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Petunia zinc finger protein gene phzfp1 and its application in improving plant cold resistance

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

Active Publication Date: 2021-01-29
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Reduced expression of CBF3 leads to reduced expression of ZAT10 / STZ, another C2H2-type zinc finger protein

Method used

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  • Petunia zinc finger protein gene phzfp1 and its application in improving plant cold resistance
  • Petunia zinc finger protein gene phzfp1 and its application in improving plant cold resistance
  • Petunia zinc finger protein gene phzfp1 and its application in improving plant cold resistance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Isolation and cloning of PhZFP1 gene

[0032] The research group 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 preliminarily screened the candidate gene PhZFP1 in the cold response pathway by using the expression profile chip of "H" under low temperature stress. Using an EST sequence obtained from the expression profile chip data as an informative probe, it was compared with the genome sequence and transcriptome sequence in the petunia database, and the CDS sequence of the zinc finger protein gene PhZFP1 was obtained by analysis. Contain introns, use PrimerPremier 5 software to design a pair of outer primers containing part of the UTR region, the primers were synthesized by Shanghai S...

Embodiment 2

[0049] Example 2 Analysis of PhZFP1 tissue expression profile and induced expression profile

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

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

Embodiment 3

[0054] Example 3 Construction of PhZFP1 overexpression vector

[0055] After connecting PhZFP1 to pMD 18-T vector, the correct plasmid was sequenced, digested with Sal1 and BamH1, and connected to the plant expression vector pCambia2300s (preserved by our laboratory, see image 3 A). The 35S primer (5'-ACGCACAATCCCACTATCCTTC-3') was paired with the outer downstream primer of the target gene: 5'-GCCTTTATCTTCATCAAGCCCTACA-3' for colony PCR detection, and the positive bacteria were picked to extract the plasmid. p2300-PhZFP1 vector (see image 3 B), and the bacterial liquid was mixed with 50% glycerol in a volume ratio of 7:3, and stored at -80°C for later use. The recombinant plasmid was then electroporated into Agrobacterium strain EHA105 (kept by our laboratory).

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Abstract

The invention discloses a petunia zinc finger protein gene PhZFP1 and its application in improving plant cold resistance. Gene cloned from cold-resistant petunia "H" PhZFP1 , whose nucleotide sequence is shown in SEQ ID.1, using the Agrobacterium-mediated genetic transformation method to transform petunia, the transgenic plants obtained, by measuring the electrical conductivity, low temperature treatment survival rate, etc., and NBT staining, it is found that PhZFP1 The cold resistance of super-table transgenic lines has been significantly improved, breaking through the barriers of traditional breeding methods, and providing important genetic resources for plant cold resistance genetic engineering.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, in particular to a zinc finger protein gene PhZFP1 which is isolated and cloned from Petunia (Petunia hybrida) and contains two typical C2H2 type zinc finger domains. Applications in plant cold resistance. Background technique [0002] Low temperature is one of the main environmental factors that limit plant growth, development, yield and geographical distribution, and it is also a natural disaster that crops often encounter in the process of growth and development. During the long evolutionary process, plants have formed a complex and effective defense mechanism to adapt to low temperature, 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 mainly passes through Ca 2+ Continue downstream, causing cytoplasmic Ca 2+ ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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