Rice Metal Tolerance Protein osmpt1 and its Encoding Gene and RNA Interference Fragment

An RNA interference, metal technology, applied in the direction of DNA/RNA fragment, genetic engineering, plant genetic improvement, etc., can solve the problem of low expression, and achieve the effect of reducing food safety hazards

Inactive Publication Date: 2011-12-28
SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Arabidopsis halleri's close relative species, Arabidopsis lyrata and Arabidopsis thaliana, cannot tolerate and accumulate heavy metals. The genomes of these two plants only contain one copy of the MTP1 gene, an

Method used

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  • Rice Metal Tolerance Protein osmpt1 and its Encoding Gene and RNA Interference Fragment
  • Rice Metal Tolerance Protein osmpt1 and its Encoding Gene and RNA Interference Fragment
  • Rice Metal Tolerance Protein osmpt1 and its Encoding Gene and RNA Interference Fragment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Cloning of the OsMTP1 gene

[0027] Take the seedling leaf parts of rice Nipponbare (seeds purchased from Guangdong Academy of Agricultural Sciences), extract total RNA from leaves with TriZol Reagent (Invitrogen Company, article number: 15596026), and detect total RNA by agarose gel electrophoresis and ultraviolet spectrophotometer Purity and quantity, take 1 μ g of total RNA to do the initial reverse transcription reaction, the reverse transcriptase used is MMLV (Pro omega company, its article number is: M1701), the step of reverse transcription reaction refers to the instructions for use of the reverse transcriptase . Use the reverse transcription product as a template and use primers:

[0028] 5'-CTCATCCCCAAGATGGACAGCC-3';

[0029] 5'-ACTACCTACCTGTCTCAAGCGGTCA-3' for conventional PCR amplification;

[0030] The PCR reaction system is: reverse transcription product 1μl, 10xBuffer 5μl, dNTP (each 2.5mM) 4μl, forward and direction primers (10μM) each 1μl,...

Embodiment 2

[0031] Example 2: Construction and genetic transformation of RNA interference vector of OsMTP1 gene

[0032] The interference vector used in the present invention is pTCK303 (A Practical Vector for Efficient Knockdown of Gene Expression in Rice (Oryza sativa L.). Wang Zhen, Chen Changbin, Xu Yunyuan, Jiang Rongxi, Han Ye, Xu Zhihong, Chong Kang. Plant Molecualr Biology Reporter.2004.22:409-417), and on this basis, the interference vector pTCK303-M1RI-F1-F2 of OsMTP1 was constructed. The construction steps of the vector are as follows: extract the plasmid of Escherichia coli DH5α transfected with the pGEM T vector containing the OsMTP1 gene according to the conventional method, use the plasmid as a template, and use pMTP1RIL:5'-CG GGATCCGAGCTC ATGGACAGCCATAACTCAGC-3' (with BamHI and SacI restriction sites) and pMTP1RIR: 5'-GG GGTACCACTAGT GCTGAGCAGCAAACATGAG-3' (with KpnI and SpeI restriction sites) was used as primer for PCR reaction. The reaction system is: plasmid 1μl (5...

Embodiment 3

[0040] Example 3: Determination of heavy metal content in RNA interference transgenic rice seeds by atomic absorption spectrometry

[0041] Seeds of the above-mentioned homozygous strains were sown into 9-em-diameter petri dishes to germinate at 37°C, and after 7 days, the seedlings were transferred to the nutrient solution (control) and the nutrient solution supplemented with 0.05mM Cd for cultivation and growth. The composition of the nutrient solution is: mother solution 1: 91.4g NH 4 NO 3 , 32.4 g MgSO 4 ·7H 2 O, add water to make up to 1L; mother liquor 2: 88.6g CaCl 2 , add water to 1L; mother liquor 3: 40.3g NaH 2 PO 4 , 71.4g K 2 SO 4 , add water to 1L; mother liquor 4: 0.943g H 3 BO 4 , 1.5g MnCl 2 4H 2 O, 0.074g (NH 4 ) 6 Mo 7 o 24 4H 2 O, 0.031g CuSO 4 ·5H 2 O, 0.035g ZnSO 4 ·7H 2 O, add water to make up to 1L; mother liquor 5: 6.9g FeSO 4 ·7H 2 O, 9.3 g Na 2 EDTA·2H 2 O, add water to make up to 0.5L. When in use, add 5mL of No. 1-5 mother l...

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Abstract

The invention discloses rice metal-tolerant protein OsMPT1 as well as an encoding gene and an RNA (Ribonucleic Acid) interference segment thereof. The amino-acid sequence of the rice metal-tolerant protein OsMPT1 is shown as SEQ ID NO.2, the nucleotide sequence of the encoding gene OsMPT1 is shown as SEQ ID NO.1, and the gene is a key gene for responding cadmium stress, which has important significance for comprehensively understanding the biological function of metal-ion transport protein in a plant. The nucleotide sequence of the RNA interference segment for the rice metal-tolerant protein OsMPT1 is shown as SEQ ID NO.3, the expressing quantity of the OsMPT1 gene in the whole rice plant can be decreased after the RNA interference segment is expressed in rice by utilizing an expressing vector, and the heavy-metal cadmium-enriching quantity of rice seeds is also decreased along with the decrease of the expressing quantity of the OsMPT1 gene. The RNA interference segment for the rice metal-tolerant protein OsMPT1 disclosed by the invention can be applied to the gene-engineering inheritance breeding of the rice, rice varieties with low-absorption enriched cadmium are cultivated, andfood safety hazards brought to grain crops by heavy-metal cadmium are lightened.

Description

Technical field: [0001] The invention belongs to the field of biogenetic engineering, and in particular relates to rice metal tolerance protein OsMPT1, its coding gene and its RNA interference fragment. Background technique: [0002] Rice is one of the most important food crops in the world and the largest food crop in my country. The discharge of industrial "three wastes" has caused huge environmental pollution, especially the problem of heavy metal pollution has become increasingly serious, and has become one of the important problems that endanger human society. In areas with serious heavy metal pollution in my country, such as the Pearl River Delta, the Yangtze River Delta and other highly industrialized areas, as well as parts of central my country where the mining industry is developed (such as Chenzhou, Hengyang, Yongzhou, Changde, Zhuzhou, etc. in Hunan), all are Rice is the main food crop, and the rice produced in these areas often has excessive levels of heavy meta...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/113C12N15/63C12N15/84A01H5/00
Inventor 张美袁连玉刘宝秀
Owner SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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