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Tobacco cadmium transporter gene NtHMA2, and cloning method and application thereof

A technology of gene transfer and cloning method, applied in the field of genetic engineering, can solve problems such as growth retardation, destruction of chloroplast structure, and reduction of chlorophyll content

Active Publication Date: 2014-08-13
YUNNAN ACAD OF TOBACCO AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Excessive cadmium in the soil can not only remain in tobacco, but also have obvious harmful effects on the growth and development of tobacco, destroying chloroplast structure, reducing chlorophyll content, yellowing leaves, weakening photosynthetic process, slow growth, short plants, Inhibited root system, stunted growth, reduced yield

Method used

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  • Tobacco cadmium transporter gene NtHMA2, and cloning method and application thereof
  • Tobacco cadmium transporter gene NtHMA2, and cloning method and application thereof
  • Tobacco cadmium transporter gene NtHMA2, and cloning method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1—— NtHMA2 gene cloning

[0050] Use the homologous gene Arabidopsis At HMA2 sequence to search the NCBI tobacco EST database to obtain the EST sequence of tobacco NtHMA2. The specific steps are: use the homologous gene Arabidopsis At HMA2 sequence to search the NCBI tobacco EST database to obtain the EST sequence of tobacco NtHMA2 ( NCBI No.: FG135138.1); Use this sequence to compare the tobacco genome database (in-house database) to obtain a gene sequence (Nsyl0129220), design RACE primers based on this sequence, RACE is performed using the kit Clontech Smart RACE cDNA Amplification Kit, and the reaction Refer to the kit instructions; amplify fragments of about 600bp at the 3′ end, and amplify fragments of about 500bp at the 5′ end; sequence the target fragment after recovery, and splicing with the core sequence to obtain the full-length cDNA sequence of NtHMA2, and design gene-specific primers Perform a PCR reaction to obtain the target product; sequence...

Embodiment 2

[0062] Embodiment 2——Plant Transformation Vector Construction

[0063] (1) Overexpression vector construction

[0064] According to the restriction site of pBI121 vector sequence, the Primer Premier5.0 software was used to design the primer pair for constructing NtHMA2 gene overexpression, and its sequence is as follows:

[0065] Forward primer: 5′-GGGT GGATCC TCTTCTCCTCCTCCTTAGAGAAG-3′

[0066] Reverse primer: 5′-TAAT GAGCTC CTACTCTATGACAATTTCTGATA-3′

[0067] Restriction sites are underlined.

[0068] by NtHMA2 Gene-positive clone DNA was used as template for PCR amplification. The amplification procedure of the PCR amplification system is the same as in Example 1. Double digestion system: the total reaction volume is 40 μL, including 10 μL of PCR purified product, 4 μL of 10×NEB Buffer1.1, 2 μL of BamHI and SacI, and 22 μL of sterilized double distilled water. Purify after digestion at 37°C overnight. Double digestion system of pBI121 vector: The total react...

Embodiment 3

[0078] Example 3 - Genetic Transformation of Tobacco

[0079] (1) Expression vector transformed into Agrobacterium

[0080] The recombinant vectors pBI121-NtHMA2 and pBI121-pQLi-NtHMA2 were transferred into Agrobacterium army GV3101.

[0081] Take out 3 tubes of Agrobacterium competent cells from the -80°C refrigerator, dissolve them on ice, and add the recombinant expression vectors pBI121-NtHMA2, pBI121-pQLi-NtHMA2 and pBI121 empty vector respectively.

[0082] Put the above mixture into liquid nitrogen for quick freezing for 1 minute, then transfer to a 37°C water bath for 5 minutes.

[0083] Add 1mL LB medium to the phase mixture, and incubate at 28°C and 220rpm for 3-4 hours.

[0084] The culture was spread on LB solid medium containing kanamycin (50mg / L) and rifampin (25mg / L), and cultured upside down at 28°C for 2-3 days. Clones containing the vector of interest were seen.

[0085] (2) Tobacco Transformation

[0086] Pick the Agrobacterium containing the target ve...

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Abstract

The invention discloses a tobacco cadmium transporter gene NtHMA2, and a cloning method and application thereof. The nucleotide sequence of the tobacco cadmium transporter gene NtHMA2 is disclosed as SEQ ID:No.1, and the coded amino acid sequence is disclosed as SEQ ID:No.2. The invention also discloses a cloning method of the tobacco cadmium transporter gene NtHMA2, which comprises the following steps: A. determining the NtHMA2 gene sequence; B. extracting tobacco RNA (ribonucleic acid), and carrying out reverse transcription to obtain a first chain cDNA (complementary deoxyribonucleic acid); C. designing and synthesizing specific primers according to the NtHMA2 gene sequence, and carrying out PCR (polymerase chain reaction) amplification by using the cDNA as the template; D recycling and purifying the PCR product; E. connecting the purified product with the vector, and transforming competent cells; and F. screening positive clone, carrying out PCR amplification on the positive clone, and sequencing. By regulating the expression of the tobacco cadmium transporter gene NtHMA2, the method can lower the operation rate of cadmium being transported from the tobacco root to leaves, thereby lowering the chromium content of the tobacco and providing a gene resource for green high-quality tobacco.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a tobacco cadmium transport gene NtHMA2 and its cloning method and application. Background technique [0002] tobacco( Nicotiana tabacum ) is an important economic crop and is an annual herb of Solanaceae. There are about 60 species of Nicotiana. The heavy metal cadmium is a non-essential element for the growth of tobacco, but it is easily absorbed and causes harm to the human body through the food chain. Cadmium enters the soil with water irrigation and sludge, and the cadmium absorbed by the soil generally accumulates in the surface soil of 0~15cm. Excessive cadmium in the soil can not only remain in tobacco, but also have obvious harmful effects on the growth and development of tobacco, destroying chloroplast structure, reducing chlorophyll content, yellowing leaves, weakening photosynthetic process, slow growth, short plants, The root system is inh...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/10C12N15/82A01H5/00
Inventor 李文正王丙武高玉龙宋中邦曾建敏张家瑞孔光辉李永平
Owner YUNNAN ACAD OF TOBACCO AGRI SCI
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