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Method for synthesizing astaxanthin by apple trees to improve photooxidation resistance

A technology of carotene ketolase and hydroxylase, which is applied in the field of genetic engineering, can solve problems that have not been reported, and achieve the effects of preventing sunburn, enhancing the ability to resist photooxidation, and improving photosynthetic efficiency

Inactive Publication Date: 2012-11-07
QINGDAO AGRI UNIV +1
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  • 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

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  • Method for synthesizing astaxanthin by apple trees to improve photooxidation resistance
  • Method for synthesizing astaxanthin by apple trees to improve photooxidation resistance
  • Method for synthesizing astaxanthin by apple trees to improve photooxidation resistance

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Experimental program
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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 ...

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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

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Application Information

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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
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