育成的品种（苏秀×春丰）；培养箱为广东省韶关鑫腾科普仪器有限公司生产的250D人工气候箱；定量PCR仪为ABI ViiA7。
样本：每隔48小时分别取6个以上雄蛹或雌蛹用于模型的构建。
（2）上蔟及数量采集。
采用常规方法饲养家蚕至上蔟，上蔟3天后采下蚕茧，削开蚕茧取出没有化蛹的毛脚蚕，待毛脚蚕化蛹后2小时，分别取6个雄蚕蛹或雌蚕蛹的血液，设3个重复，此蛹期发育阶段记为P1。采用温度25℃、湿度75%的条件保护蚕蛹至化蛾，每48小时取该6个雄蚕蛹或雌蚕蛹的血液，蛹期发育阶段分别记为P3、P5、P7、P9、P11、P13。
（3）定量PCR分析。
根据常规方法送外合成如下引物：5’-TCGCCGGATCGAAACTGAAA-3’；5’-GCACCGTTACTTTGGAAATACCTT-3’；5’-CGGCTACTCGTTCACTACC-3’；5’-CCGTCGGGAAGTTCGTAAG-3’。
血液总RNA提取、DNA酶处理和反转录合成cDNA：总RNA的提取，DNA酶处理和反转录合成cDNA处理根据分子克隆指南（第三版）进行，为常规技术。
定量PCR检测：采用荧光染料SYBR GreenI在ViiA7仪器上进行。
Real-time PCR反应体系：2×SYBR Ex Taq (2×) 10 μl; ROX Reference Dye (50×) 0.4 μl; 上游引物（10μmol/L）0.4 μl；下游引物（10μmol/L）0.4 μl；cDNA 2.0 μl; dH 2O 6.8 μl; 总体系20 μl。反应条件如下：95 ℃ 1 min；95 ℃ 5 s, 55 ℃ 10 s, 72 ℃ 10 s，45个循环。所有样品均设三个独立的重复实验，上游引物为5’-TCGCCGGATCGAAACTGAAA-3’、 5’-CGGCTACTCGTTCACTACC-3’，下游引物为5’-GCACCGTTACTTTGGAAATACCTT-3’、 5’-CCGTCGGGAAGTTCGTAAG-3’。
（4）数据统计：按照J.H. Schefe公开的方法，根据测定的Ct值制定标准曲线，溶茧酶基因的标准曲线方程为：y= -3.2488x+29.379, R 2=0.9962； Actin3基因的标准曲线方程为y= -3.213x+29.341, R 2=0.9987。根据各样本测定的Ct值，经三组平均后，分别求出对应的log值，再根据2- △△ CT法求出对应的表达量如下表。
Figure 955711dest_path_image001
。
将表1数据进行统计作图，附图1为上述苏秀×春丰品种雄蛹溶茧酶基因相对转录表达量和蛹期发育时间的相对关系曲线图，从中可以看出溶茧酶基因相对转录表达量符合多项式趋势曲线模型，方程为：y=0.3458x 3-2.9414x 2+5.6313x-1.8162, R 2=0.988。
Figure 27441dest_path_image002
。
将表2数据进行统计作图，附图2为上述苏秀×春丰品种雌蛹溶茧酶基因相对转录表达量和蛹期发育时间的相对关系曲线图，从中可以看出溶茧酶基因相对转录表达量符合多项式趋势曲线模型，方程为：y=0.3471x 3-4.8614x 2+18.322x-15.571, R 2=0.9934。
（5）应用：家蚕蛹采用苏州大学育成的品种（苏秀×春丰），在常规饲养过程中随机选择蚕蛹进行化蛾判断，家蚕性别雌雄的判断为常规手段。
应用例一：取一个需要精确判断化蛾时间的雌蛹进行定量PCR检测，采用上述相同方法，测得溶茧酶基因Ct均值为19.085， Actin3基因Ct均值为25.796，根据两个基因的标准曲线方程式，得出对应的log值分别为3.169和1.103，再根据2- △△ CT法求出对应的溶茧酶基因转录表达量为1474.1937， Actin3基因转录表达量为12.6862，该雌蛹溶茧酶基因相对转录表达量为116.2049。对照图2可以看出蚕蛹的蛹期发育时间处于第12~13天之间。根据公式y=0.3471x 3-4.8614x 2+18.322x-15.571，计算出x≈12.23，按照苏秀×春丰品种雌蛹蛹期经历时间13天，可判定雌蛹将于0.77天后化蛾。根据常规观察法判断该雄蛹于第2天化蛾。
