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Process for culturing brownness resistant sweet potatoes utilizing gene engineering technology

A sweet potato, gene technology, applied in genetic engineering, biochemical equipment and methods, horticultural methods, etc.

Inactive Publication Date: 2008-03-12
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no relevant report on the application of genetic engineering technology mentioned in this patent application to cultivate anti-browning transgenic sweet potatoes, including patents and literature

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Synthesis of core fragment of sweetpotato polyphenol oxidase gene and construction of binary expression vector and program bacteria

[0029] 1. Obtaining the core fragment of antisense polyphenol oxidase gene

[0030] According to the full-length sequence of the polyphenol oxidase gene, primers were designed between the first base and the 488th base in the polyphenol oxidase gene, and the 502bp sequence was amplified as an antisense gene fragment. For the multiple cloning enzyme cutting site of the vector pCAMB1A1304, two PCR amplification primers containing SacI and BamHI restriction endonuclease sites and protective bases were designed respectively. The primer sequences are:

[0031] P1: 5'-cgagctctatgttcagaccgattacccc-3'

[0032] P2: 5'-cggatccatggtccacattcggtcgac-3'

[0033] Extract total RNA from sweet potato tubers (RNA extraction kit from Shanghai Huashun Bioengineering Co., Ltd.), reverse transcribe into cDNA (TaKaRa RNA PCR Kit), and then perform PCR amplif...

Embodiment 2

[0042] Obtain anti-browning transgenic sweet potato with reduced PPO activity

[0043] 1. Agrobacterium LBA4404-ppoF. Take it out from the refrigerator before use, inoculate in 50ml YEB liquid (Rif + ,Str + , Kan + ), 28 degrees, 200rpm shaking culture twice;

[0044] 2. The second activation of OD 600 When it reaches 0.3, add 100 μmol / mL acetosyringone, continue to culture at 28 degrees, shake at 200 rpm, OD 600 Centrifuge at 4000r for 10min at room temperature when reaching 0.6;

[0045] 3. Discard the supernatant, suspend the bacteria with MS (100 μmol / mL acetosyringone) liquid medium, and dilute to 5-20 times the original volume to make the OD of the bacteria solution 600 = about 0.3; called the conversion liquid;

[0046] 4. Take sterile sweet potato terminal buds, side buds, leaves, stems, roots and other plant parts, cut the stems into 1cm pieces, or cut the leaves into about 1cm2, and use a sterile scalpel to draw a "+" shaped wound, Put it into the above tra...

Embodiment 4

[0051] Example 4: Comparison of transgenic sweet potato and non-transgenic sweet potato polyphenol oxidase activity and anti-browning performance

[0052] 1. Compare the activity of polyphenol oxidase in transgenic and non-transgenic sweet potato cubes, using the method established by Jiang Shaotong et al. (2001);

[0053] 2. The activity of polyphenol oxidase in transgenic sweet potato tubers was 0.09, and the activity of polyphenol oxidase in non-transgenic sweet potato tubers was 0.89;

[0054] 3. According to the method (1984) invented by V.Sciancale and V.Zongne, the browning intensity of sweet potato was measured. The browning intensity of transgenic sweet potato tubers was 0.11, and the activity of polyphenol oxidase in non-transgenic sweet potato tubers was 0.58.

[0055] SEQ ID NO: 1

[0056] Southwest Normal University

[0057] A method of cultivating anti-browning sweet potato using genetic engineering technology

[0058] 1

[0059] 1

[0060] 569

[0061] ...

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Abstract

The invention provides a process for culturing brownness resistant sweet potatoes utilizing gene engineering technology, which relates to the achievement of yam polyphenol oxidase gene core fragments and the process of cultivating brown trans-gene yam by employing antisense RNA technology, and the method comprises cloning polyphenol oxidase gene core fragment from yam, constructing plant highly effective antisense expression carrier, and leading into agrolbacterium and genetically transforming the yam.

Description

technical field [0001] The invention belongs to the fields of molecular biology, enzymology, physiology, breeding and genetic engineering, and relates to a method for cultivating anti-browning sweet potatoes using genetic engineering technology, in particular to sweet potato polyphenol oxidase gene cDNA and its antisense chain sequence , and the specific procedures for expressing the gene and antisense gene and screening transformants in plants using the constructed high-efficiency expression vector and engineering strains. The present invention also provides the transpolyphenol oxidase and antisense polyphenol oxidase transgenic cells, hairy roots and their cultured offspring, regenerated plants, plant tissues or seeds obtained by genetic engineering. Background technique [0002] Polyphenol oxidase is a class widely distributed in plants, which can catalyze the hydroxylation of unit phenols, dihydric phenols, and polyphenols to biphenols and the dehydrogenation of hydroxyp...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N15/09C12N15/63C12N15/82A01H4/00C12N5/10
Inventor 廖志华陈敏杨春贤谌容付玉凡张启堂
Owner SOUTHWEST UNIV
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