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Construction method of expression profile for high homologous degree polygene family

A technology of gene family and construction method, applied in the field of construction of expression profiles of multi-gene families, can solve the problems of boredom and duplication of northern blotting, difficulty in determining the expression of members, and difficulty in detecting expression genes.

Inactive Publication Date: 2008-01-02
SHANGHAI UNIV
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Problems solved by technology

But when the gene family size exceeds 10 genes, northern blotting becomes quite tedious and repetitive work due to the preparation of many specific hybrids (Dong et al., 2003)
Moreover, it is difficult to detect genes with very low expression levels by northern blot technology (Brown et al., 2003; Jakab et al., 2003)
Members of multigene families are usually so similar in sequence that cross-hybridization is easy to occur during northern blot (Montrichard et al., 2003), so it is difficult to identify a gene expression of a particular member of the family

Method used

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  • Construction method of expression profile for high homologous degree polygene family

Examples

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Comparison scheme
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Embodiment 1

[0019] Example 1: Obtaining the expression status and relative abundance of each member gene of the α-gliadin multigene family 24 days after pollination

[0020] In order to obtain the expression profile of the α-gliadin supergene family in the whole process of grain development, the endosperm on the 24th day after pollination was collected (every two days), the total RNA was extracted (Trizol kit, Tiangen), and the total RNA was treated with denatured agar Glycogel electrophoresis and UV spectrophotometer analysis and quantification.

[0021] The sequences of all homologous genes in each family were compared, and primers were designed in the conserved regions, see Table 1, and the amplified fragments contained polymorphic comparison sites. Using this primer, the reverse transcription product of the total RNA of the corn endosperm at various developmental times was used as a template for PCR amplification, and the number of cycles used for the amplification was the optimal num...

Embodiment 2

[0027] Example 2: Obtaining the total expression of each gene family of the α-gliadin super gene family

[0028] The endosperm was harvested from 10 days to 34 days after pollination (every two days), and total RNA was extracted (Trizol kit, Tiangen). See Figure 1. Total RNA was analyzed and quantified by denaturing agarose gel electrophoresis and UV spectrophotometer. Using the endosperm total RNA of each sample point as a template, Oligo(dT) 16 used as primers for reverse transcription. Primers were designed for each gene family according to the expressed gene sequence, see Table 1, and real-time fluorescent quantitative PCR was performed using the reverse transcription product as a template. Actin was used as an internal reference gene. The data were collected and analyzed by the software Opticon3.1, and the expression profiles of the four families during the whole process of grain development were obtained, see Figure 2.

Embodiment 3

[0029] Example 3: Obtaining the expression profile of each member gene of the α-gliadin super gene family

[0030]Using the random clone sequencing described in Example 1, the relative expression abundance of each member gene of the four families in the maize endosperm (every four days) from day 14 to day 34 after pollination was analyzed. Combining the overall expression level of each family with the relative abundance of each member gene contained in it, the expression profile of each member gene in the whole process of grain development can be obtained, see Figure 3.

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Abstract

The invention discloses a constructing method of expression spectrum of each member gene in the high-homologization gene family, which is characterized by the following: connecting RT-PCR random clone and fluorescent real-time quantitative PCR; using RT-PCR random clone to obtain expression condition of each member gene and relative abundance of the expressed gene; utilizing real-time fluorescent quantitative PCR to obtain the total expression quantity of each gene family; making the expression spectrum as new method to study the expression modulation of multi-gene family; providing theoretical guide of gene engineering modification for gliadin content of forage crop.

Description

technical field [0001] The invention relates to a method for constructing expression profiles of multigene families, in particular to a method for constructing expression profiles of multigene families with high degree of homology. Background technique [0002] A multigene family is a group of genes that originated from a common ancestor and thus have the same function and similar DNA sequences. Among the genes of Escherichia coli (E.coli), the known gene family members exceed 50% (Koonin et al., 1998), and this proportion may be even higher in eukaryotes (Chervitz et al., 1998 ; Semple and Wolfe, 1999). In plant genomes, multigene families are more of a feature. For example, 66% of the genes in Arabidopsis thaliana (A. thaliana) belong to gene families, and half of the families have at least five member genes (Analysis of the genome sequence of the flowering plant Arabidopsis thaliana, 2000), which is not the case in other ubiquitous in plants. Studying all the genes in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/00C40B50/06
Inventor 宋任涛冯玲娜朱佳王刚许政暟
Owner SHANGHAI UNIV
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