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Phosphofructokinase and application of coding gene thereof

A technology of phosphofructokinase and coding genes, which can be applied in application, genetic engineering, plant genetic improvement, etc., and can solve the problems that the enzyme activity characteristics have not been reported.

Inactive Publication Date: 2013-06-12
RUBBER RES INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the gene and enzymatic activity characteristics of the key enzyme of rubber tree glycolysis—phosphofructokinase (PFK-1)

Method used

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  • Phosphofructokinase and application of coding gene thereof
  • Phosphofructokinase and application of coding gene thereof
  • Phosphofructokinase and application of coding gene thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1. Acquisition of Hevea phosphofructokinase (PFK-1) and its coding gene

[0042] Analyze the nucleotide sequences of phosphofructokinase (PFK-1) from Arabidopsis thaliana, poplar and castor bean that have been registered in NCBI, and determine a phosphofructokinase (PFK-1) of about 900bp by searching the Hevea latex EST sequence database established by us. -1) The assembled sequence (contig) of the gene EST, design a pair of specific primers to amplify the cDNA fragment, and use the rapid amplification technology (RACE) at the end of the cDNA to finally obtain the full-length cDNA sequence including the complete reading frame .

[0043] The specific method is as follows:

[0044] cDNA fragment cloning

[0045] Specific primers were designed as follows:

[0046] F (5' end): 5'-TGA CAC TGC GGT TGA GGA GG-3';

[0047] R (3' end): 5'-AAA ATA TTT ATT CTA TTA CAA AAC ATC CTT T-3'.

[0048] Using Hevea brasiliensis Reyan 7-33-97 (cultivated by the Rubber Research...

Embodiment 2

[0053] Example 2. Analysis of HbPFK-1-2 gene expression pattern

[0054] HbPFK-1-2 gene tissue-specific expression

[0055] The tissue expression specificity of the rubber tree HbPFK-1-2 gene was analyzed in the existing transcriptome database of the laboratory (rubber tree 7-33-97 leaves, bark and latex), and the results showed that the gene was highly expressed in bark and latex. Secondly, the expression level was the lowest in leaves ( figure 1 , the relative expression level was compared with the expression level in leaves). At the same time, using the random reverse-transcribed cDNA of rubber tree 7-33-97 leaves, flowers, latex, leaf buds, seeds and bark RNA as templates, HbPFK-1-2 gene-specific primers (F: 5'-GGA AGA ACC TGC TAC CTT GT-3'; R:5'-AGG CAT TGA ACA TGA ATA ACA AAC-3') for real-time fluorescent quantitative PCR, the results also showed that the gene was highly expressed in bark, followed by latex and seeds, and in Relatively low expression in other tissues ...

Embodiment 3

[0064] Example 3. Prokaryotic expression and functional verification of HbPFK-1-2 gene

[0065] The prokaryotic expression vector of the HbPFK-1-2 gene was constructed by using the pEASY-E1 expression vector (the pEASY-E1 expression vector was purchased from TransGen Biotech Company), and the Escherichia coli phosphofructokinase-deficient strain RL257 (the strain was purchased from Yale University, ingshimer MR, Siegele D, Reinhart GD. Construction of an inducible, pfkA and pfkB deficient strain of Escherichia coli for the expression and purification of phosphofructokinase from bacterial sources. Protein Expr Purif. 2006,46(2):475-82.) Gene functional verification, the specific method is as follows:

[0066] Obtaining the recombinant vector containing HbPFK-1-2 gene coding region

[0067] Design primers for the coding region of HbPFK-1-2 gene (F: 5'-ATG GTG GAT TCC GGC GAT TCT CAG AT -3', R: 5'-TCA AAT ATT TCC ATT CAA CAA GGT AGC AGG TTC TT-3') , using pMD18-HbPFK-1-2 as a ...

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Abstract

The invention discloses a phosphofructokinase, and a coding gene and application thereof. The protein has one of the following amino acid residue sequences: 1) amino acid residue sequence disclosed as SEQ ID No.1 in the sequence table; and 2) protein with phosphofructokinase derived from SEQ ID No.1, subjected to substitution and / or deletion and / or addition of one or more amino acid residues on the amino acid residue sequence disclosed as SEQ ID No.1 in the sequence table. The transgenic experiment on the protein indicates that the gene has the function of phosphofructokinase; and after the cDNA (complementary deoxyribonucleic acid) of the gene is transfected into a phosphofructokinase deficient strain RL257, the obtained transgenic strain can normally grow in an M63 basal culture medium which uses mannitol as the unique carbon source.

Description

technical field [0001] The invention relates to a rubber tree phosphofructokinase and its encoding gene and application. Background technique [0002] Glycolysis is a common way to break down sugar in all organisms. This way decomposes glucose in the cytoplasm to generate pyruvate. During this process, a small amount of ATP and NADH are generated, and some important precursor metabolites are also produced for the organism. It is a central metabolic pathway that provides the necessary carbon and energy sources (Plaxton, WC. The organization and regulation of plant glycolysis. Annu. Rev. Plant Physiol. Plant Mol. Biol..1996;48:185–214.) . Therefore, the precise regulation of the glycolysis process plays an important role in the growth and development of organisms and biotic and abiotic stresses, so as to meet the needs of the organism and maintain the stability of the internal environment, and the regulation and regulation in this process are mainly Realized by regulating th...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12N15/54C12N15/63C12N5/10C12N1/21C12N15/70C12R1/19
Inventor 龙翔宇唐朝荣方永军阳江华戚继艳秦云霞
Owner RUBBER RES INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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