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Potassium-ammonium double-function transport molecule of rice and application thereof

A dual-function, rice technology, applied in the field of plant genetic engineering, can solve the problems of limited soil nutrients, difficult utilization of effective nutrients, water eutrophication and water environment damage.

Inactive Publication Date: 2010-09-08
CAPITAL NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Potassium and nitrogen in the soil are the main source of potassium nutrition for plants, and the potassium and nitrogen in the soil are easy to be leached and lost. Excessive leaching and loss will cause limited nutrients in the soil and make it more and more difficult to use effective nutrients. On the other hand, excessive application will also cause eutrophication of the water body and serious damage to the water environment.

Method used

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  • Potassium-ammonium double-function transport molecule of rice and application thereof
  • Potassium-ammonium double-function transport molecule of rice and application thereof
  • Potassium-ammonium double-function transport molecule of rice and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Embodiment 1: Cloning of rice OsKAT3 gene

[0035] Extract total RNA from rice roots and leaves grown in hydroponic culture for 10 days, use it as a template, and use Oligo dT as a primer for reverse transcription. After the reaction is completed, take 1 μL of the reverse transcription product as a template, and perform PCR amplification with specific primers K3F and K3R . As a result, a single product of about 2.1 kb ( Figure 1A ). After the amplified band was purified and recovered, the recombinant plasmid pTK3 connected with the pEASY-T1 vector was transformed into Escherichia coli competent cell DH5α. Single clone plasmids were picked for enzyme digestion detection ( Figure 1B ) and colony PCR identification ( Figure 1C ), positive clones were selected and sequenced.

[0036] K3F: 5′-ATGACCCAAGCTCACTCAAAATCTTGCTTCC-3′

[0037] K3R: 5′-CTACATCTCAAGAAGGAATAGATGGTCGCCA-3′

[0038] The cDNA sequence of OsKAT3 and the amino acid sequence of its encoded protein a...

Embodiment 2

[0104] Example 2: Construction and functional complementation of eukaryotic expression vector of rice OsKAT3 gene

[0105] 1. Construction of rice OsKAT3 gene expression plasmid

[0106] The rice OsKAT3 was constructed into the pYES2 vector (the vector was purchased from Invitrogen), and after transforming into Escherichia coli, the plasmid was extracted and detected ( Figure 3A ).

[0107] 2. Functional complementation experiment of recombinant plasmid pYES2-OsKAT3

[0108] The constructed pYES2-OsKAT3 vector was transformed into yeast CY162 strain, grown for 1-2 days under 10mM SD solid medium, and yeast clones were picked for detection ( Figure 3B ), cultivated for about 1 day under 10mM SD liquid medium, and then transferred to 0.1mM YNB+Gal solid medium for growth, and verified that the rice OsKAT3 gene had the activity of transporting potassium inward by observing the growth situation ( Figure 4 ).

Embodiment 3

[0109] Example 3: Electrophysiological function analysis of rice OsKAT3 gene

[0110] The rice OsKAT3 gene was constructed into the pGEMHE vector (the vector was first transformed by introducing the 3'-UTR and 5'UTR of a β-globin gene of Xenopus laevis into the pGEM-3Z vector by Liman et al. of Harvard Medical School. The vector diagram is as follows Figure 8 ), detection after transformation of Escherichia coli (such as Figure 3A ), using specific primers M13 and M13R for PCR amplification, and the product was made into Capping RNA, which was injected into Xenopus oocytes by microinjection technology for expression for about 2 days, and then OsKAT3 was expressed by TEVC (Two-electrode voltage-clamp) technology Gene for electrophysiological analysis.

[0111] 1. Potassium transport function analysis of rice OsKAT1 gene

[0112] 1) Preparation of bath liquid:

[0113] Potassium ion bath: 100mM potassium glutamate, 2mM MgCl 2 , 1mM CaCl 2 , 10mM Hepes;

[0114] Mannitol ...

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Abstract

The invention relates to the field of plant gene engineering, and in particular relates to a potassium-ammonium double-function transport molecule of rice and an application thereof. The gene order of the potassium-ammonium double-function transport molecule of rice is as shown in SEQ ID No.1. The invention firstly obtains an OsKAT3 gene by cloning in Nipponbare (Oryza sativa ssp.japonica), and verifies that the OsKAT3 gene of the rice has the function of high-efficiency K+ absorption; and furthermore, the gene also has the characteristic of high-efficiency NH4+ transport.

Description

technical field [0001] The invention relates to the field of plant genetic engineering, in particular, the invention relates to rice potassium and ammonium bifunctional transport molecules and their application. Background technique [0002] N, P, and K are the necessary macroelements for crop growth. The absorption and utilization of nutrients are the main factors that limit the yield. Improving the effective use of crop nutrients has always been the goal of breeding. Under the current conditions of increasing eutrophic soil pollution Under the circumstances, increasing the self-utilization efficiency of crop nutrients and creating green ecological agriculture is another aspect worthy of attention. Potassium and nitrogen in the soil are the main source of potassium nutrition for plants, and the potassium and nitrogen in the soil are easy to be leached and lost. Excessive leaching and loss will cause limited nutrients in the soil and make it more and more difficult to use ef...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00
Inventor 李乐攻张鹏田丽丽
Owner CAPITAL NORMAL UNIVERSITY
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