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Earthnut acetyl coenzyme A carboxylase carboxyltransferase beta subunit gene and encoding protein and cloning method thereof

A carboxylase carboxyl, acetyl coenzyme technology, applied in the field of genetic engineering, to achieve the effect of increasing oil content

Inactive Publication Date: 2009-10-07
SHANDONG PEANUT RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no report about this gene in peanut

Method used

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  • Earthnut acetyl coenzyme A carboxylase carboxyltransferase beta subunit gene and encoding protein and cloning method thereof
  • Earthnut acetyl coenzyme A carboxylase carboxyltransferase beta subunit gene and encoding protein and cloning method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0019] Example 1 Cloning of peanut acetyl-CoA carboxylase carboxyltransferase beta subunit (AhCTbeta) gene

[0020] (1) E12 peanut leaves were selected as experimental materials, and the total RNA in peanut leaves was extracted using Pbiozol Plant Tissue RNA Extraction Kit. RNA content and quality can be detected by agarose gel electrophoresis.

[0021] (2) The obtained total RNA was used as a template, and the SMARTTM RACE Amplification Kit (Clontech) was used to synthesize 5'- and 3'-RACE-Ready cDNA by reverse transcription.

[0022] (3) Design 5' and 3' end cDNA fragment primers according to the EST information in the NCBI database:

[0023] GSP1: 5'-CCCGTTTAGTTGACCTGTGCCTGTTT-3'

[0024] GSP2: 5'-CATCTCCTACTACTGGGGGGGTGAC-3'

[0025] (4) Use the SMARTTM RACE Amplification Kit (Clontech) to amplify the 5' and 3' end gene fragments of the acetyl-CoA carboxylase carboxyltransferase β subunit gene, and recover the amplified product and combine it with pMD18-T simple vector ...

Embodiment 2

[0033] Example 2 Sequence information and characteristic analysis of AhCTβ protein

[0034] The AhCTβ gene of the present invention is 1636bp long, and its open reading frame is 1479bp, located at 92-1570bp. BioXM software analysis showed that AhCTβ encoded a total of 492 amino acids. According to the analysis of the 492 amino acids encoded by the peanut AhCTβ gene according to the PROSITE database, it was found that this sequence contains a CTβ active site, located at 250-469aa, such as figure 1 shown. Using Protparam to predict the physicochemical properties of the encoded protein, it is speculated that the molecular formula is C 2464 h 3796 N 654 o 775 S 27 , the molecular weight is 55846.7, the isoelectric point is 5.14, the theoretically deduced half-life is 30h, and the instability parameter is 48.68, which belongs to the unstable protein. The relatively abundant amino acids in the protein are Ser (11.8%), Gly (7.1%), Glu (7.5%), Leu (8.5%), Asn (6.9%); the protei...

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Abstract

The present invention discloses an earthnut acetyl coenzyme A carboxylase carboxyltransferase beta subunit gene and an encoding protein and a cloning method thereof. The gene sequence is composed of a nucleotide sequence from 92th to 1570th bits of SEQ ID No.1; the protein encoded by the gene has an amino acid sequence represented by the SEQ ID No.2 in a sequence table. The nucleotide sequence of the earthnut acetyl coenzyme A carboxylase carboxyltransferase beta subunit (AhCT beta) gene and the encoding protein sequence and the gene cloning method of the invention may be used for improving the earthnut quality and the earthnut cultivation widely, especially a field where the earthnut is used for developing and using oil crops.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to peanut acetyl-CoA carboxylase carboxyltransferase beta subunit gene and its encoded protein and a cloning method. Background technique [0002] Acetyl-CoA Carboxylase (ACCase) is a biotin-containing enzyme that catalyzes the carboxylation of acetyl-CoA to form malonyl-CoA in vivo, providing substrates for the synthesis of fatty acids and many secondary metabolites (Konishi T, Shinohara K, Yamada K. Acetyl-CoA carboxylase in higher plants: most plants other than gramineae have both the prokaryotic and the eukaryotic forms of this enzyme [J]. Plant Cell Physiol, 1996, 37(1): 17-122 .). There are 2 types of ACCase in organisms. A type of ACCase called Homomeric, also known as multifunctional or eukaryotic, exists in the cytosol of animals, yeast, algae and plants, and has a biotin containing subunit with a relative molecular weight of 220~260 . The other is heterogen...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12P19/34
Inventor 迟晓元杨庆利禹山林潘丽娟和亚男曹玉良
Owner SHANDONG PEANUT RES INST
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