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Corn raffinose synthesis key enzyme gene and application thereof

A key enzyme gene, raffinose technology, applied in application, genetic engineering, glycosyltransferase, etc., to achieve the effect of improving drought resistance

Inactive Publication Date: 2019-08-13
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above reports are limited to the overexpression of the key enzyme in the first step of the raffinose synthesis pathway, and there is no report on the regulation of plant drought resistance by the key enzyme in the second step of RFOs synthesis, raffinose synthase (RAFS)

Method used

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  • Corn raffinose synthesis key enzyme gene and application thereof
  • Corn raffinose synthesis key enzyme gene and application thereof
  • Corn raffinose synthesis key enzyme gene and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0015] Example 1. The present invention cloned the coding region of the maize ZmRAFS gene by PCR method, and constructed the gene plant expression vector.

[0016] Dehydration treatment was performed on hydroponic three-leaf stage corn seedlings. RNA was extracted from leaves dehydrated for 2 hours and reverse transcribed into cDNA.

[0017] Using cDNA as a template, the coding region of ZmRAFS was amplified by PCR with the upstream primer 5'-CGCGGATCCTGGCTCCCAACCTCAGCAAGAAG-3' and the downstream primer 5'-TGCTCTAGAGGTAGACGTACTGGACGCGACACAG-3'. The amplification program was: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 60°C annealing for 30 s, 72°C extension for 1 min 20 s, 35 cycles; 72°C final extension for 10 min. The amplified product was gel-cut and recovered, and the fragment was digested with BamHI and XbaI, and then combined with the pC sGFPBT vector (for stable transformation of Arabidopsis, see figure 1 )connect. The ZmRAFS ORF fragment on the vect...

Embodiment 2

[0019] Embodiment 2, the molecular identification of transgenic strain

[0020] The mRNA expression level of transformed plants was identified by RT-PCR. The expression level of ZMRAFS protein in transformed plants was identified by Western blot.

[0021] The RT-PCR amplification program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 s, 28 cycles; final extension at 72°C for 8 min.

[0022] For Western blot steps, refer to the following prior art.

[0023] Gu,L.,et al.,ZmGOLS2,a target of transcription factor ZmDREB2A,oferssimilar protection against biotic stress as ZmDREB2A.Plant Mol Biol,2016.90(1-2):p.157-70.

[0024] The ZmRAFS primary antibody was prepared from rabbits immunized in the research group, and the dilution factor was 1:5000; the secondary antibody (goat anti-rabbit) was purchased from Kangwei Company, and the dilution factor was 1:10000.

Embodiment 3

[0025] Example 3. Drought resistance identification of maize mutant lines and Arabidopsis transgenic lines.

[0026] Drought resistance test of maize under drought stress: the zmrafs-1 mutant and co-segregated NS plants were planted in the greenhouse, and one NS and one zmrafs-1 material were planted in the same pot. The soil is mixed with the surface soil of the field and the substrate at a ratio of 1:1, and part of the soil is dried and weighed to calculate the soil moisture content. The drought treatment was started 55 days after sowing, the control group was watered normally, the water supply was stopped in the treatment group, and the soil water loss rate was recorded every day. At the same time, the phenotype changes of the leaves of the plants were observed and recorded by taking pictures.

[0027] Drought resistance test of transgenic Arabidopsis seedlings: Weigh the same weight of nutrient soil in a four-hole small pot, then absorb water from the bottom to make the so...

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Abstract

The invention discloses a corn raffinose synthesis key enzyme gene and application thereof. The nucleotide sequence of the corn raffinose synthesis key enzyme gene is shown in SEQ ID NO:1, and the protein sequence is shown in SEQ ID NO:2. The invention further discloses application of the corn raffinose synthesis key enzyme gene in the process of improving the plant drought resistance capacity. Accordingly, a corn ZmRAFS gene is overexpressd in plants, distribution of sugar components in an RFOs metabolic pathway is changed by decomposing galactinol in plant leaves, and the plant drought resistance is improved.

Description

technical field [0001] The invention belongs to the technical field of biological genetic engineering, and in particular relates to a corn raffinose synthesis key enzyme gene ZmRAFS (raffinose synthase / GRMZM2G150906) and its application. Background technique [0002] Raffinose family oligosaccharides (RFOs) are a kind of galactoside series oligosaccharides unique to plants, galactinol synthase (Galactinol Synthase; GOLS) and raffinose synthase (Raffinose Synthase; RAFS) is the key enzyme responsible for raffinose synthesis. Zhuo reported in 2013 that the overexpression of the alfalfa MfGolS1 gene in tobacco increased the content of galactinitol and raffinose in transgenic tobacco and improved cold resistance, drought resistance and salt resistance. Sun reported in 2013 that expressing the TsGolS2 gene in Arabidopsis improved the salt tolerance of transgenic plants. Gu reported in 2016 that overexpression of ZmGOLS2 in Arabidopsis improved plant drought tolerance. The abov...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/10C12N15/82A01H5/00A01H6/20
CPCC12N9/1051C12N15/8273C12N15/8245C12Y204/01082
Inventor 赵天永李涛张玉民
Owner NORTHWEST A & F UNIV
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