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Hevea Copper Ion Transporter and Its Encoding Gene

A technology encoding gene and transporter, applied in the field of genetic engineering, can solve the problem that copper ion transporter has not been reported yet

Active Publication Date: 2019-04-16
RUBBER RES INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • Hevea Copper Ion Transporter and Its Encoding Gene
  • Hevea Copper Ion Transporter and Its Encoding Gene
  • Hevea Copper Ion Transporter and Its Encoding Gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1 Genes related to copper ion transport in rubber trees HbCOPT1 and the acquisition of its coding sequence

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

[0089]The RNA of young tree roots, stems, leaves, xylem and latex was extracted using BioTeke's general plant total RNA (spin column type) extraction kit, and the RNA concentration was determined, and detected by 1.2% agarose gel electrophoresis. After integrity and concentration determination, a total of 1 μg of RNA was used to reverse transcribe the RNA into cDNA using the reverse transcription kit RevertAid First Strand cDNASynthesisKit (Fermentas).

[0090] 1.2 Screening and cloning of target genes

[0091] In this study, the copper transporter COPT in the rubber tree airthorn long-running gum transcriptome and the rubber tree genome was screened, and the complete open reading frame sequence of the copper transporter-related gene was obtained. According ...

Embodiment 2

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

[0094] 2.1 HbCOPT1 gene sequence analysis

[0095] Obtained to embodiment 1 HbCOPT1 According to the analysis of the gene, the nucleotide sequence of its open reading frame contains 426bp, and the nucleotide sequence is shown in SEQ ID NO: 2 in the sequence table, encoding 141 amino acids in total, and the encoded amino acid sequence is shown in SEQ ID NO: 3.

[0096] 2.2 HbCOPT1 Gene encoding product structure prediction analysis

[0097] Use ExPASy-Prot-Param Tool (http: / / web.expasy.org / protparam / ) to HbCOPT1 The physical and chemical properties of the encoded copper transporter were analyzed and predicted. The results showed that the copper transporter deduced by HbCOPT1 contained more amino acids Leu, Val and Ala, the isoelectric point was 8.53, the instability coefficient was 28.76, and the total average hydrophilicity The sex coefficient is 0.691, predicting HbCOPT1 The...

example 3

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

[0100] After PCR analysis of the primers and determination of the standard curve, and use the primer 5 biological 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 was selected as 18s rRNA, and the upstream primer sequence The sequence of the downstream primer is SEQ ID NO: 8, and the sequence of the downstream primer is SEQ ID NO: 9.

[0101] The relative expression level of the gene was normalized with the 18s rRNA gene as the internal reference, and the relative quantification of the gene was expressed by the 2^ΔΔCT method, and Excel and SPSS software were used for statistical analysis of the results. Analysis of tissue expression of HbCOPT1 gene sequence by fluorescent quantitative PCR, such as image 3 As shown, according to the results of fluorescent quantitative PCR, the expression l...

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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 present invention relates to the field of genetic engineering, in particular to a copper ion transporter in Hevea brasiliensis Muell.arg. and its coding gene HbcOPT1 isolation, cloning, and functional verification. Background technique [0002] Rubber tree, scientific name: Hevea brasiliensis (Willd. ex a. Juss.) Muell. arg, Euphorbiaceae Euphorbiaceae Hevea plant, deciduous tree. Native to the Amazon River Basin in Brazil, South America, the climatic conditions in this area are characterized by high temperature, small temperature difference, high humidity, abundant rainfall and uniform rainy days (Liu Jinhe, "The relationship between the moisture status and growth and rubber production of Hevea brasiliensis relationship”, Acta Ecologica Sinica, 1982). At present, in addition to the place of origin, many countries and regions such as Asia, Africa and Oceania are also widely planted; the planting of rubber trees requires an average annual rainf...

Claims

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Application Information

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Patent Type & Authority Patents(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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