Helianthus tuberosus fructan-exohydrolases (Ht-FEH) gene and application thereof

A kind of fructan, hydrolase technology, applied in the field of genetic engineering, can solve problems such as unreported

Inactive Publication Date: 2012-09-05
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The nucleotide sequence or peptide information of Jerusalem artichoke 1-FEH has not been reported so far

Method used

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  • Helianthus tuberosus fructan-exohydrolases (Ht-FEH) gene and application thereof
  • Helianthus tuberosus fructan-exohydrolases (Ht-FEH) gene and application thereof
  • Helianthus tuberosus fructan-exohydrolases (Ht-FEH) gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Cloning of Example 1 Ht1-FEH Gene

[0043] 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.

[0044] 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 (TC4077) with a full length of 1792 bp. The EST sequence was compared with the existing 1-FEH gene CDS sequence and found that the EST sequence was similar to that of chicory. The CDS sequence similarity of Ci1-FEH I (AJ242538) is 78.3%, and there are start codon ATG and stop codon TGA. Therefo...

Embodiment 2

[0061] According to the cloned Ht 1-FEH CDS sequence, the semi-quantitative expression primer qFEH was designed with Primer Premier 5.0, and the expression pattern of Ht 1-FEH in Jerusalem artichoke was analyzed using the 18S ribosome-encoded gene as an internal reference. The PCR reaction system and procedure were the same as in Example 1, and the number of cycles was 25. The primer sequences are as follows:

[0062] qFEH-F:5'-TGCGTCAAAGTCATTCTA-3' (SEQ ID NO.5)

[0063] qFEH-R:5'-CCATAACTCCCACTCGTA-3' (SEQ ID NO.6)

[0064] 18S-F:5'-TACCGTCCTAGTCTCAACCA-3' (SEQ ID NO.7)

[0065] 18S-R:5'-AACATCTAAGGGCATCACAG-3' (SEQ ID NO.8)

[0066] The transcription level of Ht 1-FEH in different organs and tuber development and germination of Jerusalem artichoke was detected by semi-quantitative RT-PCR ( image 3 ). The expression of Ht 1-FEH was the highest in germinated tubers, and the expression of Ht 1-FEH increased with time after germination; Ht 1-FEH was expressed in mature le...

Embodiment 3

[0067] The construction of embodiment 3 expression vector

[0068] According to the sequence analysis results of the fructan exohydrolase gene Ht 1-FEH, the upstream primer removed the predicted N-terminal signal peptide sequence: MVKEMAGWVLSFCILLVVNGVGVHA, a total of 25 amino acids, and the upstream primer was designed from the 26th amino acid, plus Xho I Restriction site: C|TCGAG, plus Kex2 signal cleavage sequence: AAGAGA, plus protective base: CCG, use the yeast α factor leader peptide sequence in the vector as the signal peptide to construct a secretory expression vector to The recombinant fructan exohydrolase can be secreted outside the cells 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 restriction site: T|CTAGA , plus the protective ...

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Abstract

The invention belongs to the field of gene engineering and discloses a helianthus tuberosus fructan-exohydrolases (Ht-FEH) gene and application of the Ht-FEH gene. The gene Ht 1-FEH has a nucleotide sequence shown in the SEQ ID NO.1 and is used for coding Ht 1-FEH with an amino acid sequence shown in the SEQ ID NO.2. The gene Ht 1-FEH can be applied to production of fructose substances through hydrolysis of inulin or cultivation of transgenic salt-tolerant plants.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a Jerusalem artichoke fructan exohydrolase gene and application thereof. Background technique [0002] Plant fructans are mainly carbohydrates composed of multiple fructosyl groups connected by glycosidic bonds. Although fructans can be found in 15% of angiosperms, the content of fructans in plants is generally low, only in the tubers of Jerusalem artichoke (Helianthus tuberosus); chicory (Cichorium intybus), burdock (Arctium lappal) and chicken Roots of footgrass (Dactylis glomeratus); bulbs of lilies (Lilium brownii) and onions (Allium cepa); tissues of a few plants such as stems and leaves of ryegrass (Lolium perenne), barley (Hordeum vulgare), and wheat (Triticum aestivum) Relatively high levels in medium (Ritsema and Smeekens 2003a; Van Laere and Van Den Ende 2002). [0003] Sucrose is a precursor for fructan synthesis (Van Laere and Van Den Ende 2002). There are mainly tw...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N9/24C12N15/81C12N1/19C12P19/14A01H5/00C12R1/84
Inventor 梁明祥康健隆小华刘兆普
Owner NANJING AGRICULTURAL UNIVERSITY
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