Screening and application of internal reference genes in pitaya fruit at different developmental stages
An internal reference gene and dragon fruit technology, applied in the field of molecular biology, can solve problems such as errors and low accuracy, and achieve the effects of wide application, authentic and reliable data, and high sensitivity
Active Publication Date: 2022-07-26
GUIZHOU UNIV
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Problems solved by technology
A large number of studies in recent years have shown that the expression of internal reference genes is specific, and there is no internal reference gene that can be stably expressed at different developmental stages of the same species and under different culture conditions. Without screening, a housekeeping gene is blindly used as an arbitrary Internal reference under experimental conditions, it is easy to get low accuracy or even wrong results
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Embodiment 1
[0032] Embodiment 1: the screening of the most stable internal reference gene in the different developmental stages of dragon fruit, comprises the steps:
[0033] 1. Experimental materials: The red-fleshed dragon fruit variety—Zihonglong and the white-fleshed dragon fruit—Jinghonglong were taken from the Luodian dragon fruit planting base (Xingguoyuan) in Guizhou Province. On the 25th, 30th, 35th, and 40th days after artificial pollination, the pulp samples were collected, followed by the early coloration period (H1), color breaking period (H2), coloring period (H3), ripening period (H4) and white flesh B1 of the red flesh fruit. , B2, B3, B4, 3 samples were taken each time, each sample was set up with 3 biological replicates, and samples were taken according to Cheng et al.
[0034] 2. Extraction of total RNA: Extract total RNA from materials of different periods of dragon fruit according to the instructions of total RNA extraction kit (Tiangen).
[0035] 3. cDNA synthesis: ...
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The invention discloses an internal reference gene related to different developmental stages of dragon fruit and its application. The internal reference gene is: TBP2 and YLS8 ; wherein the internal reference gene TBP2 The nucleotide sequence is shown in SEQ ID NO.1, the internal reference gene YLS8 The nucleotide sequence is shown in SEQ ID NO.2. In the present invention, 17 relatively stable candidate internal reference genes are screened from the dragon fruit transcriptome data and previous studies, and the qRT-PCR technology is used to evaluate the gene stability through geNorm and NormFinder software. TBP2 and YLS8 It is an internal reference gene that is stably expressed during the development of dragon fruit. The internal reference gene combination provided by the invention provides strong support for the research of pitaya pigment metabolism and key genes in fruit development, and has important guiding significance for the selection and breeding of excellent fruit quality.
Description
technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to an internal reference gene related to different developmental stages of dragon fruit and its application. Background technique [0002] Dragon fruit (Hylocereus undatus) belongs to the genus Hylocereus of the family Cactaceae. According to the color of dragon fruit peel and flesh, it can be divided into red skin and white flesh, red skin red flesh, yellow skin and white flesh. Among them, the red skin and red flesh of dragon fruit contain a lot of natural pigments, and the content of beet pigment is the key factor for the color of dragon fruit. At present, many studies have used dragon fruit as a raw material to extract the pigments, and it has become a hot spot to study the expression levels of genes related to the regulation of beta pigment metabolism pathways and to use molecular biology methods to study the pitaya beta pigment metabolism pathway and its regulatory...
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IPC IPC(8): C12Q1/6895C12N15/11
Inventor 文晓鹏崇慧影
Owner GUIZHOU UNIV