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Clone of corn phosphatidylinositol transportprotein gene ZmSEC14p and application thereof

A technology of phosphatidylinositol and transporter, applied in the field of molecular biology and biology, to achieve the effect of improving cold resistance

Inactive Publication Date: 2015-08-05
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Despite these evidences that phosphatidylinositol transporters are induced by stress, there are no reports of using these biological stress-related genes to improve plant cold tolerance

Method used

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  • Clone of corn phosphatidylinositol transportprotein gene ZmSEC14p and application thereof
  • Clone of corn phosphatidylinositol transportprotein gene ZmSEC14p and application thereof
  • Clone of corn phosphatidylinositol transportprotein gene ZmSEC14p and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Cloning of maize phosphatidylinositol transporter gene ZmSEC14

[0049] 1. Extraction of RNA

[0050] Total RNA was extracted from maize leaves using RNAiso Reagent (Takara, Japan) kit.

[0051] (1) When the corn grows to the three-leaf stage, put it in an artificial climate box, treat it at low temperature (4°C) for 12 hours, take 3 leaves of the same part (the young part of the third leaf) and mix (about 40mg) Put it into a 1.5mL centrifuge tube, quickly add liquid nitrogen to freeze and grind it into powder.

[0052] (2) Quickly add 1mL RNAiso reagent (Takara) reagent to the tube, and let stand at room temperature for 5 minutes until the powder melts, flick the tube wall to spread the powder evenly in the reagent, let stand at room temperature for 10 minutes.

[0053] (3) 12,000×g, 4°C, centrifuge for 15 minutes.

[0054] (4) Aspirate the supernatant, add 0.2ml chloroform, shake vigorously, and let stand at room temperature for 5 minutes.

[0055] (5) ...

Embodiment 2

[0098] Example 2: Bioinformatics analysis of maize ZmSEC14 gene

[0099] The ZmSEC14p gene encodes a maize phosphatidylinositol transporter, its open reading frame has 888 bp nucleotides, encodes 295 amino acids, and has a protein size of 33.978 KD. Using the NCBI website to analyze the functional domains of the ZmSEC14 protein, it was found that the protein contained a conserved SEC14 domain (86-241aa) and a CRAL-TRIO-N domain (19-65aa). Using DNAMAN software to perform multiple comparisons of the SEC14p amino acid sequences of various plants (rice, barley, soybean, Arabidopsis, millet, sorghum, wheat, grape) (such as figure 1 shown), the results showed that these genes had high homology in the conserved SEC14 domain. Therefore, this gene belongs to one of the members of the SEC14 superfamily.

[0100] The NCBI database combined with the maizegdb database searched for homologous SEC14 amino acid sequences in different species, including: rice (NP_001051120.1), barley (MLOC_...

Embodiment 3

[0101] Example 3: Response of maize ZmSEC14p gene to abiotic stress

[0102] When the corn grows to the three-leaf and one-heart stage, it is treated with low temperature (4°C), Nacl (250mM), and ABA (100uM) for 0h, 2h, 4h, 6h, 12h, and 24h, respectively. RNA was extracted from maize leaves and roots for analysis of relative gene expression. ABI7500RealTime PCR System (Applied Biosystems) and 7500System software version 1.3.1 were used for Real-time RT-PCR amplification. RNA extraction and cDNA first-strand synthesis were the same as above. Afterwards, the cDNA was diluted 5 times, and Real-time PCR was performed according to the instructions of the kit SYBR Green RealMasterMix with ROX. The primers used are as follows:

[0103] YGSEC14F: 5'-AAAATCTGCACTTGGTCCTT-3'

[0104] YGSEC14R: 5'-GTCCATCCCGTGAAGTCTAT-3'

[0105] ActinF: 5'-CGATTGAGCATGGCATTGTCA-3'

[0106] ActinR:5'-CCCACTAGCGTACAACGAA-3'

[0107] GAPDHF; 5'-CCCTTCATCACCACGGACTAC-3'

[0108] GAPDHR; 5'-ACCTTCTTG...

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Abstract

The invention belongs to the clone of corn phosphatidylinositol transportprotein gene ZmSEC14p and an application thereof and belongs to the technical field of molecular biology and biotechnology. The nucleotide sequence of the cold-tolerant corn selfing line W9816 phosphatidylinositol transportprotein gene ZmSEC14p is shown in SEQ ID NO:1; the cold-tolerant corn selfing line W9816 phosphatidylinositol transportprotein is coded by the ZmSEC14p gene and the amino acid sequence is shown in SEQ ID NO:2; a plant expression vector contains the cold-tolerant corn selfing line W9816 phosphatidylinositol transportprotein gene ZmSEC14p; the plant expression vector is constructed and transformed into arabidopsis thaliana, and the life states of the transgenic Arabidopsis, such as the phenotype and the survival rate, are observed through low temperature and freeze stress; the experiment result shows that the cold-tolerant corn selfing line W9816 phosphatidylinositol transportprotein gene ZmSEC14p is capable of increasing the cold resistance of the arabidopsis.

Description

technical field [0001] The invention belongs to the field of molecular biology and biotechnology, and specifically relates to the cloning and application of a phosphatidylinositol transporter gene ZmSEC14p of a cold-tolerant corn inbred line W9816. Background technique [0002] Crops are often subject to various abiotic stresses, such as drought, salt, low temperature, etc., which seriously restrict the planting area of ​​crops and even lead to a decline in yield [1] . Low temperatures affect seed germination, seedling growth, and agricultural yields. In order to resist or adapt to adversity, the plant senses the changes in the external environment and transmits signals to the cells through a variety of channels, induces the expression of some response genes, produces some functional proteins, osmotic adjustment substances and transmission Transcription factors that signal and regulate gene expression. Many genes that respond to low temperature have been reported, such as...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/00
Inventor 原亚萍王晓宇单晓辉苏胜忠吴颖李世鹏刘宏魁韩俊友薛春梅王帅
Owner JILIN UNIV
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