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Full-length cDNA (complementary Deoxyribose Nucleic Acid) of switchgrass lignin biosynthetic enzyme gene PvCCoAOMT and cloning method of full-length cDNA

A technology of lignin synthase and cloning method, which is applied in the field of switchgrass molecular breeding, and can solve problems such as primer design for unfavorable non-coding regions and construction of fusion expression vectors

Active Publication Date: 2013-10-09
CHINA AGRI UNIV
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  • Abstract
  • Description
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Problems solved by technology

However, regarding the cloning of the switchgrass PvCCoAOMT gene, it was found that the CCoAOMT1b sequence uploaded by Shen et al. on Genbank contained only the open reading frame (ORF) without 5' and 3' non-coding regions, which was not conducive to primer design in the non-coding regions. Construction of fusion expression vectors, etc.

Method used

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  • Full-length cDNA (complementary Deoxyribose Nucleic Acid) of switchgrass lignin biosynthetic enzyme gene PvCCoAOMT and cloning method of full-length cDNA
  • Full-length cDNA (complementary Deoxyribose Nucleic Acid) of switchgrass lignin biosynthetic enzyme gene PvCCoAOMT and cloning method of full-length cDNA
  • Full-length cDNA (complementary Deoxyribose Nucleic Acid) of switchgrass lignin biosynthetic enzyme gene PvCCoAOMT and cloning method of full-length cDNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Embodiment 1 Homologous cloning technique obtains switchgrass PvCCoAOMT gene core fragment

[0087] The flowchart of cloning switchgrass PvCCoAOMT full-length cDNA in the embodiment of the present invention is as follows figure 1 shown.

[0088] 1. Extract the total RNA from the stem of switchgrass, test the integrity and purity of the RNA, and reverse transcribe it into cDNA

[0089] The switchgrass total RNA is extracted from the stem node tissue at the base of switchgrass, and the operation is performed according to the instructions of the total RNA extraction kit.

[0090] The integrity of the extracted switchgrass total RNA was detected by agarose gel electrophoresis. The absorbance values ​​of the extracted total RNA at 260nm and 280nm were measured with a UV spectrophotometer to determine the purity and concentration of the RNA. The RNA concentration was 224ng / μl, and the OD260 / 280 was 2.1. The qualified and quantified total RNA was stored in a -80°C freezer ...

Embodiment 25

[0134] Embodiment 25' RACE cloning technique obtains the cDNA fragment of switchgrass PvCCoAOMT gene 5' RACE

[0135] 1. Extract switchgrass total RNA and perform RNA integrity and purity detection

[0136] The switchgrass total RNA is also extracted from the stem node tissue at the base of switchgrass, and the detection method of RNA integrity and purity is the same as that in Example 1.

[0137] 2. Synthesis of 5'RACE first-strand cDNA

[0138] The synthetic method of described first-strand cDNA, its operation method is as follows:

[0139] ① Prepare 4 μL buffer mixture, the system is:

[0140]

[0141]

[0142] ② Preparation of 5'RACE Ready cDNA, the system is:

[0143]

[0144] Among them, 5'-CDS Primer A (12μM): 5'-(T) 25 VN-3' (N=A or C or G or T; V=A or G or C) (SEQ ID No. 10).

[0145] After mixing, incubate in a PCR instrument: 72°C, 3min; 42°C, 2min, after cooling the centrifuge tube, centrifuge at 14000 rpm for 10sec. Add 1 μL of SMARTer IIA oligo to ...

Embodiment 3

[0164] Example 3 Sequence splicing

[0165]Using DNAMAN software to compare the core fragment of the switchgrass PvCCoAOMT gene obtained by the homologous cloning method with the cDNA fragment obtained by the 5'RACE cloning method, it was found that the overlapping sequence size of the two was 261 bp. After splicing, the full-length cDNA of the gene The size is 1005bp, including a complete open reading frame (ORF) of 777bp, the size of its 5'UTR is 96bp, and the size of 3'UTR is 132bp, encoding 258 amino acids. The nucleotide sequence of the full-length cDNA of the switchgrass lignin synthase gene PvCCoAOMT is shown in SEQ ID No.1, and the amino acid sequence of the switchgrass lignin synthase encoded by it is shown in SEQ ID No.2.

[0166] The homology of the nucleotide and encoded amino acid sequence of the full-length cDNA of the switchgrass lignin synthase gene PvCCoAOMT was compared by online Blast of the NCBI database. Nucleotide comparison results: The full-length swit...

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Abstract

The invention discloses a full-length cDNA (complementary Deoxyribose Nucleic Acid) of a switchgrass lignin biosynthetic enzyme gene PvCCoAOMT. The full-length cDNA has a nucleotide sequence as shown in (1) or (2): (1) a nucleotide sequence shown as SEQ ID No.1, and (2) a nucleotide sequence with the same function and formed by replacing, missing or adding one or more nucleotide to the nucleotide sequence as shown in SEQ ID No.1. The invention also provides a cloning method of the full-length cDNA of the switchgrass lignin biosynthetic enzyme gene PvCCoAOMT. The cloning method provides convenience for the subsequent study on primer design, vector construction and genetic transformation by utilizing the sequence of the full-length cDNA of the switchgrass lignin biosynthetic enzyme gene PvCCoAOMT, and also provides the foundation for developing the study on fluorescence quantitative expression, transgenosis, gene function identification and the like in switchgrass in future.

Description

technical field [0001] The invention belongs to the technical field of switchgrass molecular breeding, in particular to full-length cDNA of switchgrass lignin synthase gene PvCCoAOMT and a cloning method thereof. Background technique [0002] Switchgrass is a perennial Gramineae, Grass genus, C4 herbaceous plant, which originated in the United States. As one of the model plants for cellulosic ethanol transformation, it has wide adaptability, high yield, tolerance to barrenness, high photosynthetic utilization rate and high water and nitrogen utilization rate, etc. advantage. However, its high lignin content and complex structure limit the hydrolysis of cellulose and hemicellulose, reduce the saccharification efficiency, and increase the production cost of cellulose conversion to ethanol. The proposed solution is to use biotechnology to down-regulate the related genes that control lignin synthesis, reduce the lignin content, and change the lignin composition. [0003] CCoAO...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N9/10C12N15/10C12N15/70C12N1/21C12R1/19
Inventor 张蕴薇刘斯佳杨富裕方程李洪超黄艳华杜娟
Owner CHINA AGRI UNIV
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