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Helianthus tuberosus fructan 1-exo-hydrolase gene Ht1-FEH II and application thereof

A technology of fructan and hydrolase, which is applied in the field of genetic engineering and can solve the problems of no public reports of amino acid or nucleotide sequences

Inactive Publication Date: 2015-01-21
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although as early as 1964, Edelman and Jefford found at least two hydrolases (A and B) that hydrolyze linear fructans in Jerusalem artichoke tubers (Edelman and Jefford 1964), Stefan P.Marx also passed preliminary SDS-PAGE has found a protein component of hydrolyzed fructan in Jerusalem artichoke, but there is no public report of the amino acid or nucleotide sequence of the protein

Method used

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  • Helianthus tuberosus fructan 1-exo-hydrolase gene Ht1-FEH II and application thereof
  • Helianthus tuberosus fructan 1-exo-hydrolase gene Ht1-FEH II and application thereof
  • Helianthus tuberosus fructan 1-exo-hydrolase gene Ht1-FEH II and application thereof

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Experimental program
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Embodiment 1

[0031] The cloning of embodiment 1Ht1-FEH II gene

[0032] The young leaves of the Jerusalem artichoke variety "Nanyu No. 1" at the 4th leaf stage were taken, and the total plant RNA was extracted according to the instructions of the OMEGA plant RNA extraction kit, and the cDNA template was obtained by two-step reverse transcription with the TaKaRa reverse transcription kit.

[0033] Blast the existing 1-FEH gene coding sequence (coding sequence, CDS) sequence of other species into Jerusalem artichoke (NCBI) and sunflower EST database (http: / / compbio.dfci.harvard.edu / tgi / plant.html), The EST sequence with high similarity was screened and spliced ​​with DNAstar software to obtain an EST sequence with a full length of 2061bp. The EST sequence was compared with the existing 1-FEH gene CDS sequence and found that the EST sequence was similar to the Ci1-FEH of chicory. The CDS sequence similarity of IIa (AJ295033) is 80.3%, and that of Ci1-FEH IIb (AJ295034) is 79.6%, and there are...

Embodiment 2

[0048] Example 2 Construction of pPICZαC-picFEH II expression vector

[0049] According to the sequence analysis results of the fructan exohydrolase gene Ht1-FEH II, the upstream primer removed the predicted N-terminal signal peptide sequence: MNQLLSSFLTLCFLVLVFDTPTSNA, a total of 25 amino acids, starting from the 26th amino acid to design the upstream primer, and adding Xho I Restriction site: C|TCGAG, plus Kex2 signal cleavage sequence: AAAAGA, plus protective base: CCG, using the yeast α factor leader peptide sequence that comes with the vector as the signal peptide, construct a secreted expression vector, so that Recombinant fructan exohydrolase can be secreted into the extracellular of Pichia pastoris. The downstream primer removes the stop codon of the sequence itself, and adds a fusion 6×His tag after the expression frame to make the recombinant protein a fusion protein, which is convenient for purification and detection after protein expression, plus an Xba I restricti...

Embodiment 3

[0057] Embodiment 3 Expression vector transforms Pichia pastoris

[0058] (1) Linearization of plasmids

[0059] The restriction endonuclease SacI was used to linearize the vector pPICZαC-picFEH II, so that the linearized plasmid DNA could be integrated with the host genome DNA through homologous recombination.

[0060] The digestion system is as follows: 10×NEB Buffer 415 μL, DNA≤6 μg 50 μL, 100×BSA 1.5 μL, Sac I 3 μL, ddH 2 O 80.5 μL, total volume 150 μL.

[0061] Enzyme digestion at 37°C overnight, 1% agarose gel electrophoresis to detect the linearization effect, and use a nucleic acid recovery kit (PCR clean Kit) to recover the linearized product.

[0062] (2) Competent cells of Pichia pastoris X-33 (INVITROGEN company) were prepared.

[0063] (3) Electrotransform Pichia pastoris X-33 with the linearized product prepared in step (1), and obtain positive transformants through YPDS+ZeocinTM (100 μg / mL) resistance plate screening.

[0064] ⑷ Detection of positive clones ...

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Abstract

The invention discloses a helianthus tuberosus fructan 1-exo-hydrolase gene Ht1-FEH II and an application thereof. The helianthus tuberosus fructan 1-exo-hydrolase gene Ht1-FEH II has a nucleotide sequence as shown in SEQ ID NO. 1. A protein encoded by the gene has an amino acid sequence as shown in SEQ ID NO. 2. The helianthus tuberosus fructan 1-exo-hydrolase gene Ht1-FEH II can be applied to producing fructan type substances from helianthus tuberosus hydrolysis inulin and can also be applied in improvement of drought resistance of plants.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to Jerusalem artichoke fructan 1-exohydrolase gene Ht1-FEH II and its application. Background technique [0002] Plant fructans are polymers linked by sucrose to one or more fructosyl groups, mainly distributed in Gramineae (such as wheat (Triticum aestivum) and barley (Hordeum vulgare)), Liliaceae (onion (Allium cepa) and garlic (Allium sativum) and Asteraceae (Cichorium intybus and Helianthus tuberosus) (Livingston III et al. 2009; Vijn and Smeekens 1999). About 15% of angiosperms have fructan as their major carbohydrate (Hendry 1993). Fructans in nature can be divided into five categories: linear inulin-type fructans (Inulin), Timothy-type fructans (Levan), mixed-type fructans (Branched), and inulin-type fructans. series (Inulin neoseries) and Timothy grass sugar-type fructan newborn series (Levan neoseries). Linear fructans are composed of fructosyl groups connected only by β(...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/24C12N15/81C12N1/19C12P19/14C12N15/82A01H5/00C12R1/84
Inventor 梁明祥许欢欢
Owner NANJING AGRICULTURAL UNIVERSITY
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