Hexosaminidase and coding gene related to strawberry softening, preparation and application thereof

A technology of hexosaminidase encoding and hexosaminidase, which is applied in the field of hexosaminidase encoding gene and its preparation and application, can solve the problems such as unclear change rules, and achieve strong pertinence, high practical value and application prospect Effect

Active Publication Date: 2019-07-05
辽宁精细化工产业技术发展有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the enzyme and coding gene of β-hex in strawberry have not been reported yet, and how it changes in the strawberry softening process is still unclear, and there is no relevant report on its application in the screening of strawberry preservatives

Method used

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  • Hexosaminidase and coding gene related to strawberry softening, preparation and application thereof
  • Hexosaminidase and coding gene related to strawberry softening, preparation and application thereof
  • Hexosaminidase and coding gene related to strawberry softening, preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1 Strawberry hexosaminidase β-hex1 and β-hex2 full-length gene cloning

[0045] Perform multiple sequence alignment analysis on the hexosaminidase gene sequence in the NCBI database, and design RF cloning primers:

[0046] β-hex1-FR-F:5'GGGTATCTCTCGAGAAAAGAAAAGTCAAAAGCCCCCGTCACGTA-3'

[0047] β-hex1-FR-R:5'-TCAATGATGATGATGATGATGTTGAACGTAACACGAGC-3'

[0048] β-hex2-FR-F:5'-GGGTATCTCTCGAGAAAAGATCCATCAATGTGTGGCCC-3'

[0049] β-hex2-FR-R:5'-TCAATGATGATGATGATGATGAGCCACATTAACTGTG-3'

[0050] The mRNA of strawberry (Fragaria×ananassa qing xiang) was extracted, and the gene sequence encoding hexosaminidase β-hex1 and β-hex2 (not including the signal peptide gene) was amplified using the inverted cDNA as a template. The PCR reaction conditions were as follows: 94°C for 3 min, 1 cycle; 94°C for 30 s, 65°C for 30 s, 0.5°C for each cycle, 72°C for 3 min, 30 cycles; 72°C for 5 min, 1 cycle. PCR products were analyzed by agarose gel electrophoresis (see figure 1 ), an...

Embodiment 2

[0051] Example 2 Construction of recombinant plasmids pPICZαA-β-Hex1 and pPICZαA-β-Hex2

[0052] The purified PCR product was TA-ligated with the T vector pMD19-T, and the ligated product was transformed into E.coli Top10. After screening and colony PCR identification, positive clones were obtained, and the plasmid was extracted for sequencing. The correctly sequenced plasmid and the empty vector pPICZαA (purchased from Invitrogen) were double-digested with restriction endonucleases Xho I and Not I, respectively, and the digested products were purified by the gel recovery kit. Under the action of T4 DNA ligase, the The purified enzyme-cut product was ligated, and after the ligated product was transformed into Escherichia coli Top10 competent cells, it was spread on LLB (yeast powder 5 g / L, tryptone 10 g / L) containing 25 μg / ml bleomycin (Zeocin, Invitrogen Company). , sodium chloride 5g / L, agar powder 15g / L) on a solid medium, cultured at 37°C for 12h, and verified by colony PC...

Embodiment 3

[0053] Example 3 Electrotransformation of recombinant plasmids into Pichia pastoris X-33

[0054] The recombinant plasmids with correct sequencing were named pPICZαA-β-Hex1 and pPICZαA-β-Hex2, respectively, and the plasmids were extracted using a plasmid extraction kit. The extracted recombinant plasmid was linearized with the restriction endonuclease SacI. The purified linearized recombinant plasmids pPICZαA-β-Hex1 and pPICZαA-β-Hex2 were respectively electrotransformed into Pichia pastoris recipient strain X-33 and cultured. After a single colony appeared, they were picked for colony PCR verification. PCR amplification products were detected by 1 agarose electrophoresis.

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Abstract

The present invention discloses two hexosaminidase gene sequences derived from strawberries (Fragaria x ananassa qing xia) and a preparation method and an application thereof, and a technical method using genetic engineering. The hexosaminidase gene is cloned into a pichia pastoris expression vector to obtain a pichia pastoris recombinant strain heterologously expressing hexosaminidase, abd the hexosaminidase prepared by the heterologous expression of the strain can uses p-nitrophenyl-N-acetyl-beta-D-amino glucosamine or p-nitrophenyl-N-acetyl-beta-D-aminogalactosamine as a substrate for reaction. The present invention also provides the application of the hexosaminidase in controlling postharvest fruit softening and preservative activity screening, and belongs to the field of fruit preservation.

Description

technical field [0001] The invention belongs to the field of fruit preservation, and specifically relates to two hexosaminidase coding genes related to strawberry softening, as well as their preparation and application. The invention provides two recombinant plasmids of strawberry hexosaminidase and recombinant genetic engineering strains, and also provides the application of strawberry hexosaminidase in screening strawberry preservatives and degrading sugar chains. Background technique [0002] Strawberry is a perennial herbaceous plant belonging to the family Rosaceae. It is a small berry with high economic value. The fruit is soft and juicy. Because it is rich in various essential nutrients, it is known as the "Queen of Fruits" in my country. However, strawberries have high water content, thin peel, and poor storage resistance. They are easily rotted and deteriorated due to mechanical damage and germ infection after harvesting. They can only be stored at room temperature ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56C12P19/26A23B7/153
CPCA23B7/153C12N9/2402C12P19/26C12Y302/01052
Inventor 尹恒王文霞何艳秋刘同梅
Owner 辽宁精细化工产业技术发展有限公司
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