Plant expression vector of arabidopsis thaliana cytosolic malate dehydrogenase gene and application thereof

A technology of plant expression vector and malate dehydrogenase, which is applied in application, genetic engineering, plant gene improvement, etc., to improve the ability to resist aluminum toxicity and detoxify the effect

Inactive Publication Date: 2009-11-25
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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There is no report of a plant expression vector containing

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  • Plant expression vector of arabidopsis thaliana cytosolic malate dehydrogenase gene and application thereof
  • Plant expression vector of arabidopsis thaliana cytosolic malate dehydrogenase gene and application thereof
  • Plant expression vector of arabidopsis thaliana cytosolic malate dehydrogenase gene and application thereof

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Experimental program
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Effect test

Embodiment 1

[0046] AMDH gene cDNA amplification and TA cloning:

[0047] First search the full-length gene sequence of AMDH from GenBank, and design a pair of primers, the sequence is as follows:

[0048] AMDH5:5'-CA CCATGG CGAAGGAACCAGTTCGTG-3'

[0049] AMDH3: 5'- CTCGAG TTAAGAGAGGCATGAGTAAGCG-3'

[0050] The CACC characteristic sequence was added to the end of the primer AMDH5 at the 5' end, thereby forming an NcoI restriction site; the end of the primer EMDH3 at the 3' end was added with an XhoI restriction site.

[0051] Use TRIzoL Reagent (Invitrogen) to extract total RNA from Arabidopsis thaliana seedlings, take about 0.1 g of young leaves of the plant, add 1 ml of TRIzoL extract, grind in a mortar, let stand at room temperature for 5 minutes, transfer to a centrifuge tube, and then Add 0.2ml of chloroform, shake and mix, centrifuge for 15min (12000rpm), transfer the supernatant to a new tube, add 0.5ml of isopropanol, mix and place at room temperature for 10min, centrifuge at ...

Embodiment 2

[0053] Construction of Gateway entry cloning vector pENTR*-PrbcS-AMDH of AMDH gene:

[0054] Cut the purified plasmid vector pENTR*-PrbcS-*T-GFP and pUCm-AMDH with NcoI and XhoI ( image 3 ), separated the cut vector and insert by agarose gel electrophoresis, and recovered the vector fragments pENTR*-PrbcS (4.0kb) and pUCm- The DNA fragment (1.0kb) of the AMDH gene generated by cutting AMDH, and then use the ligase kit of TaKaRa to connect the DNA fragment of pENTR*-PrbcS and AMDH gene to generate the entry vector pENTR*-PrbcS-AMDH( image 3 ). 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 kanamycin (Km, 50 μg / ml), and cultivate overnight at 37 ° C. Screen the Km-resistant recombinant colony, extract the plasmid from the Km-resistant recombinant colony, select the successfully connected plasmid vector pENTR*-PrbcS-AMDH, and ...

Embodiment 3

[0056] Construction of AMDH gene plant expression vector pH2-35S-PrbcS-AMDH:

[0057] PrbcS-AMDH was subcloned into the plant expression vector pH2GW7 (Gateway's destination vector, Belgium VIB / Gent company) by the LR reaction of Gateway technology ( Figure 5 ). The specific method is: use the plasmid extraction kit to purify Gateway’s target vector pH2GW7, add pENTR*-PrbcS-AMDH and pH2GW7 each 150ng, 1μl LR Clonase II Enzyme Mix (Invitrogen) to the LR reaction system of Gateway, and mix well in React overnight at 25°C, and integrate PrbcS-AMDH into pH2GW7 through the action of integrase to obtain the plant expression vector plasmid pH2-35S-PrbcS-AMDH of AMDH ( Figure 5 ). 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 spectinomycin (Spe, 50 μg / ml), cultivate overnight at 37°C, and screen Spe-resistant recombinant sub-colo...

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Abstract

The invention in particular relates to a plant expression vector pH2-35S-PrbcS-AMDH for improving the aluminum toxicity resistance of plants, a construction method and application thereof, which belong to the field of plant gene engineering. The special vector pH2-35S-PrbcS-AMDH for improving the aluminum toxicity resistance of the plants is the plant expression vector containing a photoinducible promoter (PrbcS) of a rubulose-1, 5-bisphosphate carboxylase (RubIsco) small subunit gene and an arabidopsis thaliana cytosolic malate dehydrogenase gene (AMDH). The AMDH gene is cloned from arabidopsis thaliana, the photoinducible promoter is used to control the overexpression of the AMDH gene in tobacco, malic acid is synthesized, and the malic acid is secreted out of cells so as to strengthen the resistance of the plants on aluminum toxicity in acid soil. Experimental results show that the activity of malate dehydrogenase of trans-AMDH genic tobacco leaves is 1.4 times of that of wild tobacco. Under the stress of 30 mu M of aluminum toxicity, trans-AMDH genic tobacco can secrete more organic acid, and has better root system growth; and the growth condition under the stress of the aluminum toxicity shows that the plant height and the green leaf number of the trans-AMDH genic tobacco are higher than those of the wild tobacco.

Description

Technical field: [0001] The invention belongs to the field of plant genetic engineering, and specifically relates to a plant expression vector pH2-35S-PrbcS-AMDH for improving the ability of plants to tolerate aluminum toxicity, its construction method and application. Background technique: [0002] Aluminum is the most abundant metal element in the earth's crust, and its average content accounts for about 8% of the earth's crust. Experiments have shown that an appropriate amount of aluminum can promote the growth and development of tea trees and wild peony. For example, aluminum in a certain range (≤400μmol / L.) increases the number and length of lateral roots of tea trees, and aluminum below 50μmol / L can promote the growth of rice seedlings , but most crops are susceptible to aluminum poisoning, and usually nanomolar concentrations of aluminum can affect the growth of many crop roots in a short period of time (MA J F, PETER R R, DELHAIZE.2001.Aluminum tolerance in plants an...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/53A01H1/00
Inventor 陈丽梅王奇峰玉永雄李昆志易琼
Owner KUNMING UNIV OF SCI & TECH
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