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Internal reference gene of loofah and its primer and application

A technology of internal reference gene and loofah, which is applied in the field of molecular biology, can solve the problems of lack of internal reference gene, etc., and achieve the effect of authenticity and reliability of data, improvement of detection efficiency and detection results, and high stability

Active Publication Date: 2021-11-02
INST OF VEGETABLES GUANGDONG PROV ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, there is a lack of internal reference genes stably expressed in different tissues of loofah

Method used

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  • Internal reference gene of loofah and its primer and application
  • Internal reference gene of loofah and its primer and application
  • Internal reference gene of loofah and its primer and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] The screening of embodiment 1 candidate reference gene

[0041] The roots, stems, leaves, growth points, male flower petals, female flower petals, stamens, stigmas, ovary, and fruit 10 days after pollination were taken from loofah P93075, and RNA was extracted and reverse-transcribed into cDNA. A total of 11 candidate reference genes were screened out in this study: Lc18SRNA, ACT7, β-ACTIN, DNAJ, EF1α, EIF4A, GAPDH, PP2A, RPL2, TUA, UBQ, including a reference gene Lc18SRNA used in the previous literature, specifically See Table 1 for information.

[0042] Table 1 Candidate reference gene information

[0043]

[0044]

[0045] Further, qRT-PCR was performed by agarose gel electrophoresis and qRT-PCR melting curve (using QuantStudio 6Flex (ABI, USA) in a 10 μl reaction volume containing 0.5 μl cDNA, 5 μl SYBR Premix Ex TaqII (Takara, Japan), 0.2 μl forward and reverse primers (10 μM) and 4.1 μl ddH 2 O. PCR conditions are: 95°C, 30 seconds; 95°C, 10 seconds, 40 ...

Embodiment 2

[0046] The expression analysis of embodiment 2 candidate reference genes

[0047] The expression levels of 11 candidate reference genes were detected by qRT-PCR, and all the test results can be found in image 3 . It can be seen that the Ct values ​​of candidate reference genes in all samples range from Lc18SRNA (14.3) to TUA (33.2). Lc18SRNA has the lowest Ct value, indicating the highest expression level, while TUA has the highest Ct value, implying the lowest expression level. The expression variation of Lc18SRNA was the least, and that of TUA was the highest.

Embodiment 3

[0048] The expression stability analysis of the candidate internal reference gene of embodiment 3

[0049] The expression stability of 11 candidate reference genes in 10 tissues were evaluated by BestKeeper, comparative delta-Ct method, geNorm and NormFinder programs, respectively.

[0050] 1. BestKeeper sorts the candidate reference genes according to the standard deviation (SD) and coefficient of variation (CV) values, see the results Figure 4 Middle panel A and Table 2. BestKeeper analysis results showed that the lowest CV±SD was Lc18SRNA at 3.25±0.50, indicating that this gene was the most stable reference gene, followed by DNAJ at 3.76±0.82, and TUA at 17.78±4.34, with the highest CV±SD being the least stable reference gene. The stable gene, followed by GAPDH was 7.23 ± 1.58.

[0051] Table 2 Candidate gene stability analysis

[0052]

[0053] 2. According to the comparative delta-Ct method, genes with lower SD values ​​show higher expression stability, see the res...

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Abstract

The invention discloses internal reference genes of different tissues of loofah, which are PP2A and / or β-ACTIN. The sequence of the internal reference gene PP2A is shown in SEQ ID No.1; the sequence of the internal reference gene β-ACTIN is shown in SEQ ID No. 2. The internal reference genes PP2A and β-ACTIN of the present invention have higher stability than other internal reference genes, and these two internal reference genes are screened out, and are the most stable internal reference genes in different tissues of loofah, and the data are true and reliable. Given the fact that there is no internal reference gene in loofah gene expression analysis, it can also improve detection efficiency and detection results, and has high practical value. It provides strong support for obtaining accurate quantitative data of functional gene expression in different tissues of loofah, and provides a basis for gene function of loofah. Research provides a theoretical basis.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to an internal reference gene of loofah and its primer and application. Background technique [0002] Luffa cylindrica is an important vegetable crop widely grown in my country, India and Southeast Asia. The unripe loofah fruit is a good source of carbohydrates, vitamins, and various minerals. Recent studies have shown that winter melon has antibacterial, anticancer, antioxidative and other effects, and has been widely used in medicine. The mature loofah compound can be used for washing, filtering, and combining with graphene nanosheets to make a new type of electromagnetic shielding composite material. [0003] Quantitative real-time PCR (qRT-PCR) is widely used in the quantitative analysis of gene expression because of its sensitivity, accuracy and rapidity. However, the accuracy of qRT-PCR is easily affected by several factors, including RNA content and q...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/6809C12Q1/686C12N15/11
CPCC12Q1/6809C12Q1/686C12Q1/6895C12Q2600/166C12Q2531/113C12Q2521/107C12Q2545/101C12Q2537/165
Inventor 赵钢军吴海滨罗剑宁龚浩李俊星郑晓明刘小茜
Owner INST OF VEGETABLES GUANGDONG PROV ACAD OF AGRI SCI
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