Gene BjCET2 of Indian mustard cation transport protein family and its protein

A technology of Indian mustard and protein, which is applied in the field of plant metal cation transporters, to achieve the effect of improving human health, increasing crop yield, and improving heavy metal resistance

Inactive Publication Date: 2005-01-26
GRADUATE SCHOOL OF THE CHINESE ACAD OF SCI GSCAS
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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 metal cation transmembrane transporter gene of CE family in Indian mustard

Method used

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  • Gene BjCET2 of Indian mustard cation transport protein family and its protein
  • Gene BjCET2 of Indian mustard cation transport protein family and its protein
  • Gene BjCET2 of Indian mustard cation transport protein family and its protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Preparation of a new metal cation transmembrane transporter gene of Indian mustard CE family

[0043] Step 1: Total RNA Extraction

[0044] The 3-week-old Indian mustard plants were transferred to 1 / 2×Hoagland liquid medium (without sucrose and agar) for pre-culture, and the fresh culture medium was changed every two days. After one week, 200 mM CdCl was added to the medium 2 Stress was carried out, fresh medium was changed once a day, and roots and leaves were collected (harvested) two days later. The control was Indian mustard grown under the same conditions, but no CdCl was added to the medium 2 Do coercion. The RNA extraction kit (RNeasy Plant Mini Kit) from QIAGEN was used to carry out control and CdCl 2 Extraction of total RNA from stress-treated samples. The contaminating DNA was removed with RNase-free DNase I (final concentration 80,000 U / L), and its integrity was identified by formaldehyde denaturing gel electrophoresis. The amount of 28s and 18...

Embodiment 2

[0073] Example 2 Heavy metal resistance analysis of yeast transformed with a new gene of metal cation transmembrane transporter of Indian mustard CE family

[0074] Step 1: Construction of yeast expression vector

[0075] Based on the multi-cloning restriction site HindIII / BamHI sequence on the shuttle vector pYES2 (see Figure 15), PCR primers for the ORF region of the BjCETs (1-4) sequence were designed (see Table 1), and pGEM-T Easy vector The full-length cDNA sequences of BjCETs (1-4) above were used as templates to amplify their ORF regions, and the amplified DNA fragments were digested with HindIII / BamHI double enzymes, and then circularized with the pYES2 vector cut with HindIII / BamHI double enzymes. Transform competent Escherichia coli DH5α, select on LB medium containing 50 μg / mL ampicillin, and culture overnight at 37°C. Pick 5-10 single clones respectively, culture and amplify, extract the plasmid and carry out HindIII / BamHI double enzyme digestion, take the clone c...

Embodiment 3

[0093] Example 3 Analysis of Heavy Metal Resistance and Accumulation in Tobacco Transformed with New Genes of Metal Cation Transmembrane Transporters of Indian Mustard CE Family

[0094] Step 1: Construction of plant expression vector and Agrobacterium transformation

[0095] Based on the polyclonal restriction site XbaI / BamHI sequence on the bivalent vector pBIN-mGFP5-ER (see Figure 16), the PCR primers (see Table 2) for the ORF region of the BjCETs (1-4) sequence were designed to The full-length cDNA sequence of BjCETs (1-4) on the pGEM-T Easy vector was used as a template to amplify its ORF region respectively. After the amplified DNA fragment was digested with XbaI / BamHI, it was digested with XbaI / BamHI double digested pBIN-mGFP5 -ER vector was circularized and ligated, transformed into competent Escherichia coli DH5α, and screened on LB medium containing 50 μg / mL kanamycin. Incubate overnight at 37°C. Pick 5-10 single clones, culture and amplify, extract plasmids and ca...

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Abstract

The present invention relates to a new CE gene family member. The invention provides a cDNA sequence of protein gene BiCET1 carried through membrane by metallic ion of CE family of India mustard, and a sequence of hydrogen radical acid of coded protein molecule inferred from cDNA sequence. The said sequence of gene or sequence of amino acid of the invention can be applied to development of transgene in genetic engineering products. The invention provides important application values: improving resistance of heavy metal for plant; reducing accumulation of toxic heavy metal in eatable part of plant; enriching needed mineral matter of trace elements such as Zn, Ni, Se by plants in developing foodstuff enriched by mineral matter.

Description

field of invention [0001] The invention belongs to the field of plant metal cation transporters, in particular to gene sequences and amino acid sequences of Zn, Ni, Cd, Co and other heavy metal ion transmembrane transporters of Indian mustard (Brassica juncea) CE (cation-efflux transporters) family. Background technique [0002] The metal cation transmembrane transporter of the CE (cation-efflux transporters) family plays a role in the extracellular export of metal ions and the regional isolation of intracellular vacuoles in biological cells, improving the resistance and enrichment of metal ions plays a key role. Nies and Silver (1995) first identified the CDF (cation diffuse facilitor proteins) family in bacteria, and later found such carrier proteins in fungi, plants and animals. Its typical structural features are 6 transmembrane (TM, transmembrane) domains (TM1-6), 1 N-terminal characteristic amino acid sequence (SLAI / V M / L TDAA / LHL / MLT / SD) and 1 C-terminal Cation expo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H1/00C07K14/415C12N15/29
Inventor 柴团耀朗明林
Owner GRADUATE SCHOOL OF THE CHINESE ACAD OF SCI GSCAS
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