Rubber tree copper ion transport protein and encoding gene thereof

A technology for encoding genes and transporters, applied in the field of genetic engineering, can solve problems such as copper ion transporters that have not been reported yet

Active Publication Date: 2016-08-24
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

[0007] At present, the research on copper transporters mainly focuses on mammals, yeast and the model plant Arabidopsis thaliana, and the research on copper ion transporters mainly focuses on yeast and the dicotyledonous plant Arabidopsis thaliana. Based on the existing literature, it can be seen that copper ion transport Protein research has not been reported in Hevea brasiliensis

Method used

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  • Rubber tree copper ion transport protein and encoding gene thereof
  • Rubber tree copper ion transport protein and encoding gene thereof
  • Rubber tree copper ion transport protein and encoding gene thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1 Obtaining of the gene HbCOPT1 related to copper ion transport in rubber tree and its coding sequence

[0088] 1.1 Extraction of total RNA from Hevea Reyan 7-33-97 and reverse transcription of cDNA

[0089] Use BioTeke's universal plant total RNA (spin column type) extraction kit to extract RNA from young tree roots, stems, leaves, xylem and latex, determine the concentration of RNA, and perform 1.2% agarose gel electrophoresis detection. After completeness and concentration determination, a total of 1μg of RNA was taken to reverse transcription of RNA into cDNA using the RevertAid First Strand cDNA Synthesis Kit (Fermentas).

[0090] 1.2 Screening and cloning of target genes

[0091] In this study, the copper transporter COPT in the rubber tree air thorn long gum transcriptome and the rubber tree genome were screened, and the complete open reading frame sequence of the copper transport protein related gene was obtained. According to the obtained open reading frame seq...

Embodiment 2

[0093] Example 2 HbCOPT1 gene sequence and coding product structure prediction analysis

[0094] 2.1 Sequence analysis of HbCOPT1 gene

[0095] The HbCOPT1 gene obtained in Example 1 was analyzed. The open reading frame nucleotide sequence contained 426 bp. The nucleotide sequence is shown in SEQ ID NO: 2 in the sequence table. It encodes a total of 141 amino acids, and the encoded amino acid sequence is shown in SEQ ID. NO: shown in 3.

[0096] 2.2 Structure prediction analysis of HbCOPT1 gene coding product

[0097] Using ExPASy-Prot-Param Tool (http: / / web.expasy.org / protparam / ) to analyze and predict the physical and chemical properties of the copper transporter encoded by HbCOPT1, the results show that the copper transporter deduced by HbCOPT1 contains more amino acids For Leu, Val and Ala, the isoelectric point is 8.53, the instability coefficient is 28.76, and the total average hydrophilicity coefficient is 0.691. It is predicted that the copper transporter encoded by HbCOPT1 i...

example 3

[0099] Example 3 Tissue-specific expression analysis of HbCOPT1 gene

[0100] After primer PCR analysis and standard curve determination, and using primer 5 biology software to determine the fluorescent quantitative PCR primers, the upstream primer SEQ ID NO: 6 and the downstream primer SEQ ID NO: 7; the internal reference gene is selected as 18s rRNA, the upstream primer sequence It is SEQ ID NO: 8, and the downstream primer sequence is SEQ ID NO: 9.

[0101] The relative expression level of the gene was normalized with the 18s rRNA gene as an internal reference, the relative quantification of the gene was expressed by the 2^ΔΔCT method, and the results were statistically analyzed by Excel and SPSS software. Use fluorescence quantitative PCR to analyze the HbCOPT1 gene sequence for tissue expression, such as image 3 As shown, according to the results of fluorescence quantitative PCR, the expression level of genes in leaves is calculated as 1, HbCOPT1 is expressed in the roots, st...

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Abstract

The invention provides a segment of protein composed of the amino acid residue sequence as shown in SEQ ID NO:3 in the sequence table and an encoding gene HbCOPT1 thereof. The total length of the open reading frame nucleotide sequence is 426 bp, 141 amino acids are encoded, and the protein belongs to Ctr/COPT copper transporter family, contains three membrane spanning domains, and contains no signal peptide sequence; the HbCOPT1 sequence is cloned into a yeast expression vector to construct a yeast expression vector carrying the gene, it is verified through repeated yeast mutant verification tests that the HbCOPT1 encoded copper transporter protein has specific high compatibility to copper ions and has a copper transportation function, and therefore the gene is a copper transportation gene; the protein and the encoding gene thereof have important significance in promoting copper ion absorption and accumulation of plants, especially in rubber tree latex quality genetic improvement and high-yield new species cultivation.

Description

Technical field [0001] The invention relates to the field of genetic engineering, and is not specifically the isolation, cloning and functional verification of a copper ion transporter and its coding gene HbcOPT1 in Hevea brasiliensis Muell. arg. Background technique [0002] Rubber tree, scientific name: Hevea brasiliensis (Willd. ex a. Juss.) Muell. arg, Euphorbiaceae rubber tree genus, deciduous tree. It is native to the Amazon River Basin in Brazil, South America. The climatic conditions in this area have the characteristics of high temperature, small temperature difference, high humidity, abundant rainfall and uniform rainy days (Liu Jinhe, "The moisture status and growth of the Brazilian rubber tree and the amount of rubber production Relationship", Acta Ecologica Sinica, 1982). At present, in addition to the place of origin, it is also widely planted in many countries and regions such as Asia, Africa and Oceania; the planting of rubber trees requires an average annual rai...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29
CPCC07K14/415
Inventor 王聪魏芳校现周罗世巧仇键杨文凤吴明高宏华
Owner RUBBER RES INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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