Plant expression vector for improving aluminum-tolerance of plant

A plant expression vector and carrier technology, applied in the field of plant genetic engineering, can solve the problems of no tissue specificity, etc., and achieve the effects of improving resistance, improving aluminum toxicity tolerance, and good root growth

Inactive Publication Date: 2008-07-30
KUNMING UNIV OF SCI & TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The existing plant expression vectors of malate dehydrogenase gene all adopt ...

Method used

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  • Plant expression vector for improving aluminum-tolerance of plant
  • Plant expression vector for improving aluminum-tolerance of plant
  • Plant expression vector for improving aluminum-tolerance of plant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Embodiment 1, construction of intermediate vector pUC118-PrbcS-T

[0068] Purify (operate according to the kit instructions) pUC118-PrbcS-T-rbcS-3C (constructed and provided by Sugita et al., Sugita et al.1987, MGG, 209: 247-256) with a plasmid extraction kit (Broadtech Corporation) , the rbcS-3C in pUC118-PrbcS-T-rbcS-3C was cut out with restriction endonuclease SphI (Fermentas), and the cut vectors pUC118-PrbcS-T and rbcS-3C were separated by agarose gel electrophoresis Fragment, recover the 4.6kb vector pUC118-PrbcS-T, and then use the ligase kit of TaKaRa to ligate (operate according to the kit instructions) the vector DNA fragment without rbcS-3C to generate an intermediate vector pUC118-PrbcS -T (Figure 1), conversion of high efficiency (10 8 ) Escherichia coli competent cells (DH5α, purchased from Tiangen Biochemical Technology Co., Ltd.), spread the transformed Escherichia coli on a plate added with ampicillin (Amp, 100 μg / ml), cultivate overnight at 37°C, and ...

Embodiment 2

[0069] Example 2, using point mutation technology to introduce NcoI site in the intermediate vector pUC118-PrbcS-T

[0070] Using the purified plasmid pUC118-Prbcs-T as a template, a pair of complementary primers (NcoI5 and NcoI3, Figure 1) for point mutations were designed according to the chloroplast localization sequence, and TaKaRa was commissioned to synthesize them. Add 25ng of purified plasmid pUC118-PrbcS-T to the point mutation reaction mixture as a template, and at the same time add 125ng of point mutation primers NcoI5 and NcoI3, 1μl dNTP (2.5mM), 5μl of 10×KOD reaction buffer and 1μl of KOD polymerization Enzyme (Toyobo Japan), add double distilled water to make the final reaction volume 50 μl. Heated at 95°C for 30 seconds on a PCR instrument, followed by 15 cycles of reaction at 95°C for 30 seconds, 55°C for 1 minute, 68°C for 10 minutes, and finally extended the reaction at 68°C for 10 minutes to synthesize The child chain of the mutation site. After the react...

Embodiment 3

[0071] Example 3. Using point mutation technology to change XmnI in Gateway's entry vector pENTR-2B multiple cloning site to HindIII

[0072] Using the purified plasmid pENTR-2B (purchased from Invitrogen) as a template, a pair of complementary primers (HindIII5 and HindIII3) for point mutation were designed according to the sequence near XmnI (Figure 2), and commissioned to Shanghai Shenneng Bocai Company to synthesize. Add 25ng of purified plasmid pENTR-2B as a template to the point mutation reaction mixture, and add 125ng of point mutation primers HindIII5 and HindIII3, 1μldNTP (2.5mM), 5μl of 10×KOD reaction buffer and 1ul of KOD polymerase (Japan Toyobo), add double distilled water to make the final volume of the reaction 50 μl. Heated at 95°C for 30 seconds on a PCR instrument, followed by 15 cycles of reaction at 95°C for 30 seconds, 55°C for 1 minute, 68°C for 10 minutes, and finally extended the reaction at 68°C for 10 minutes to synthesize The child chain of the mut...

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Abstract

The invention discloses a plane expression vector for increasing the ability of resisting the aluminum toxicity and the application thereof, which belongs to plant genetic engineering field. The vector contains the Escherichia coli malate dehydrogenase (EMDH); the upstream of the malate dehydrogenase (EMDH) is Rubisco small subunit photoinduced promoter. The test shows that, the activity of EMDH of transgene tobacco obtained by transforming the tobacco by the vector is 1 to 3.3 times of that of the wild tobacco. Under the stress of 100 to 300uM aluminum toxicity, the EMDH transferred tobacco can secrete more malic acid; the root grows well, which has an enhanced tolerance for aluminum toxicity. The specific vector of the invention can increases the tolerance for aluminum toxicity of the plant, and has a great potential of application in improving the crops in particular in the acid soil of South China.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and specifically relates to a component and application of a plant expression vector for improving the ability of plants to tolerate aluminum toxicity. Background technique [0002] Most of the soil in southern my country is red soil, which is poor in nutrition and acidic, making the phosphorus in the soil and the phosphorus fertilizer applied to the soil insoluble, so it is difficult for plants to absorb and utilize, and the growth is hindered and the growth is low. In acidic soils, phosphorus forms very insoluble compounds with aluminum and iron. In addition, in acidic soil, aluminum becomes soluble and easily enters the roots of plants, inhibiting the growth and development of roots, making the roots unable to absorb water and nutrients, resulting in a decrease in crop yield, so it grows in acidic soil Plants are often poisoned by aluminum. Acidic soil is a typical low-yielding soil,...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/53C07H21/04A01H1/00
Inventor 陈丽梅玉永雄李昆志刘迪秋赵玥王奇峰
Owner KUNMING UNIV OF SCI & TECH
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