Zinc finger protein gene PhZFP1 of petunia hybrida and application of zinc finger protein gene PhZFP1 in improving cold resistance performance of plants

A petunia, cold resistance technology, applied in the field of plant genetic engineering, can solve the problem of ZAT10/STZ expression reduction and achieve the effect of stable resistance

Active Publication Date: 2019-05-21
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Reduced expression of CBF3 leads to reduced expres...

Method used

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  • Zinc finger protein gene PhZFP1 of petunia hybrida and application of zinc finger protein gene PhZFP1 in improving cold resistance performance of plants
  • Zinc finger protein gene PhZFP1 of petunia hybrida and application of zinc finger protein gene PhZFP1 in improving cold resistance performance of plants
  • Zinc finger protein gene PhZFP1 of petunia hybrida and application of zinc finger protein gene PhZFP1 in improving cold resistance performance of plants

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Experimental program
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Effect test

Embodiment 1

[0031] Example 1 Isolation and cloning of PhZFP1 gene

[0032] The research team evaluated the cold resistance of the preserved petunia inbred lines in the early stage, and screened out the more cold-resistant line "H" (Li B, Ning L, Zhang J, Bao M, Zhang W. Transcriptional profiling of Petuniaseedlings reveals candidate regulators of the cold stress response. Frontiersin Plant Science. 2015; 6: 118.), and the candidate gene PhZFP1 in the cold response pathway was preliminarily screened using the expression profile chip of "H" under low temperature stress. Using an EST sequence obtained from the expression profile microarray data as an information probe, the petunia database was compared with the genome sequence and transcriptome sequence respectively, and the CDS sequence of the zinc finger protein gene PhZFP1 was obtained by analysis, and it was found that the gene was not Containing introns, use PrimerPremier 5 software to design a pair of outer primers containing part of t...

Embodiment 2

[0049] Example 2 PhZFP1 tissue expression profile and induced expression profile analysis

[0050] According to the gene sequence obtained in the petunia database and the primer requirements for real-time quantitative PCR, a pair of specific primers were designed with PrimerPremier 5 software, forward primer: 5'-CCTCCACCTCTGCCACCACTT-3', reverse primer: 5'-CACCGTTGCCGCCATCATAA-3 '. RT-PCR test method was used to analyze the tissue expression profile and induced expression profile of the candidate gene PhZFP1.

[0051] The extraction of total RNA and the reverse transcription reaction of total RNA were carried out using the EASYspin Plant RNA Rapid Extraction Kit produced by Adelaide Company and the PrimeScript produced by TaKaRa Company respectively. TM RTReagent Kit with gDNA Eraser Reverse Transcription Kit, the specific steps follow the instructions. The real-time quantitative PCR reaction was carried out in the ABI 7500fast fluorescence detection system, and the operati...

Embodiment 3

[0054] Example 3 Construction of PhZFP1 overexpression vector

[0055] After connecting PhZFP1 to the pMD 18-T vector, the sequenced correct plasmid was digested by Sal1 and BamH1, and connected to the plant expression vector pCambia2300s (preserved by our laboratory, see image 3 A). Use the 35S primer (5'-ACGCACAATCCCACTATCCTTC-3') and the outer downstream primer of the target gene: 5'-GCCTTTATCTTCATCAAGCCCTACA-3' to perform colony PCR detection, pick the positive bacteria and shake the plasmid to extract the plasmid, and obtain the successful construction after enzyme digestion verification p2300-PhZFP1 vector (see image 3 B), and mix the bacterial solution with 50% glycerol at a volume ratio of 7:3, and store at -80°C for later use. Then the recombinant plasmid was electroporated into Agrobacterium strain EHA105 (preserved by our laboratory).

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Abstract

The invention discloses a zinc finger protein gene PhZFP1 of petunia hybrida and application of the zinc finger protein gene PhZFP1 in improving cold resistance performance of plants. The gene PhZFP1is coned from cold-resistant petunia hybrida 'H', the nucleotide sequence of the gene PhZFP1 is shown in SEQ ID. 1, and an agrobacterium-mediated genetic transformation method is utilized for transforming the petunia hybrida to obtain a transgenic plant. Through measurement of conductivity, low-temperature treatment survival rate and the like and NBT staining, it is discovered that the cold resistance performance of a PhZFP1 overexpression transgenic plant line is significantly improved, obstacles of a traditional seed culturing means are broken through, and an important gene resource is provided for cold resistance gene engineering of plants.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and specifically relates to a zinc finger protein gene PhZFP1 containing two typical C2H2 zinc finger domains isolated and cloned from Petunia hybrida, and also relates to the improvement of PhZFP1 gene Application in plant cold resistance. Background technique [0002] Low temperature is one of the main environmental factors limiting plant growth, development, yield and geographical distribution, and it is also a natural disaster that crops often encounter during growth and development. During the long evolutionary process, plants have formed a complex and effective defense mechanism to adapt to low temperature, mainly including the perception of external low temperature signals, signal transduction and transcriptional regulation (Hannah, et al., 2005). After low temperature is sensed by the plant cell membrane, the low temperature signal is mainly through Ca 2+ continue to pass downst...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/82
Inventor 张蔚张慧琳包满珠宁露云李蓓
Owner HUAZHONG AGRI UNIV
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