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Functional expression of yeast nitrate transporter (ynt1) in maize to improve nitrate uptake

A technology of nitrate transport and maize, applied in the field of molecular biology, to achieve the effect of reducing fertilizer costs, increasing yield, and reducing environmental impact

Inactive Publication Date: 2012-07-04
PIONEER HI BRED INT INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Improving nitrogen use efficiency and nitrate uptake in plants would be desirable; however, attempts to improve nitrate uptake by overexpressing tobacco's endogenous high-affinity nitrate transporter have failed (Fraisier et al., Plant J., ( 2000) 23: 489-496)

Method used

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  • Functional expression of yeast nitrate transporter (ynt1) in maize to improve nitrate uptake
  • Functional expression of yeast nitrate transporter (ynt1) in maize to improve nitrate uptake
  • Functional expression of yeast nitrate transporter (ynt1) in maize to improve nitrate uptake

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0225] Example 1: Cloning of the yeast nitrate transporter (YNT1) coding sequence from Pichia angus sequenced and tested for functionality in Pichia pastoris

[0226] According to published data (Perez MD et al., Biochem.J., (1997) 15:397-403), PCR was used to obtain the coding sequence of the YNT1 gene from Pichia angus genomic DNA, restricted by BamH I and EcoRI sex sites at the 5' and 3' ends, respectively. This fragment was cloned into pCR-Blunt TOPO vector for sequencing. The functionality of YNT1 was verified using the Pichia pastoris system developed at Pioneer Hi-Bred Int'l (patent application serial number 12 / 136,173). The clone with the correct sequence was used to prepare the yeast expression vector pGAPZA-YNT1 through the BamHI and EcoRI sites. Pichia pastoris strain KM71 (Invitrogen) carrying p3.5GAP-YNR1 (yeast nitrate reductase driven by a promoter integrated into the His4 locus) was integrated into the pGAP promoter region with pGAPZA-YNT1 Transfor...

Embodiment 2

[0227] Example 2: Modification of the YNT1 coding sequence for maize expression

[0228] To enhance the expression potential in maize, the codons of the YNT1 coding sequence were optimized for maize expression. Remove rare codons. The GC composition was set at 60% and distributed relatively evenly over the entire length of the open reading frame. At the same time, several unwanted features were removed, including cryptic intron donor or acceptor sites, RNA instability sites, long homologous base stretches, and unwanted restriction enzymes. The sequence of maize codon-optimized YNT1 (YNT1MO) is shown in SEQ ID NO:3. Maize expression constructs for YNT1MO driven by root-preferred promoters such as the ZM-RM2 promoter and the BSV(TR) promoter were prepared by standard cloning techniques.

Embodiment 3

[0229] Embodiment 3: the preparation of plant expression vector

[0230] Two expression vectors for the YNT1 gene driven by the maize UBI promoter (PHP26091) or the maize ZM-RM2 promoter (PHP27279) were prepared by standard cloning techniques for maize GapexGS3 transformation.

[0231] Further constructs of YNT1 driven by different promoters were made for elite maize line transformation. They are PHP32095 (ZM-RM2: YNT1), PHP32100 (ZM-RM2: ADHI intron: YNT1), PHP38318 (BSV(TR): ADHI intron: YNT1), PHP38506 (ZM-NAS2: YNT1), new PHP (BAV(FL):YNT1).

[0232] Stacked constructs containing nitrate transporter genes, nitrate reductase genes and / or root genes for improved nitrate uptake and assimilation were prepared for elite line transformation. They are PHP32372(ZM-RM2:ADHI Intron:YNT1 / / UBI:YNR1), PHP32267(ZM-RM2:YNT1 / / UBI:YNR1), PHP38942(ZM-PEPC:PPNR A551G / / ZM-RM2:ADHI Intron: YNT1), PHP38943 (ZM-PEPC: PPNR A551G / / BSV(TR): YNT1), PHP38945 (BSV(TR): ZM-CKXg / / ZM-RM2: A...

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Abstract

The present invention provides methods and compositions relating to altering NT activity, nitrogen utilization and / or uptake in plants. The invention relates to a method for the production of plants with maintained or increased yield under low or normal nitrogen fertility. The invention provides isolated nitrate transporter (NT) nucleic acids and their encoded proteins. The invention further provides recombinant expression cassettes, host cells, and transgenic plants. Plants transformed with nucleotide sequences encoding the NT enzyme show improved properties, for example, increased yield.

Description

technical field [0001] The field of the present disclosure relates primarily to molecular biology. In particular, the present invention relates to methods and compositions for improving nitrogen use efficiency and / or nitrogen uptake in plants. Background technique [0002] Nitrate is the main source of nitrogen that plants absorb from the soil. To meet the demands of the global supply of food, feed, fiber and fuel, farmers often apply excess nitrogen fertilizers to increase grain yield in crops such as maize. To avoid nitrate pollution and reduce farming costs, plants, especially maize, that can absorb / utilize nitrate more efficiently are needed to maintain food supply and protect our environment. [0003] Nitrate uptake from soil into plant root cells is an active process against the electrochemical potential gradient of the plasma membrane. Once in root cells, nitrate is: 1) reduced to nitrite by the cytoplasmic enzyme nitrate reductase, then reduced to ammonia by nitri...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/82A01H5/00C07K14/415
CPCC07K14/415C12N9/0038C12N15/8243C12N15/8261C12N15/8271C12N15/8262Y02A40/146
Inventor D·F·卢塞尔王海音
Owner PIONEER HI BRED INT INC
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