应用例二：取一个需要精确判断化蛾时间的雄蛹进行定量PCR检测，采用上述相同方法，测得该雄蛹溶茧酶基因相对表达量为50.4033。对照图1可以看出蚕蛹的蛹期发育时间处于第8~9天之间。根据公式y=0.3458x 3-2.9414x 2+5.6313x-1.8162，计算出x≈8.64，按照苏秀×春丰品种雄蛹蛹期经历时间11天，可判定雄蛹将于2.36天后化蛾。根据常规观察法判断该雄蛹于3天后化蛾。
（6）检测结果的验证：将应用例一、二中同批次的雌蛹、雄蛹继续保护至羽化，结果观察到雄蛹于2.5日后化蛾，而雌蛹于当日内化蛾，证明本发明的检测方法是准确的；同时，检测方法精确度也比传统方法要高，天数精确至小数点后。
（7）重复应用：根据上述应用例一、应用例二的方法，再随机检测50颗雌蛹、50颗雄蛹，具有不同时间间隔，根据图1、图2得出的化蛾时间都与实际化蛾时间一致。
（8）对比应用：将（2）中的毛脚蚕化蛹后2小时更改为毛脚蚕化蛹后1小时，其余不变，得到雄蛹蛹期发育时间与溶茧酶基因相对转录表达量关系模型，将应用例二带入该关系模型，计算得到x≈7.38。
将（3）中的引物更换为：5’-ATGAGTTCCTCACCTGTCGC-3’；5’-ATCGAACCTCCGCATTGGAA-3’；5’-CGGCTACTCGTTCACTACC-3’；5’-CCGTCGGGAAGTTCGTAAG-3’。
其余不变，得到雄蛹蛹期发育时间与溶茧酶基因相对转录表达量关系模型，将应用例二带入该关系模型，计算得到x≈9.55。

Claims (8)

  1. 一种判断家蚕化蛾时间的方法，其特征在于，包括以下步骤：
    （1）分别构建家蚕雄蛹、雌蛹的溶茧酶基因的相对转录表达量与蛹期发育时间的关联模型；
    （2）测定待检测家蚕的蚕蛹溶茧酶基因相对表达量，根据性别雌雄对照步骤（1）所得模型，从而确定蛹期发育时间，进而完成家蚕化蛾时间的判断。
  2. 根据权利要求1所述判断家蚕化蛾时间的方法，其特征在于，步骤（1）、步骤（2）的家蚕品种一致。
  3. 根据权利要求1所述判断家蚕化蛾时间的方法，其特征在于，步骤（1）中，蛹期发育时间起点为化蛹2~3小时，终点为化蛾；取每个蛹期发育时间下蚕蛹的血液，提取总RNA，经DNA酶处理后，反转录制备cDNA；以肌动蛋白基因3、溶茧酶基因为模板，通过实时荧光定量PCR反应进行扩增，分别得到肌动蛋白基因3转录表达量、溶茧酶基因转录表达量；溶茧酶基因相对转录表达量为溶茧酶基因的转录表达量/肌动蛋白基因3的转录表达量。
  4. 根据权利要求3所述判断家蚕化蛾时间的方法，其特征在于，荧光定量PCR反应时，引物如下：
    5’-TCGCCGGATCGAAACTGAAA-3’
    5’-GCACCGTTACTTTGGAAATACCTT-3’
    5’-CGGCTACTCGTTCACTACC-3’
    5’-CCGTCGGGAAGTTCGTAAG-3’。
  5. 根据权利要求3所述家蚕化蛾时间的检测方法，其特征在于：步骤（1）中，在常规环境条件下保护蚕蛹由化蛹至化蛾；相邻蛹期发育时间间隔为48小时。
  6. 根据权利要求1所述判断家蚕化蛾时间的方法，其特征在于，步骤（2）中，取待检测家蚕的蚕蛹的血液，提取总RNA，经DNA酶处理后，反转录制备cDNA；以肌动蛋白基因3、溶茧酶基因为模板，通过实时荧光定量PCR反应进行扩增，分别得到肌动蛋白基因3转录表达量、溶茧酶基因转录表达量；溶茧酶基因相对转录表达量为溶茧酶基因的相对表达量/肌动蛋白基因3的转录表达量。
  7. 根据权利要求6所述判断家蚕化蛾时间的方法，其特征在于，荧光定量PCR反应时，引物如下：
    5’-TCGCCGGATCGAAACTGAAA-3’
    5’-GCACCGTTACTTTGGAAATACCTT-3’
    5’-CGGCTACTCGTTCACTACC-3’
    5’-CCGTCGGGAAGTTCGTAAG-3’。
  8. 根据权利要求1所述判断家蚕化蛾时间的方法，其特征在于，关联模型为溶茧酶相对转录表达量与蛹期发育时间的曲线方程。
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