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

Method of creating high-oil cotton material by using cotton GhPEPC gene

A gene and cotton technology is applied in the field of using cotton GhPEPC gene to create cotton high-oil materials, which can solve the problems of low silencing efficiency, and achieve the effects of reducing protein content, increasing content and reducing synthesis.

Inactive Publication Date: 2017-05-31
HUAZHONG AGRI UNIV
View PDF1 Cites 9 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In terms of silencing efficiency, the RNA silencing structure with an intron structure has the highest efficiency, up to 90%-100%; the silencing efficiency without an intron structure is low

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
  • Method of creating high-oil cotton material by using cotton GhPEPC gene
  • Method of creating high-oil cotton material by using cotton GhPEPC gene
  • Method of creating high-oil cotton material by using cotton GhPEPC gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Cloning of Example 1 GhPPC Gene

[0061] The applicant cloned a 308bp sequence from upland cotton (Gossypium hirsutum) [The primers used are: GhPPC-F: (5'-TTGAATACTTCCGCCTAGCA-3'), GhPPC-R: (5'-AGCGATTCCAGGGTCTCC-3') 】. Perform TA cloning and sequencing on the obtained DNA fragments, and compare with the sequence on NCBI to verify whether it is the GhPPC gene sequence (see figure 1 Figure A in the figure), and at the same time, we carried out evolutionary analysis on the homologous genes of the GhPPC gene in cotton, and found that there were multiple copies, and we selected two of the copies as the targets of RNA interference, namely the GhPPC1 and GhPPC2 genes (see figure 1 in panel B).

[0062] 1. Extraction of total RNA from upland cotton leaves (improved guanidine isothiocyanate method) and cDNA acquisition:

[0063] Modified guanidine isothiocyanate method:

[0064] 1.1 Grind fresh leaves or leaves frozen at -70 degrees into powder with liquid nitrogen, weigh 0...

Embodiment 2

[0077] Embodiment 2: the construction of GhPPC gene plant interference vector

[0078] According to the obtained SEQ ID NO: 1, primers were designed for constructing expression vectors, and linker bases for BP reaction were added to both ends of the primers respectively. The primer sequences were GhPPC-BP-F: (5'-GGGG ACA AGT TTG TAC AAA AAA GCA GGC TNNTTGAATACTTCCGCCTAGCA-3'), GhPPC-BP-R: (5'-GGGG AC CAC TTT GTA CAA GAAAGC TGG GTNAGCGATTCCAGGGTCTCC-3'). The pGEM-T-GhPPC plasmid was used as a template for PCR amplification. The PCR conditions were 95°C for 5 min; 95°C for 30 sec, 55°C for 30 sec, 72°C for 20 sec, and 32 cycles; 72°C for 10 min. The correct GhPPC fragment containing BP linker was obtained by PCR amplification. The PCR product was connected to the pHellsgate 4 carrier by BP reaction (BP enzyme was purchased from Invitrogen, the United States, 25°C for 4 hours), and then transformed into Escherichia coli Top10 competent cells, and GhPPC-specific primers (GhPPC-F ...

Embodiment 3

[0079] Embodiment 3 Genetic transformation and screening identification of GhPPC gene

[0080] 1. Preparation of sterile vaccine

[0081] The test material is wild type upland cotton YZ1 (Yuzao 1, Cotton Research Institute, Chinese Academy of Agricultural Sciences). Sowing cotton aseptic seedlings under in vitro culture conditions: sterilize the shelled seed kernels with 0.1% mercuric chloride for 8 minutes on the ultra-clean workbench, and then wash them with sterile water for 3-4 times, and the resulting sterile seedlings Kernels (or explants) are inoculated in the aseptic seedling medium, the seedlings are supported after 2 days, the seed coat is removed, and hypocotyls can be obtained after 5 days.

[0082] 2. Activation and infection of Agrobacterium

[0083] Take out the stored expression vector p35S-GhPPC containing the target gene from the ultra-low temperature refrigerator, transform Agrobacterium LBA4404 by electric shock, and apply spe + Resistance, single clones...

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 belongs to the technical field of plant gene engineering, in particular to a method of creating a high-oil cotton material by using a cotton GhPPC gene. The invention relates in particular to the gene GhPPC cloned from upland cotton, and functional verification and an application of the gene. Part of a conservative cDNA (complementary deoxyribonucleic acid) sequence of the obtained encoding gene is as shown as SEQ ID NO: 1 (sequence identifier number 1) in a sequence table. The invention further discloses an application of the gene GhPPC in genetic modification of the upland cotton, and a cottonseed oil content of the upland cotton can be increased by regulating expression of the cloned gene.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a method for creating cotton high-oil material by utilizing cotton GhPEPC gene. Background technique [0002] At present, there is a shortage of edible oil in my country, and the proportion of dependence on foreign countries has reached more than 60%. If the oil content of seeds of oil crops can be improved, the shortage of edible oil in my country will be alleviated. Cotton is an important strategic economic crop in my country. It not only provides fiber to humans, but also contains rich oil and protein in cottonseed. Cotton has always been considered as a fiber crop. However, cotton is also one of the five major oil crops in my country, namely: rapeseed, soybean, sesame, peanut, and cottonseed. The cottonseed oil content of most cotton species in my country is about 30%. If the oil content can be increased by one percentage point, the economic ben...

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): C12N15/60C12N15/82C12Q1/68A01H5/00A01H4/00
Inventor 金双侠郭小平许忠平张献龙
Owner HUAZHONG AGRI UNIV
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