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A group of pear pulp qrt-PCR reference genes and their primers and applications

A technology of pulp and internal reference, applied in the field of plant genetic engineering, can solve the problems of no expression stability comparison verification, unstable expression of traditional internal reference genes, lack of combined application of multiple internal references, etc., and achieve the best effect of gene expression homogenization

Active Publication Date: 2022-07-15
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] 1) The application of these traditional internal reference genes in the study of pear pulp development is only a reference to other species, and there is no systematic comparative verification of expression stability within the genome, and many studies have shown that these traditional internal reference genes in the tissue development process of other species expression is unstable;
[0009] 2) At present, only a single internal reference is used for homogenization in the study of gene expression during pear pulp development, and there is a lack of more accurate combination of multiple internal references;
[0010] 3) There is a lack of a suitable internal reference gene system suitable for the study of gene expression in pulp development in a wide range of pear varieties
[0011] Therefore, it is necessary to establish a suitable internal reference gene system on a genome-wide scale based on extensive transcriptome data as soon as possible to solve the problems of poor stability of traditional internal references and unsatisfactory gene expression equalization effects in pear pulp.

Method used

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  • A group of pear pulp qrt-PCR reference genes and their primers and applications
  • A group of pear pulp qrt-PCR reference genes and their primers and applications
  • A group of pear pulp qrt-PCR reference genes and their primers and applications

Examples

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Embodiment 1

[0071] This example provides a set of pear pulp qRT-PCR internal reference genes, including: the internal reference genes are RABF2b gene, ALFIN-like1 gene, BPS1 gene, ICDH1 gene; RABF2b gene transcript is shown in SEQ ID NO: 1 in the sequence table; ALFIN The -like1 gene transcript is shown in SEQ ID NO: 2 in the sequence listing; the BPS1 gene transcript is shown in SEQ ID NO: 3 in the sequence listing; the ICDH1 gene transcript is shown in SEQ ID NO: 4 in the sequence listing.

[0072] The above four genes can be used as new reference genes for gene expression analysis during the pulp development of the broad pear cultivar. Among them, BPS1 and ICDH1 are recommended as the optimal internal reference combination for the gene expression analysis of the broad pear variety during the pulp development.

Embodiment 2

[0074] The present embodiment provides a group of methods for screening internal reference genes during the development of pear pulp, comprising the following steps:

[0075] From a botanical point of view, pear fruit is composed of seeds, receptacle (flesh), endocarp, mesocarp and exocarp, and is a false fruit developed from the receptacle. The swollen receptacle becomes the fleshy part of the pear fruit and is its main edible part ( figure 1 ), the present invention relates to the screening of internal reference genes during the development of pear pulp.

[0076] 1. The candidate internal reference genes (RGs) provided by the present invention are obtained based on the analysis of the Dangshansu pear (P.bretschneideri) genome and the transcriptome related to pear pulp development.

[0077] 1.1) At present, in pear pulp qRT-PCR research, the six most commonly used reference genes (RGs) include: Actin, EF1α, TUB, UBI, GAPDH and 26-18S rRNA, which are defined as commonly used ...

Embodiment 3

[0095] The present embodiment provides the application of a group of internal reference genes in the development of pear pulp, including the following steps:

[0096] The present invention uses the expression pattern of PbrCAD1 in pear pulp to verify the practicability of the internal reference genes (RGs) recommended by the above analysis

[0097] 1.1) Cinnamyl alcohol dehydrogenase (CAD) is involved in the biosynthesis of stone cell lignin during pear fruit ripening. Combining the pear genome and RNA-seq data, the present invention identified that the expression profile of a CAD gene (PbrCAD1) was closely related to the lignin content of different pear varieties. Detailed observation found that the expression of PbrCAD1 first increased in the split stage of pear fruit, reached a peak in the fruit expansion stage, and gradually decreased after the ripening stage ( Figure 21 ). These results suggest that PbrCAD1 promotes lignin biosynthesis during fruit ripening in differen...

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Abstract

The present invention provides a set of pear pulp qRT-PCR internal reference genes, primers and applications thereof, which are RABF2b, ALFIN-like1, BPS1 and ICDH1 respectively, wherein BPS1 and ICDH1 can be used as an optimal internal reference gene combination for gene expression analysis of pear pulp. The expression stability of the four new internal reference genes screened based on the genome and transcriptome of the present invention is better than that of traditional internal reference genes (Actin, EF1α, TUB, UBI, GAPDH and 26-18S rRNA) during the development of pear pulp; A pair of BPS1 and ICDH1 internal reference combinations have been developed in the study, which are used to homogenize gene expression during the development of pear pulp. Process for gene expression studies.

Description

technical field [0001] The invention relates to the field of plant genetic engineering, in particular to 4 new internal controls that can be used for gene expression analysis in the development of pulp of a wide range of pear varieties, wherein BPS1 and ICDH1 are recommended as optimal internal reference gene combinations for gene expression analysis of the development of pulp of a wide range of pear varieties . Background technique [0002] Pear (Pyrus L.), one of the most economically valuable fruits in the world, can be roughly divided into two categories: Western pears and Eastern pears. Since the genome of P. bretschneideri has been sequenced (~512Mb), it has become a model plant for the study of pears. The development process of pear fruit is divided into fruit set stage, division stage, expansion stage, maturity stage and senescence stage. From a botanical point of view, pear fruit is composed of seeds, receptacle (flesh), endocarp, mesocarp and exocarp, and is a fa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12N15/11
CPCC12Q1/6895C12Q1/686C12Q2600/166C12Q2561/113C12Q2563/107C12Q2545/114C12Q2521/107
Inventor 陈建清宋娟娟陈昌妹刘悦滢陈之一周金玉陈清西
Owner FUJIAN AGRI & FORESTRY UNIV