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Cucurbita moschata internal reference gene and application thereof

An internal reference gene, Chinese pumpkin technology, applied in the field of molecular biology, to achieve the effect of improving detection efficiency, stability and reliability

Active Publication Date: 2015-07-22
CROP RES INST OF FUJIAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the cloning of the 18S rRNA gene of Chinese pumpkin and its use as an internal reference gene for Chinese pumpkin.

Method used

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  • Cucurbita moschata internal reference gene and application thereof
  • Cucurbita moschata internal reference gene and application thereof
  • Cucurbita moschata internal reference gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] The acquisition of internal reference gene of embodiment 1

[0023] Step (1), design a pair of primers according to the 18S rRNA gene of watermelon, muskmelon and zucchini announced by Genbank, and utilize the clustalx software to carry out sequence alignment to the primer pair designed, and by Platinum Biotechnology (Shanghai) Co., Ltd. The pair of primers were synthesized, specifically, the pair of primers (as shown in SEQ ID NO: 1, 2): forward primer 5'-CCAATCATACTCAAAAAGAAGAGTT-3', reverse primer 5'-AGGATTCAATCCAGCCACAGGTT-3'.

[0024] Step (2), DNA extraction: Weigh 0.5 g of Chinese pumpkin leaves in a mortar, add liquid nitrogen and 0.1 g of PVP to quickly and fully grind to powder, transfer the powder to a 1.5 mL centrifuge tube; add 600 μL pre- Heat up to 65°C 2% CTAB extraction buffer, add 7 μL 2-mercaptoethanol at the same time, mix well, put in a water bath at 65°C for 30 minutes, and invert once every 10 minutes; take out the centrifuge tube, cool to room te...

Embodiment 2

[0029] Embodiment 2 real-time fluorescent quantitative PCR design and conventional PCR detection

[0030] Based on the nucleotide sequence of the internal reference gene obtained in Example 1, using Primer Premier5.0 software, and following the principles of real-time fluorescent quantitative PCR primer design, a pair of fluorescent quantitative specific primers were designed, and the amplified fragment was 132bp. The specific primers for fluorescence quantification are the real-time fluorescent quantitative PCR primers (as shown in SEQ ID NO: 4, 5): forward primer 5'-AAACCTTACCAGCCTTGAC-3', reverse primer 5'-CGCTCGTTATAGGACTTGACC-3'.

[0031] Extract the total RNA of Chinese pumpkin, and synthesize the first strand of cDNA according to the method of PrimeScriptTM 1st Strand cDNA Synthesis Kit, that is, reverse transcribe the RNA into cDNA; then use the obtained cDNA as a template and real-time fluorescent quantitative PCR primers as primer pairs for PCR Amplification, and the...

Embodiment 3

[0035] Embodiment 3 real-time fluorescent quantitative PCR primer verification

[0036] Extract the total RNA of Chinese pumpkin, and synthesize the first strand of cDNA according to the method of PrimeScriptTM 1st Strand cDNA Synthesis Kit, that is, reverse transcribe the RNA into cDNA; then use the obtained cDNA as a template and follow the Power Green PCR Master Mix Instructions Carry out the PCR reaction on the ABI7500 real-time quantitative PCR instrument. The reaction system and reaction procedure of the PCR reaction are as follows:

[0037] The reaction system is: the total volume of the reaction system is 25 μL, 12.5 μL Power Green PCR Master Mix, 1 μ L template, 0.5 μ L (concentration is 10 μ mol / L) of the forward primer of real-time fluorescent quantitative PCR primer in embodiment 2, the reverse primer 0.5 μ L (concentration is 10 μ mol / L) of real-time fluorescent quantitative PCR primer among the embodiment 2 / L), add distilled water to a total volume of 25 μL. ...

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Abstract

The invention discloses a cucurbita moschata internal reference gene 18 S rRNA gene sequence, a quantitative specific primer is designed on the basis, the cucurbita moschata 18 S rRNA gene can be stably expressed in each growth and development stage of cucurbita moschata and all abiotic stress conditions, and is suitable for using as an internal reference gene in study of cucurbita moschata gene expression. For the first time, the 18 S rRNA gene as the internal reference gene is used for cucurbita moschata gene expression analysis, helps to improve the stability, reliability and repeatability of the cucurbita moschata gene expression analysis, the detection primer disclosed in the invention is specific, greatly improves the test efficiency, and improves the credibility of the test results.

Description

【Technical field】 [0001] The invention belongs to the technical field of molecular biology, and specifically relates to a Chinese pumpkin internal reference gene and its application. Specifically, a pair of fluorescent quantitative specific primers are designed based on the nucleotide sequence of the Chinese pumpkin internal reference gene. 【Background technique】 [0002] Chinese squash (Cucurbita moschata Duch.) is one of the three main cultivars in Cucurbitaceae (Cucurbitaceae) Cucurbita spp. Chinese pumpkin has strong adaptability, wide distribution, various varieties and rich nutrition. It is a traditional vegetable loved by our people. With the continuous deepening and development of its molecular biology research, gene expression analysis is gradually being applied to reveal the gene regulation mechanism of Chinese squash. In the analysis of gene expression, in order to eliminate the deviation of initial template amount, RNA quality, enzymatic reaction efficiency, etc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 朱海生温庆放王彬张前荣陈敏氡蓝新隆
Owner CROP RES INST OF FUJIAN ACAD OF AGRI SCI
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