Arabidopsis thaliana AtPP2CA2 gene and application thereof

A technology of Arabidopsis, gene, applied in the field of genetic engineering

Inactive Publication Date: 2011-07-27
HUNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the role of AtPP2CA2 gene in regulating plant grow...

Method used

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  • Arabidopsis thaliana AtPP2CA2 gene and application thereof
  • Arabidopsis thaliana AtPP2CA2 gene and application thereof
  • Arabidopsis thaliana AtPP2CA2 gene and application thereof

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Embodiment 1

[0022] Embodiment 1, construction of AtPP2CA2 gene GFP fusion expression vector

[0023]Total RNA was extracted from Arabidopsis thaliana seedlings using RNAeasy Mini Kit (Ambiogen Biotechnology Co., Ltd.) according to the instructions, reverse transcription was performed according to the Invitrogen M-MLV Reverse Transcriptase Instruction, and 2 μg of total RNA treated with DNaseI was used for cDNA Synthesis of the first strand, and the product was used as a template for RT-PCR. PCR amplification of AtPP2CA2 gene: Design a pair of specific primers according to the mRNA sequence of AtPP2CA2 predicted by TAIR (http: / / www.arabidopsis.org / ), and add attB1 and attB2 sites at the 5' ends of the upstream and downstream primers, respectively , using Gateway technology for cloning. The primers for the amplification of AtPP2CA2 full-length cDNA were as follows: the 5’-end primer was 5’-GGGGACAAGTTTGTACAAAAAGCAGGCTTCATGGCTGAGATTTGTTACGAGAA-3’; the 3’-end primer was 5’-GGGGACCACTTTGTACAA...

Embodiment 2、35

[0024] Example 2, 35S:GFP:AtPP2CA2 fusion expression vector bombardment by particle gun

[0025] Gold powder embedding of DNA follows the method of Biolistic PDS-1000 / He Particle Delivery System. Take 50 μL of gold powder suspension (60g L -1 ), add 50 μL of CaCl 2 (2.5mol· -1 ), 20 μL spermidine (0.1mol L -1 ), 3 μg of 35S:GFP:AtPP2CA2 vector plasmid were mixed thoroughly, centrifuged, washed with absolute ethanol, and resuspended in 50 μL of absolute ethanol. The Bio-RadPDS-1000 / He gene gun was used for bombardment, the rupture pressure was 1.3kPa, and onions (small pieces of about 1cm×1cm) were placed in 10g·L -1 On the agar medium, the distance to the cleavable membrane was 6 cm, and the culture was continued for 16-24 hours after transformation, and slices were made, and the green fluorescence in the cells was observed under a fluorescent microscope (OLYMPUS-BH2). Choose B (IF-490) excitation filter, and use PM-30 automatic photomicrograph to take pictures. The expe...

Embodiment 3

[0026] Embodiment 3, wild-type Arabidopsis thaliana is subjected to the induction expression situation of ABA and NaCl

[0027] 2-week-old Arabidopsis wild-type seedlings were treated with 100mM NaCl and 10μM ABA, and the materials were harvested after treatment at different times (0h, 1h, 2h, 4h), and total RNA was extracted for semi-quantitative RT-PCR analysis. The primers of PP2CA2 gene are: PP2CA2F (5'-CAGCGGGTGGTCGTGTTA-3'), PP2CA2R (5'-CGCAAGCCTCGTCAGCAA-3'). The primers for the control Actin-2 are as follows: Actin-2F: 5'-CACTGTGCCAATCTACGAGGGT-3'; Actin-2R: 5'-CACAAACGAGGGCTGGAACAAG-3'. 100mM NaCl and 10μM ABA induced the increase of PP2CA2 gene expression ( figure 2 ).

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Abstract

The invention discloses protein phosphatases 2C of arabidopsis thaliana and encoding gene and application thereof. The invention aims at providing the protein phosphatases 2C of the arabidopsis thaliana, the encoding gene thereof, and application thereof to controlling growth and development and stress resistance of plants. The AtPP2CA2 gene disclosed by the invention is found through screening from a cDNA database of arabidopsis thaliana processed by ABA (Abscisic Acid), salt and drought induction by using a gene chip method, the number of the AtPP2CA2 gene in GenBank is At5g59220, the length of cDNA of the gene is 1242bp, and the gene encodes 413 aa proteins. The gene disclosed by the invention and the encoding protein thereof have great theoretic and practical significance in research of inverse resistance mechanism of plants and improvement of inverse resistance such as drought endurance, salt tolerance and the like and relevant properties, and play an important role in the improvement of inverse-resistance gene engineering of plants, and have broad application prospect.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to the application of an Arabidopsis protein phosphatase 2C gene. Background technique [0002] Organisms need to make timely and accurate adjustments to the expression of their genes at different stages of development and when adapting to changing external environments. For example, plants are often affected by various environmental factors in the process of growth and development, among which there is a complex process of information perception, recognition and transmission, and corresponding physiological and biochemical responses, and an important cell biological event involved in this process is protein production. reversible phosphorylation. Studies (Kerk et al. 2002, Luan 2003) have shown that reversible phosphorylation of proteins plays an important role in various signal transduction pathways of life activities, and the cascade and transduction of signals are realized th...

Claims

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Application Information

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IPC IPC(8): C12N15/55A01H5/00
CPCY02A40/135
Inventor 郭新红刘选明王婕
Owner HUNAN UNIV
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