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Function identification and application of delta-5 desaturase gene PinD5 of Phytophthora infestans in potato

A technology of Phytophthora infestans and desaturase, which is applied in the fields of molecular biology and biology, and can solve problems such as the inability to synthesize ultra-long-chain polyunsaturated fatty acids.

Inactive Publication Date: 2011-10-19
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Higher plants can synthesize a large amount of linoleic acid (LA, 18:2Δ 9，12 ) and linolenic acid (ALA, 18:3Δ 9，12，15 ), but cannot synthesize very long chain polyunsaturated fatty acids

Method used

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  • Function identification and application of delta-5 desaturase gene PinD5 of Phytophthora infestans in potato
  • Function identification and application of delta-5 desaturase gene PinD5 of Phytophthora infestans in potato
  • Function identification and application of delta-5 desaturase gene PinD5 of Phytophthora infestans in potato

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0130] Example 1: Acquisition of Phytophthora infestans Δ5 desaturase gene PIND5

[0131] 1. Cultivation of Phytophthora infestans potato: Inoculate Phytophthora infestans potato into fresh oat medium, cultivate in dark at 20 degrees.

[0132] 2. Extraction of total RNA: the mycelia grown for about 15 days were collected, and the total RNA was extracted by using the TRIZOL method (invitrogen).

[0133] 3. Take 2 micrograms of total RNA and use MMLV Reverse Transcriptase (Promega) to reverse transcribe into single-stranded cDNA.

[0134] 4. Using the obtained cDNA of Phytophthora infestans as a template, use primers 5'-ATGACCACGGATGACATCAC-3' and 5'-CTCGAGTTAGCCCATGTGGACGGT-3'; PCR system: ddH 2 O 35.5μl; 10xPCRbuffer (containing Mg 2+ ) 5 μl; dNTP (2.5 mM) 4 μl; each primer (5 μM) 2 μl; template 1 μl; Taq enzyme 0.5 μl. Reaction conditions: pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 30 seconds, annealing at 65°C for 30 seconds, extension at 72°C for 1 ...

Embodiment 2

[0137] Example 2: Phytophthora infestans Δ5 desaturase gene PinD5, the following sequence:

[0138] (1) Information of SEQ ID NO.1

[0139] (a) Sequential features

[0140] * Length: 1551 bp

[0141] *Type: nucleic acid

[0142] * Chain type: double chain

[0143] *Topology: Linear

[0144] (b) Molecular type: cDNA

[0145] (c) Assumption: No

[0146] (d) Antisense: no

[0147] (e) Original source: Phytophthora infestans

[0148] (f) Sequence description: SEQ IN NO.1

[0149] 1 ATGACCACGG ATGACATCAC ACTCCAATTC GCACTCACGG CCGGAAAGAC CATCGGAACC

[0150] 61 TCTGCTTCCC CTGCATTGTC ACCACTAATT GACGAAGTAG AAGCTGCCAT GGCCCCTATC

[0151] 121 GAGACCGAGA AGACCATCGT TAACGAGGGT CTTTACCAGC GCAAGACTGC TGCTGATGCA

[0152] 181 GCTACTAACA AGTCTGCCGC CACCTACACG TGGCAGGACG TGGCCAAGCA CAACACAGAC

[0153] 241 AACAGCGCTT GGGTCATCAT CCGCGGAATC GTATACGACG TAACTGAATG GGCGGATCGC

[0154] 301 CACCCTGGAG GCCGTGAACT CGTGCTGCTG CACTCTGGTC GCGAGTGCAC CGACACATTC

[0155] 361 GACTCGTACC ACCCATTCTC...

Embodiment 3

[0253] Embodiment 3: Construction of yeast expression vector pYES2-PinD5

[0254] 1. Use EcoRI and XhoI to excise the fragment from the pMD18-T vector containing the PinD5 fragment constructed in Example 1, and connect it to the yeast expression vector pYES2 digested with the same restriction endonuclease. The ligation product was transformed into Escherichia coli XL10 competent cells, and then cultured on LB solid plates containing ampicillin. The colonies were screened by PCR, identified and digested with plasmid DNA to obtain the yeast expression vector pYES2-PinD5 with PinD5.

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Abstract

The invention relates to function identification and an application of delta-5 desaturase gene PinD5 of Phytophthora infestans in a potato. The delta-5 desaturase gene PinD5 relating to fish oil synthesis is separated form the Phytophthora infestans in the potato by an RT-PCR (reverse transcription polymerase chain reaction) method, and the invention belongs to the field of molecular biology and biological technologies. TtThe new delta-5 desaturase gene is determined through yeast expression identification, and coded delta 5 desaturase which allows dohono-gamma-linolenic acid to be converted into arachidonic acid can be used for fish oil production in plants especially oil crops.

Description

(1) Technical field [0001] The invention relates to the functional identification and application of Δ5 desaturase gene PinD5 in the synthesis and metabolism pathway of ultra-long-chain polyunsaturated fatty acid of Phytophthora infestans, belonging to the fields of molecular biology and biotechnology. (2) Background technology [0002] Very long-chain polyunsaturated fatty acids, generally containing 20-22 carbons, 4-6 cis double bonds, such as arachidonic acid (AA, 20:4Δ 5，8，11，14 ), EPA (20:5Δ 8，14，14，17 ) and DHA (22:6Δ 4，7，10，13，16，19 ) play an important role in human nutrition and health. Regular supplementation of fatty acids such as EPA and DHA not only plays an important role in the development of fetal and infant cerebral cortex and neurons (Crawford, 2000), but also can reduce the incidence of cardiovascular and cerebrovascular diseases, hypertension and inflammation (Thies et al., 2003; Kinsella et al., 1990; Yamazaki et al., 1992). At present, such fatty aci...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N15/10C12N15/82A01H5/00C12R1/645
Inventor 李新征亓宝秀孙全喜
Owner SHANDONG AGRICULTURAL UNIVERSITY
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