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Internal reference gene for trichosanthes kirilowii maxim seedling stage male and female plant gene expression research and application thereof

A gene expression and internal reference gene technology, applied in the field of molecular biology, can solve problems such as uncontrollability, failure to find stable indicators, imbalance of male and female plant ratios, etc., and achieve the effect of improving accuracy and reliability

Pending Publication Date: 2021-01-29
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to increase the planting area and benefit of Trichosanthes chinensis, it is an economical and practical method to propagate by seeds, but studies have shown that only 10-30% of the female plants are used for seed propagation, and the ratio of male and female plants is seriously out of balance and uncontrollable
Predecessors have failed to find stable indicators for the identification of male and female plants of Trichosanthes seedlings through morphological, physiological and molecular markers.
At present, there is no report of internal reference genes used in the study of gene expression in male and female plants of Trichosanthes seedlings

Method used

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  • Internal reference gene for trichosanthes kirilowii maxim seedling stage male and female plant gene expression research and application thereof
  • Internal reference gene for trichosanthes kirilowii maxim seedling stage male and female plant gene expression research and application thereof
  • Internal reference gene for trichosanthes kirilowii maxim seedling stage male and female plant gene expression research and application thereof

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Effect test

Embodiment 1

[0038] The fluorescent quantitative PCR primer design of embodiment 1 candidate internal reference gene

[0039] The present invention selects 12 candidate internal reference genes from the transcriptome data of the leaves of the seedling stage male and female plants of the three existing species Cuilou (Wanlou No. 9, Wanlou No. 13, and Wanlou No. 17), and the described internal reference genes are 18S ribosomal RNA (18SrRNA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin (UBQ10), elongin (EF1α1, EF1α2, EF1α3), actin (Actin, Actin 7, Actin 97) , tubulin (α-tubulin, α-tubulin3, β-tubulin), each gene sequence according to Actin, GAPDH, 18SrRNA (marked as 18S in the figure), UBQ10, EF1α1, EF1α2, EF1α3, Actin7, Actin97, αTUB, αTUB3, The sequence of βTUB is shown in SEQ ID NO.1-SEQ ID NO.12 in turn. Using the above 12 candidate internal reference gene sequences as templates, Primer 5 was used to design fluorescent quantitative PCR-specific primers for each internal r...

Embodiment 2

[0044] Embodiment 2 real-time fluorescence quantitative PCR experiment

[0045] The experimental materials are the leaves of male and female plants of three Trichosanthes cultivars (Wanlou No. 9, Wanlou No. 13, and Wanlou No. 17) at the seedling stage. All samples were quickly frozen in liquid nitrogen immediately after sampling, and then stored in a -80°C refrigerator.

[0046] The RNA of each sample was extracted using Takara's miniBEST Plant RNA Extraction kit, and Takara's PrimerScript reverse transcriptase kit was used for reverse transcription. Using the 10-fold dilution of the cDNA obtained by reverse transcription as a template, the real-time fluorescent quantitative PCR experiment was carried out with specific primers for fluorescent quantitative PCR of each candidate internal reference gene.

[0047] Using fluorescent quantitative PCR primers of each candidate internal reference gene to amplify the cDNA of male and female plant leaf samples of three varieties of Tri...

Embodiment 3

[0049] Software evaluation of internal reference genes under all samples in embodiment 3

[0050] GeNorm software

[0051] Among the present invention, described geNorm software is geNorm_3.4, and its steps and standard are as follows:

[0052] First calculate the ΔCt value: find the minimum Ct value of each candidate internal reference gene in all samples, and then subtract the minimum Ct value from the Ct value of each sample to obtain the ΔCt value. Then use the function in Excel to calculate the 2 of the corresponding gene of the corresponding sample -ΔCt value, which is the relative quantitative data of each candidate internal reference gene, which is also the data to be used in geNorm software analysis. In the present invention, geNorm first realizes the sorting of expression stability of 12 candidate internal reference genes by calculating the expression stability mean value (M) ( figure 2 ), the results show that under all samples, the two most stable internal refe...

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Abstract

The invention discloses an internal reference gene for trichosanthes kirilowii maxim seedling stage male and female plant gene expression research and application thereof, and belongs to the technicalfield of molecular biology. The reference gene for trichosanthes kirilowii maxim seedling stage male and female plant gene expression research is an Actin gene and / or a GAPDH gene; the nucleotide sequence of the Actin gene is as shown in SEQ ID NO. 1; the nucleotide sequence of the GAPDH gene is as shown in SEQ ID NO. 2. By adopting the internal reference gene, the accuracy and reliability of trichosanthes kirilowii maxim seedling stage male and female plant gene expression research can be improved; a foundation is laid for digging differential marker genes of male and female plants in the trichosanthes kirilowii maxim seedling stage.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and specifically relates to an internal reference gene for gene expression research of male and female plants of Trichosanthes seedling stage and application thereof. Background technique [0002] In plant research, in order to understand gene function, the analysis of gene expression pattern is one of the important research approaches. Real-time fluorescent quantitative PCR is the most commonly used nucleic acid quantitative technology in the study of gene expression patterns, which has higher sensitivity, better specificity and wider detection range. However, there may be some differences in the RNA concentration, RNA quality, and cDNA synthesis efficiency of different samples, which will affect the accuracy of real-time fluorescent quantitative PCR results. In fluorescent quantitative PCR experiments, the use of internal reference genes can effectively correct these differences, so ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6851C12N15/11
CPCC12Q1/6895C12Q1/6851C12Q2600/158C12Q2600/166C12Q2531/113C12Q2545/101C12Q2563/107
Inventor 束红梅郭书巧徐筋燕何晓兰
Owner JIANGSU ACAD OF AGRI SCI