Method for rapidly detecting expression of ANS (Anthocyanidin Synthetase) genes from different sources in rape seed capsule and application thereof

A technology of gene expression and detection method, applied in the fields of biotechnology and molecular breeding

Inactive Publication Date: 2012-05-02
HUNAN UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Existing methods for detecting the expression of the ANS gene are specific primer RT-PCR and Northern analysis methods, but these methods cannot distinguish whether the expressed ANS gene is from the A chromosome group, the B chromosome group or the C chromosome group, wherein Chinese cabbage (Brassica campestris, AA , 2n=20) is the A chromosome group; black mustard (Brassica nigra, BB, 2n=16) is the B chromosome group; cabbage (Brassica oleracea, CC, 2n=18) is the C chromosome group
Due to the incomplete sequence information of the ANS genes of the three basic species of rapeseed (Chinese cabbage, black mustard, and cabbage) and three allotetraploids (Brassica napus, Brassica napus, and Ethiopian mustard), RT-PCR and The Northern analysis method cannot distinguish whether the expressed ANS gene is from the A chromosome group, the B chromosome group or the C chromosome group, and cannot be used for large-scale use in the breeding process

Method used

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  • Method for rapidly detecting expression of ANS (Anthocyanidin Synthetase) genes from different sources in rape seed capsule and application thereof
  • Method for rapidly detecting expression of ANS (Anthocyanidin Synthetase) genes from different sources in rape seed capsule and application thereof
  • Method for rapidly detecting expression of ANS (Anthocyanidin Synthetase) genes from different sources in rape seed capsule and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] (1) According to the published Arabidopsis ANS gene and rape ANS gene sequence (Abrahams S, Lee E, Walker A R, Tanner G J, Larkin P J, Ashton AR. The Arabidopsis TDS4 gene encodes leucoanthocyanidin dioxygenase (LDOX) and is essential for proanthocyanidin synthesis and vacuole development. Plant J, 2003,35:624-636; Mingli Yan , Xianjun Liu, Chunyun Guan, Xinbo Chen, Zhongsong Liu. Cloning and expression analysis of anthocyanidin synthase gene homolog from Brassica juncea , Mol Breeding, 2011, 28:313–322), through bioinformatics analysis, design a pair of degenerate primers to amplify the full-length cDNA of ANS gene, black mustard (BB), cabbage (CC), Chinese cabbage (AA) , Ethiopian mustard (BBCC) and Brassica napus (AABB) seed coat cDNA were used as templates for PCR amplification. The amplified products were separated by agarose gel electrophoresis, recovered from the gel, connected to the pMD18-T vector, and then the vector was transferred to competent E. The res...

Embodiment 2

[0039] (1) Using cDNA as a template, PCR amplification was performed with specific primers (upstream primer 5'-TTAGCAAAGAGCGGAATCGAA-3', downstream primer 5'-CCACGGGCAAACCGAAGAA-3') from the ANS gene of the B chromosome group. The total volume of the PCR reaction system was 20 μL, including 2.0 μL of 10× PCR buffer, 0.3 μL of 10 mmol L?1 dNTP mix, 1 μL of 10 mmol L?1 forward primer, 1 μL of 10 mmol L?1 reverse primer, 1 U Taq DNA polymerase, 2.5 mmol L?1 MgCl2 2 μL, 1 μL reverse transcription product, sterile deionized water (make up to 20 μL). PCR amplification conditions were as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 25 s, annealing at 60°C for 30 s, extension at 72°C for 20 s, and 30 cycles; finally, extension at 72°C for 6 min.

[0040] (2) Amplified products were separated by 1.5% agarose gel electrophoresis and stained with ethidium bromide. Then take pictures and analyze through the gel analysis system, the results are as follows figu...

Embodiment 3

[0043] (1) Using cDNA as a template, PCR amplification was performed with specific primers from the ANS gene of the C chromosome group (upstream primer 5'-TAGCAATGGGAAGTTTAAGAGTA-3', downstream primer 5'-GTGGTTCATAGAGCATATCTCG-3'). The total volume of the PCR reaction system was 20 μL, including 2.0 μL of 10× PCR buffer, 0.3 μL of 10 mmol L?1 dNTP mix, 1 μL of 10 mmol L?1 forward primer, 1 μL of 10 mmol L?1 reverse primer, 1 U Taq DNA polymerase, 2.5 mmol L?1 MgCl2 2 μL, 1 μL reverse transcription product, sterile deionized water (make up to 20 μL). PCR amplification conditions were as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 25 s, annealing at 57°C for 30 s, extension at 72°C for 30 s, and 30 cycles; finally, extension at 72°C for 6 min.

[0044] (2) Amplified products were separated by 1.5% agarose gel electrophoresis and stained with ethidium bromide. Then take pictures and analyze through the gel analysis system, the results are as follows i...

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Abstract

The invention discloses a method for rapidly detecting expression of ANS (Anthocyanidin Synthetase) genes from different sources in rape seed capsules. In the method, a specific primer capable of distinguishing ANS genes from chromosome sets A, B and C by cloning and analyzing sequences of ANS genes in three elementary species of rape (cabbage: the chromosome set is AA, and 2n=20; brassica oleracea: the chromosome set is CC, and 2n=18; and brown mustard: the chromosome set is BB, and 2n=16) and three allotetraploids (mustard type rape: the chromosome set is AABBA, and 2n=36; cabbage type rape: the chromosome set is AACC, and 2n=38; brassica carinata: the chromosome set is BBCC, and 2n=34) according to the nucleotide polymorphic loci of ANS genes from the chromosome sets A, B and C, and the ANS gene types expressed in the seed capsules of rape of different chromosome set types are identified in combination with an RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction). The method plays a role in further researching the molecular adjusting mechanism for the color formation of rapeseed capsules and accelerating the variety breeding of yellow seed rape; and meanwhile, a novel methodfor detecting the source of a rape ANS gene is provided.

Description

technical field [0001] The invention relates to the fields of biotechnology and molecular breeding, in particular to a method for detecting ANS gene expression from A chromosome group, B chromosome group and C chromosome group in rapeseeds by using nucleotide polymorphism and allele-specific PCR technology. Background technique [0002] Rapeseed is an important oil crop in the world. One of the main goals of rapeseed breeding is to breed yellow-seeded rapeseed varieties on the basis of double low. Under the same genetic background, yellow rapeseed has the advantages of thin seed coat, high oil content and low lignin content compared with black seed rapeseed. The embryos of both yellow rapeseed and black rapeseed are yellow, the seed coat of yellow seed is transparent, and the seed coat of black seed is black. The color of yellow seed is the embodiment of the color of its embryo, and the color of rapeseed The color difference is determined by the seed coat. [0003] Studie...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 严明理刘丽莉舒佳宾
Owner HUNAN UNIV OF SCI & TECH
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