Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Functional expression of shuffled yeast nitrate transporter (YNT1) in maize to improve nitrate uptake under low nitrate environment

A technology of nitrate transport and nitrate, applied in the field of molecular biology

Inactive Publication Date: 2012-07-04
PIONEER HI BRED INT INC
View PDF83 Cites 3 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Functional expression of shuffled yeast nitrate transporter (YNT1) in maize to improve nitrate uptake under low nitrate environment
  • Functional expression of shuffled yeast nitrate transporter (YNT1) in maize to improve nitrate uptake under low nitrate environment
  • Functional expression of shuffled yeast nitrate transporter (YNT1) in maize to improve nitrate uptake under low nitrate environment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0276] Example 1: Cloning of the yeast nitrate transporter (YNT1) coding sequence from Pichia angus sequenced and tested for functionality in Pichia pastoris

[0277] 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, at the 5'- and 3 The ' ends have BamHI and EcoRI restriction sites, 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 (US Patent Application Serial No. 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 pGAP promoter integrated into the His4 locus) was integrated into the pGAP promoter region with pGAPZA-YNT...

Embodiment 2

[0278] Example 2: YNT1 DNA shuffling and functional screening in Pichia pastoris

[0279] Multiple rounds of YNT1 shuffled libraries were constructed by incorporating oligo spiked diversity from existing fungal and plant high affinity nitrate transporter genes. Expression of wild-type and shuffled YNT1 variants was under the control of the Pichia pastoris promoter (pYPT). (GenBank Accession No. AF027960) This shuffled library was transformed into a Pichia pastoris host where YNR (Pichia pastoris nitrate reductase) was expressed. (GenBank accession number Z49110) Incorporation of YNR will enable the conversion of nitrate to nitrite. Nitrite can be quantified by a colorimetric assay known as the Griess reaction. Variants from the shuffled library were screened for enhanced nitrate uptake activity in intact cell suspensions using the Griess reaction. Through three rounds of shuffling and screening, we identified several variants with significantly enhanced nitrate uptak...

Embodiment 3

[0281] Example 3: Modification of the YNT1R3FS coding sequence for maize expression

[0282] To enhance the expression potential in maize, the codons of one of the variant YNT1R3FS coding sequences were optimized. Remove rare codons. The GC composition was determined to be less than 60%, preferably less than 54-58%, relatively evenly distributed over the 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 the maize codon-optimized YNT1R3FS (YNT1R3FS MO) is shown in SEQ ID NO:9. Briefly, maize expression constructs for YNT1R3FS MO driven by root-preferred promoters such as the ZM-RM2 promoter and the BSV(TR) promoter were prepared by standard cloning techniques.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The present invention provides methods and compositions relating to altering NT activity, nitrogen utilization efficiency and / or uptake in plants. The invention relates to a method for the production of plants with maintained or increased yield under low nitrogen fertility. The invention provides isolated nitrate transporter variant (NT variant) 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 variant 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C07K14/415C12N9/02C12N15/82C07K14/39C12N15/10
CPCC07K14/415C12N9/0036C12N15/8261Y02A40/146C12N15/8243
Inventor 刘璐H·吉昂D·F·卢塞尔王海音
Owner PIONEER HI BRED INT INC
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com