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

A method for making apple tree synthesize astaxanthin to improve its photo-oxidation resistance

A technology of anti-photooxidation and astaxanthin, applied in the field of genetic engineering, can solve problems that have not been reported, and achieve the effects of preventing sunburn, enhancing anti-photooxidation ability, and improving photosynthetic efficiency

Inactive Publication Date: 2015-08-12
QINGDAO AGRI UNIV +1
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no reports at home and abroad on the use of astaxanthin key enzymes to synthesize genes to transform apples and improve the photooxidation resistance of apple fruits and leaves.

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
  • A method for making apple tree synthesize astaxanthin to improve its photo-oxidation resistance
  • A method for making apple tree synthesize astaxanthin to improve its photo-oxidation resistance
  • A method for making apple tree synthesize astaxanthin to improve its photo-oxidation resistance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1b

[0026] Cloning of embodiment 1bkt and crtR-B

[0027] 1. Total RNA was extracted from Haematococcus pluvialis

[0028] 1.1 Extraction of total RNA

[0029] Before RNA extraction, Haematococcus pluvialis cells were treated with 100 μmol·m -1 ·s -1 The light intensity was treated for 3 days, and the total RNA was extracted using an RNA extraction kit (RNeasy Plant Mini Kit). Refer to the manual for specific steps.

[0030] 1.2 DNA and RNA purity and integrity testing

[0031] For the extracted total RNA, take 2 μL, use a UV-Vis Spectrophotometer to measure the concentration of DNA and RNA, and measure its absorbance at 260nm and 280nm wavelengths, as well as OD260 / OD280 and OD260 / OD230. When OD260 / OD280 is less than 1.8, it indicates that there is more protein; when OD260 / OD280 is 1.8-1.9, the DNA purity is considered to be high; when OD260 / OD280 is 2.0, the RNA purity is considered to be good; OD260 / OD280 is greater than 2.2, indicating that the RNA has been degraded ; OD...

Embodiment 2

[0087] The construction of embodiment 2 bivalent plant expression vector pCAMBIA1301-bkt-crtR-B

[0088] Specific operation methods such as plasmid extraction, digestion, connection, and transformation were carried out according to conventional molecular biology techniques.

[0089] 1. Construction of intermediate vector pBI221-bkt

[0090] Using the plasmid pMD-18T-bkt as a template, primer combination B1 (SEQ ID NO.7, forwardprimer: 5'-AAA GGATCC ATGCACGTCGCATC-3') / B2 (SEQprimer: 5'-AAC GAGCTCTCATGCCAAGGCAG-3') and La Taa DNA polymerase for PCR amplification (pre-denaturation at 95°C for 5 min; denaturation at 94°C for 1 min, annealing at 60°C for 1 min, extension at 72°C for 1 min, cycle 35 times; extension at 72°C for 10 min) to obtain BamH The bkt gene fragment of about 963 bp at the I and Sac I restriction sites was recovered after double digestion with BamH I and Sac I. Use T 4 DNA ligase was used to connect and transform Escherichia coli TOP10 competent cells to ...

Embodiment 3

[0095] Transformation of embodiment 3 apples and regeneration of transgenic plants

[0096] 1. Preparation of engineering strain EHA105 / pCAMBIA1301-bkt-crtR-B

[0097] The pCAMBIA1301-bkt-crtR-B plasmid was extracted, and the competent Agrobacterium EHA105 was transformed by freeze-thaw method. Spread on YEB solid medium containing rifampicin (Rif, 100mg / L) and kanamycin (Km, 50mg / L), and culture in dark at 28°C for 1-2d. A single colony was picked and cultured in YEB liquid medium with the same antibiotics, the bkt gene and crtR-B gene were detected by bacterial liquid PCR, plasmid PCR and double enzyme digestion identification method, and double positive clones were screened to obtain double-valent Agrobacterium engineering strain EHA105 / pCAMBIA1301-bkt-crtR-B of plant expression vector.

[0098] 2. Preparation of infection solution

[0099] The engineering strain EHA105 / pCAMBIA1301-bkt-crtR-B was cultured in YEB liquid medium (adding Rif50mg / L, Kan 50mg / L) in a constant ...

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 provides a DNA (deoxyribonucleic acid) sequence represented by SEQ ID NO.5 for coding beta-carotene hydroxylase, a recombinant expression vector and a host cell, wherein the recombinant expression vector is constructed by the DNA sequence, a DNA sequence represented by SEQ ID NO.6 for coding beta-carotene ketoIase and a plasmid, and the host cell is transformed by the abovementioned recombinant expression vector. The invention also provides a method for synthesizing astaxanthin by apple trees to improve photooxidation resistance, which comprises the following steps: a) constructing the recombinant expression vector pCAMBIA1301-bkt-crtR-B; b) transforming competent agrobacterium tumefaciens CHA105 by the recombinant expression vector pCAMBIA1301-bkt-crtR-B; and c) transforming the apple tissue culture seedlings to obtain the transgenic plants via the agrobacterium tumefaciens. According to the invention, the key enzymic genes bkt and crtR-B synthesized by astaxanthin are transferred into an apple genome by the genetic transformation, the apples rich in astaxanthin are cultivated by regulating and controlling the metabolic pathways, thereby improving photosynthetic efficiency and preventing sunburn, and dramatically overcoming the shortcomings in the prior art.

Description

【Technical field】 [0001] The invention relates to the technical field of genetic engineering, in particular to a method for making astaxanthin synthesized by apple trees through a plant genetic transformation method to improve their light oxidation resistance. 【Background technique】 [0002] Apple trees are prone to photooxidative damage in the growing season of high temperature and strong light, which is manifested in two aspects. On the one hand, the photosynthetic efficiency of leaves will be reduced due to photooxidative damage during photosynthesis; on the other hand, apple fruits often appear Sunburn phenomenon, usually apple sunburn is caused by the photooxidative damage of the fruit under temperature adversity and strong light. It occurs to varying degrees in the world's apple producing areas every year. quality, causing great losses to production. With the popularization of dwarf cultivation system and global warming, apple sunburn tends to increase year by year. ...

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 Patents(China)
IPC IPC(8): C12N15/53C12N15/63C12N1/21C12N1/15C12N5/10C12N15/84A01H5/00
Inventor 原永兵贾东杰秦松李富超刘成连
Owner QINGDAO 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