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Application of ClCAC gene and ClSAND gene as reference genes in analysis of gene expression of watermelon fruits

A gene expression and internal reference gene technology, applied in the field of plant molecular biology, can solve the problems of lack of internal reference genes, affecting the accuracy of quantitative analysis of gene expression, unreasonable internal reference genes, etc., and achieving the effect of wide applicability.

Inactive Publication Date: 2015-02-11
HUAZHONG AGRI UNIV
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Problems solved by technology

Obviously it is unreasonable to use so many internal reference genes, especially when the number of target genes is very small
Moreover, more and more studies believe that there is no universally applicable reference gene
Watermelon is considered to be an ideal model crop for studying the development of non-respiratory climacteric fruits. However, 18SrRNA, whose expression stability is not systematically verified, is still used as an internal reference gene in the expression analysis of watermelon fruit genes, which seriously affects the process of watermelon fruit development. Accuracy in Performing Gene Expression Quantification

Method used

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  • Application of ClCAC gene and ClSAND gene as reference genes in analysis of gene expression of watermelon fruits
  • Application of ClCAC gene and ClSAND gene as reference genes in analysis of gene expression of watermelon fruits
  • Application of ClCAC gene and ClSAND gene as reference genes in analysis of gene expression of watermelon fruits

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] (1) Plant material and treatment: Watermelon inbred line 97103 and F1 hybrid 8424 were selected as materials, planted in plastic greenhouses, and the ovaries that were about to bloom were bagged and isolated one day before flowering. On the day of flowering, artificial pollination and CPPU treatment were used to induce watermelon fruit setting. CPPU (forchlorfenuron 0.1%; Sichuan Guoguang Agrochemical Co., Ltd.) was used at a concentration of 20 mg / L, and the paper bag was removed 8 hours in the morning, sprayed evenly on the watermelon ovary, and then bagged for isolation to prevent pollination. Each treatment was repeated 3 times and arranged randomly. Fruits were sampled at 0, 1, 3, 5, 7, 10, 12, 18, 23, 27, 30 and 35 days after pollination and CPPU treatment, and 3 fruits were randomly selected for each treatment as each sampling point biological replicates. After pollination and CPPU treatment, the ovary was taken 0, 1, 3 days after, and the center pulp was taken...

Embodiment 2

[0032](1) Plant material: the watermelon F1 generation hybrid 8424 was planted in a plastic greenhouse, and the branches were artificially pollinated and marked on the day of flowering. The fruits were sampled at 10, 18, 23, 27, 30 and 35 days after artificial pollination, and two fruits were randomly selected at each time point as a biological repeat, and three biological repeats were set up. The central pulp of the watermelon fruit was taken, quick-frozen in liquid nitrogen, and stored at -80°C.

[0033] (2) Isolation of total RNA, synthesis of first-strand cDNA and isolation of DNA were carried out according to the method described in Example 1.

[0034] (3) Target genes: Phytoene synthase gene (ClPSY2), carotene dehydrogenase gene (ClZDS) and β-carotene hydroxylase gene (ClCHYB1) in the lycopene synthesis pathway were selected as target genes , to analyze its expression during watermelon fruit ripening. The information of these genes is obtained from literature (Stefania...

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Abstract

The invention belongs to the technical field of analysis of gene expression and particularly relates to application of ClCAC gene and ClSAND gene as reference genes in analysis of gene expression of watermelon fruits. According to the invention, specific primers are designed for ClCAC gene and ClSAND gene. The specific primers cross over joint loci of two exons; no amplification product is generated when a genome DNA is used as a template; a target segment can be obtained through amplification when cDNA is used as a template; the nucleotide sequences of the primers are shown in SEQ ID No.1-4. Real-time fluorescence quantification PCR detection shows that both the ClCAC gene and the ClSAND gene can be expressed stably in different development stages of watermelon fruits which are different in gene type and fruit bearing mode and thus the ClCAC gene and the ClSAND gene can be used as a reliable reference gene combination in analysis of gene expression by real-time fluorescence quantification PCR detection. The reference gene combination has the advantages of good expression stability and the like and is not influenced no matter whether the genome DNA contamination exists in a cDNA sample or not.

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 gene expression analysis process. Background technique [0002] At present, real-time fluorescence quantitative PCR (qRT-PCR for short) has been widely used in the quantitative analysis of gene expression, as well as the verification of RNA-seq and gene chip results. This technique is simple and fast, but the reliability of the results depends on the selection of genes with stable expression as internal references for data standardization. The role of internal reference genes is mainly to remove the interference of abiotic variation during sample preparation and qRT-PCR analysis, such as the quality of RNA extraction, the efficiency of reverse transcription, and the amplification efficiency in qRT-PCR analysis. Therefore, the accuracy of qRT-PCR results is largely affected by the expression stability of internal reference g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 别之龙孔秋生袁静贤高凌云黄远程菲
Owner HUAZHONG AGRI UNIV
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