Application of CmEF1 alpha gene and CmRAN gene used as reference genes in analysis of genetic expression of Cucumis melo L. fruits

A gene expression and internal reference gene technology, applied in the field of plant molecular biology, can solve the problem of lack of internal reference genes, and achieve the effect of wide applicability

Active Publication Date: 2015-02-18
HUAZHONG AGRI UNIV
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  • Abstract
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  • Claims
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Problems solved by technology

[0007] In order to solve the problem of the lack of systematically verified and stable expression internal reference genes in the quantitative analysis of gene expression during the development of melon fruit, the present invention provides a reliable internal reference gene specially used for gene expression analysis during the development of melon fruit

Method used

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  • Application of CmEF1 alpha gene and CmRAN gene used as reference genes in analysis of genetic expression of Cucumis melo L. fruits
  • Application of CmEF1 alpha gene and CmRAN gene used as reference genes in analysis of genetic expression of Cucumis melo L. fruits
  • Application of CmEF1 alpha gene and CmRAN gene used as reference genes in analysis of genetic expression of Cucumis melo L. fruits

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Effect test

Embodiment 1

[0020] (1) Plant material and treatment: the commercial muskmelon species Elizabeth was selected as the material, planted in a plastic greenhouse, and the ovary that was about to bloom was bagged and isolated one day before flowering. On the day of flowering, artificial pollination and CPPU treatment were used to induce fruit setting of muskmelon. CPPU (forchlorfenuron 0.1%; Sichuan Guoguang Agrochemical Co., Ltd.) was used at a concentration of 2.5mg / L. The paper bag was removed at 8:00 a.m., sprayed evenly on the melon ovary, and then bagged for isolation. Fruits were sampled at 1, 3, 5, 7, 10, 15, 20, 25, 28, 30 and 32 days after pollination and CPPU treatment, and three fruits were randomly selected at each sampling point, and each fruit was used as a biological Learn to repeat. The ovary was taken 1 and 3 days after pollination and CPPU treatment, and the mesocarp was taken from the fruit after 3 days, quick-frozen in liquid nitrogen, and stored at -80°C for subsequent R...

Embodiment 2

[0034] (1) Plant material: the commercial muskmelon species Elizabeth was planted in a plastic greenhouse, artificially pollinated and marked on the day of flowering. Fruits were sampled at 7, 10, 15, 20, 25, 28 and 32 days after artificial pollination, and 2 fruits were randomly selected at each time point as a biological repeat, and three biological repeats were set up. The mesocarp of melon fruit was taken, quick-frozen in liquid nitrogen, and stored at -80°C.

[0035](2) Isolation of total RNA: the synthesis of first-strand cDNA and the isolation of DNA were carried out according to the method described in Example 1.

[0036] (3) Target gene: The sucrose phosphate synthase gene CmSPS1 ((MELO3C010300)), which is closely related to the accumulation of sucrose in melon fruit, was selected as the target gene to analyze its expression during the ripening process of melon fruit. The information of this gene was obtained from the literature (Dai, N.; Cohen, S.; Portnoy, V.; Tzur...

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Abstract

The invention belongs to the technical field of analysis of gene expression, and discloses application of a CmEF1 alpha gene and a CmRAN gene used as reference genes in analysis of genetic expression of Cucumis melo L. fruits. The nucleotide sequence of the CmEF1 alpha gene is shown in SEQ ID No:1; the nucleotide sequence of the CmRAN gene is shown in SEQ ID NO:2. The invention further discloses a method for analyzing genetic expression of Cucumis melo L. fruits through adopting real-time fluorescence quantification PCR, and the method includes the step that the geometric mean of the gene combination of the CmEF1 alpha gene and the CmRAN gene is used as the reference genes in analysis of genetic expression of Cucumis melo L. fruits. According to the invention, the CmEF1 alpha gene and the CmRAN gene can be stably expressed in Cucumis melo L. fruits adopting different fruit bearing manners and at different developmental stages, so as to serve as a reliable reference gene combination in analysis of genetic expression during the development process of Cucumis melo L. fruits adopting the real-time fluorescence quantification PCR technology for detection.

Description

technical field [0001] The invention belongs to the technical field of plant molecular biology and relates to an internal reference gene required in the process of analyzing muskmelon fruit gene expression by using a real-time fluorescent quantitative PCR method. Background technique [0002] Melon (Cucumis melo L.) is a Cucurbitaceae crop widely cultivated in the world and has important economic value. Melon fruits exhibit a rich diversity in size, color, sweetness, flavor, and texture. In addition, based on the different trends of respiration rate and ethylene during fruit ripening, muskmelon can be further divided into climacteric and non-respiratory climacteric types. Therefore, muskmelon is considered as an ideal model plant in the study of fruit development. [0003] Analysis of gene expression in muskmelon fruit development is an important means to study its fruit expansion and ripening mechanism. Real-time fluorescent quantitative PCR (qRT-PCR for short) has becom...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6895C12Q2600/166
Inventor 别之龙孔秋生高凌云袁静贤黄远程菲
Owner HUAZHONG AGRI UNIV
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