Check patentability & draft patents in minutes with Patsnap Eureka AI!

Inserted-intron-modified soy amino acid transport protein fusion gene and application thereof

A technology of fusion gene and transporter, applied in the field of plant genetic engineering, can solve the problems of excessive nitrogen fertilizer application, low nitrogen use efficiency of crops, etc., to achieve enhanced absorption, crop yield and quality improvement, low nitrogen tolerance and nitrogen The effect of improving the efficiency of nutrient utilization

Pending Publication Date: 2018-06-01
NANKAI UNIV
View PDF3 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to solve the problems of excessive application of nitrogen fertilizer and low nitrogen utilization efficiency of crops in the process of crop cultivation, the present invention creates a new type of crop nitrogen "source"-"sink" transfer efficiency that can be significantly improved and used for cultivating new varieties of nitrogen efficient and high-yield crops. fusion gene

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
  • Inserted-intron-modified soy amino acid transport protein fusion gene and application thereof
  • Inserted-intron-modified soy amino acid transport protein fusion gene and application thereof
  • Inserted-intron-modified soy amino acid transport protein fusion gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Creation of soybean amino acid transporter fusion gene NKNUE1 modified by inserting an intron

[0053] Using soybean genomic DNA as a template, fragment A of the NKNUE1 fusion gene was amplified by PCR; using soybean cDNA as a template, fragment B of the NKNUE1 fusion gene was amplified by PCR; after fusion PCR, the amplified product was recovered and TA cloned. Specific steps are as follows:

[0054] (1) PCR amplification of the target fragment

[0055] ①PCR amplification of target gene composition fragment A

[0056] According to the soybean genome sequence, specific primer 1 and primer 2 were designed, the sequences of which were shown in SEQ ID No.2 and SEQ ID No.3, and an Apa I restriction site was introduced into primer 1.

[0057] The fragment A of the NKNUE1 fusion gene was prepared by using the CTAB method to extract soybean genome DNA as a template, and carrying out PCR amplification using the above primers.

[0058] PCR reaction system:

[0059...

Embodiment 2

[0093]Example 2: The pCAMBIA 1301 vector (containing the CaMV 35S promoter) was used to construct a binary expression vector driven by the CaMV 35S promoter to express the fusion gene NKNUE1.

[0094] (1) First obtain the intermediate carrier

[0095] Design specific primer 5 and primer 6 according to the sequence of known eYFP, as shown in SEQ ID No.6 and SEQ ID No.7, introduce Apa I restriction site, Nco I restriction site and SpeI in primer 5 Restriction site, primer 6 introduces BstP I restriction site.

[0096] The vector pSAT6-eYFP-N1 plasmid was extracted from Escherichia coli, and the plasmid was used as a template to perform PCR amplification with the above primers to prepare the eYFP gene fragment.

[0097] PCR reaction system:

[0098]

[0099] PCR reaction program:

[0100] 94°C for 5 minutes;

[0101] 94°C for 30 seconds, 58°C for 30 seconds, 72°C for 1 minute, 30 cycles;

[0102] 72°C for 10 minutes;

[0103] After the PCR reaction, the target gene was r...

Embodiment 3

[0120] Embodiment 3: Preparation of transgenic Arabidopsis

[0121] (1) Transform Arabidopsis thaliana with the binary expression vector constructed in Example 2 driven by the CaMV 35S promoter to express the fusion gene NKNUE1. The specific transformation method adopts the bud soaking method mediated by Agrobacterium (Clough and Bent, 1998), to obtain The seeds were screened for resistance to 30mg / L hygromycin, and the plants that grew normally were transferred to soil for culture.

[0122] (2) PCR detection of transgenic plants: cut the leaves of transgenic plants and wild-type plants respectively, and extract genomic DNA from leaves with reference to the method of "Molecular Cloning Experiment Guide (Third Edition)" (Huang Peitang et al., 2002), and use primer 1 (SEQ ID No.2) and primer 4 (SEQ ID No.5) were used for PCR reaction, and the reaction system was the same as in Example 1.

[0123] The PCR product was subjected to agarose gel electrophoresis, and the NKNUE1 gene ...

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 invention relates to an inserted-intron-modified soy amino acid transport protein fusion gene and application thereof. A section of intron sequence is inserted into a soy amino acid transporter protein coding sequence lethal by an agrobacterium, and a fusion gene is manually spliced, named as NKNUE1, and has a sequence as shown in SEQ ID No.1. The NKNUE1 can be normally expressed in a plant cell, and is sheared through an intron so as to be translated into broad-spectrum amino acid transport protein; the agrobacterium is lack of a corresponding intron shearing mechanism, so that the aim ofobtaining an NKNUE1 transgenic plant by utilizing genetic transformation mediated by the agrobacterium is achieved. An NKNUE1 expression vector driven by a 35S promoter or other promoters can be constructed, and a target plant is transformed. The invention provides two embodiments, which prove that the NKNUE1 is beneficial to remarkably enhancing an amino acid absorption capacity and a source-library conveying capacity of transgenic soybeans and the like, the low-nitrogen resistance is enhanced, and the contents of total nitrogen and important amino acid in seeds are remarkably increased.

Description

【Technical field】: [0001] The invention belongs to the field of plant genetic engineering, in particular to obtaining a soybean amino acid transporter coding gene modified by inserting an intron by using molecular biology technology and its application in the cultivation of new varieties of transgenic plants / crops. 【Background technique】: [0002] Nitrogen is one of the important elements necessary for plant growth, and the supply of nitrogen directly affects the quality and yield of crops. Nitrogen fertilizer is the direct source of nitrogen supply for crops. However, the excessive application of nitrogen fertilizer not only has a very limited contribution to crop yield, but also causes many problems such as energy waste and environmental pollution (Ju Xiaotang et al., 2014a, 2014b). Therefore, improving the nitrogen use efficiency (NUE) of plants is of great significance to the sustainable development of agriculture. The approaches to cultivating crop varieties that effic...

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
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/82C12N15/11A01H5/00A01H6/54A01H6/20
CPCC07K14/415C07K2319/92C12N15/8251C12N15/8271
Inventor 王宁宁刘生王丹夏铜梅徐伟
Owner NANKAI UNIV
Features
  • R&D
  • Intellectual Property
  • Life Sciences
  • Materials
  • Tech Scout
Why Patsnap Eureka
  • Unparalleled Data Quality
  • Higher Quality Content
  • 60% Fewer Hallucinations
Social media
Patsnap Eureka Blog
Learn More

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

© 2025 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com