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Plant expression vector of citric acid synthesis gene and application thereof

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

Inactive Publication Date: 2008-09-17
KUNMING UNIV OF SCI & TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing plant expression vectors of the cs gene all use a constitutive promoter (CaMV35S), and the effect of CaMV35S has no tissue specificity

Method used

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  • Plant expression vector of citric acid synthesis gene and application thereof
  • Plant expression vector of citric acid synthesis gene and application thereof
  • Plant expression vector of citric acid synthesis gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1, construction of intermediate vector pUC118-PrbcS-T

[0058] Purify (operate according to 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) , 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 ), converting the high efficiency (10 8 ) Escherichia coli competent cells (DH5α, Tiangen Biochemical Technology), spread the transformed Escherichia coli on a plate added with ampicillin (Amp, 100 μg / ml), cultivate overnight at 37°C, and screen Amp-resistant recombinants ...

Embodiment 2

[0059] Example 2. Using point mutation technology to introduce NcoI site into the intermediate vector pUC118-T-PrbcS

[0060] Using the purified plasmid pUC118-Prbcs-T as a template, a pair of complementary primers (NcoI5 and NcoI3, NcoI3, figure 1 ), commissioned TaKaRa to synthesize. 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, 1μldNTP (2.5mM), 5μl of 10×KOD reaction buffer and 1μl of KOD polymerase (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 reaction was completed, the reaction mixture was cooled on ice for 2 minutes, 1 μl of restriction endon...

Embodiment 3

[0061] Embodiment 3, the construction of plant expression vector pPZP211-PrbcS-cs

[0062] The construction strategy of the plant expression vector pPZP211-PrbcS-cs is as follows: figure 2 As shown, the first choice is to search the full-length gene sequence of tobacco cs from GenBank, and design a pair of specific primers to amplify the first-strand cDNA of tobacco as a template to obtain the full-length cDNA of cs, recover and purify the cs full-length gene fragment, Connect it to pMD18-T vector to obtain pMD-cs. Digest pMD-cs and pUC118-PrbcS-*T with NcoI and XbaI, recover and purify the cs gene fragment and the large vector fragment pUC118-PrbcS, and then connect and obtain the recombinant plasmid pUC118-PrbcS-cs. Digest pUC118-PrbcS-cs and pPZP211 with HindIII and XbaI, recover and purify the small fragment PrbcS-cs and large fragment pPZP211, and then connect to obtain the recombinant vector pPZP211-PrbcS-cs, which contains the promoter PrbcS, followed by cs Gene. Th...

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Abstract

The invention discloses special vector pPZP211-PrbcS-cs which can enhance the aluminum toxicity resistance ability of plants. The vetor is plant expression vector containing tobacco citrate synthetase gene cs. The invention clones cs gene from the tobacco, a photoinduction promoter (the promoter of Rubisco small subunit) is used for controlling excessive expression of the gene cs in tobacco. Citric acid is synthesized and excreted out of cells. The citric acid enters the soil by roots and wraps the aluminium ion in soil with chemical bonds. Therefore, the damage to the plants, caused by high concentration aluminum in soil, is alleviated. The laboratory result shows that citrate synthase of the tobacco lamina with transferring cs gene is 1 to 5.5 times the activity of wild tobacco. Under the intimidation of the aluminum toxicity which is 100 to 300 micrometers, the tobacco with the transferring cs gene can excrete more organic acid, so that the roots grow better and the resistance ability against aluminum toxicity is strengthened remarkably. The special vector provided by the invention can enhance the aluminum toxicity resistance ability of the plants; the special vector has wide application potential in genetic improvement of plant varieties, more particularly for the crop varieties planted in the acid soil of southern China.

Description

Technical field: [0001] The invention belongs to the field of plant genetic engineering, and relates to a plant expression carrier of a citric acid synthesis gene, which can improve the ability of plants to resist aluminum toxicity. Background technique: [0002] Aluminum is one of the most common elements in the earth’s crust. It is rich in reserves, accounting for about 7% of the weight of the earth’s crust, second only to oxygen and silicon, ranking third. Aluminum is also the most abundant metal element in the earth’s crust (Zhao Hetao. 1996. The relationship between aluminum element and tea plant growth and variety. Tropical crop science and technology. (3): 33-35). In a neutral or slightly acidic environment, aluminum mainly exists in the soil in the form of fixed silicates and oxides. In an acidic environment, the insoluble aluminum compound is transformed into dissolved aluminum, which is toxic to plants when it enters the soil (Hideaki M.2000.Cell biology of alumin...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/52A01H1/00
Inventor 陈丽梅玉永雄李昆志刘迪秋胡清泉赵玥
Owner KUNMING UNIV OF SCI & TECH
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