Cloning of poncirustrifoliata mitogen-activated protein kinase (PtrMAPK) and application of PtrMAPK to improvement of drought resistance of plant

A gene and plant technology, applied in plant products, genetic engineering, plant genetic improvement, etc., can solve problems such as no drought resistance, protein kinase function to be further verified, no gene cloning and functional verification reports, etc.

Inactive Publication Date: 2012-07-04
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] It must be pointed out that although MAPKs have been cloned from different plants, the functions of most protein kinases under drought stress still need to be further verified
On the other hand, it is worth noting that most of the information about MAPK comes from Arabidopsis, rice, tobacco, and tomato, but there is very little information about MAPK in fruit trees.
Studies have shown that although the MAPK signaling pathway has a certain degree of conservation in the evolution of eukaryotes, the composition of this cascade and the functions of specific components are somewhat different (Morris, 2001; et al., 2010). Hovenia trifoliate is a widely used rootstock in the citrus industry, but it is susceptible to drought, which leads to its use in areas where water resources are limited and droughts occur periodically
Although many studies have shown that the MAPK gene has been cloned in some plants, there is no report on the cloning and functional verification of the gene in Citrus trifoliate, let alone the literature report on the application of the MAPK gene in Citrus trifoliate to transform plants to improve drought resistance

Method used

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  • Cloning of poncirustrifoliata mitogen-activated protein kinase (PtrMAPK) and application of PtrMAPK to improvement of drought resistance of plant
  • Cloning of poncirustrifoliata mitogen-activated protein kinase (PtrMAPK) and application of PtrMAPK to improvement of drought resistance of plant
  • Cloning of poncirustrifoliata mitogen-activated protein kinase (PtrMAPK) and application of PtrMAPK to improvement of drought resistance of plant

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1, PtrMAPK gene isolation cloning and expression analysis

[0028] Search the citrus EST database (HarvEST: Citrus ver.0.51) with MAPK as the keyword, and get 7 similar sequences. These are composed of DNAstar (public software) to form a unigene, and the sequence analysis software (ORF Finder) finds that the gene MAPK lacks the 5-end For the coding sequence, a complete ORF sequence was amplified using the 5'RACE kit (Clontech, PaloAlto, CA, USA), and the primer GSP1 (GSP, 5'-CCACACATCTATTGCAGCAGTGTAGTCAG-3') was designed with Primer Premier 5.0 for the sequence, and RT -PCR method to amplify the 5-terminal sequence. The amplified 5-terminal sequence and the existing MAPK unigene were overlapped into a sequence using the software DNAstar. RNA was extracted and reverse-transcribed from leaves of Hovenia trifoliata under dehydration at 25°C, and the obtained first-strand cDNA was used to amplify the full-length PtrMAPK gene. RNA was extracted using a Trizol ki...

Embodiment 2

[0030] Example 2, PtrMAPK gene subcellular localization

[0031] In this example, onion epidermis was used to study the subcellular localization of PtrMAPK gene. The entire ORF of the PtrMAPK gene was amplified by RT-PCR, and two restriction sites of NcoI and SpeI were added at both ends of the amplification primer. First, the amplified product is loaded on the pMD18-T vector to obtain a pMD18-T N / S -PtrMAPK recombinant vector. Simultaneously use NcoI and SpeI to cut PMD18-T N / S - PtrMAPK and pCAMBIA1302 (see image 3 ), the product was recovered and ligated to construct a recombinant vector, thereby obtaining the pCAMBIA1302-PtrMAPK-GFP recombinant vector. After confirming that the sequence is correct, heat shock the pCAMBIA1302-PtrMAPK-GFP recombinant vector and the control vector (pCAMBIA1302) (reference: Sam Brook, translated by Huang Peitang, "Molecular Cloning Experiment Manual" third edition, Science Press, 2002) Transformed into Agrobacterium EHA105 respectively. ...

Embodiment 3

[0032] Embodiment 3, plant transformation vector construction

[0033] According to the multiple cloning site of the pMV vector (in the plant binary transformation vector pBI121 with GUS gene excised, donated by Professor Ye Zhibiao, School of Horticulture and Forestry, Huazhong Agricultural University) and the sequence of the coding region of the PtrMAPK gene, primers were designed according to the general principles with Primer Premier 5.0 The software designs the upstream and downstream PCR primers for amplifying the entire coding region of the PtrMAPK gene (the pair of primers is the pair of primers for amplifying the cDNA sequence of the PtrMAPK gene of the present invention). Its sequence is as follows:

[0034] Forward primer: 5’-CG GTC GAC GGAGAGAGAGTAAAATGGCTGACG-3'

[0035] Reverse primer: 5’-AA GGTACC GAAAGATTGTCGGCCACCTGCAG-3'

[0036] The clone of PtrMAPK gene was used as template for PCR amplification. The annealing temperature for PCR amplification was 60°...

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Abstract

The invention belongs to the field of plant genetic engineering, and particularly relates to a poncirustrifoliata mitogen-activated protein kinase (PtrMAPK) gene which is obtained from poncirustrifoliata by separation and cloning. The nucleotide sequence of the gene is shown as a sequence table SEQ ID NO:1; the corresponding amino acid sequence of the gene is shown as a sequence table SEQ ID NO:2; and the gene comprises 1128bp of open reading frame, and encodes 375 amino acids, an isoelectric point is 5.51, and predicted molecular weight is 43kD. The PtrMAPK gene is imported into tobacco for functional verification, and the drought resistance capacity of the obtained transgenic tobacco plant can be improved obviously. New gene resources are provided for anti-abiotic stress molecular design and breeding, new genetic resources are provided for implementing green and environment-friendly agriculture, and the development and utilization of the genetic resources contribute to reducing agriculture production cost and achieving environment-friendliness.

Description

technical field [0001] This patent belongs to the field of plant genetic engineering. Specifically, it involves isolating and cloning the mitogenic protein kinase gene PtrMAPK from Poncirus trifoliata, and then introducing the gene into the model plant Nicotiana tabacum for functional verification, and the obtained transgenic plants have significantly improved drought resistance. Background technique [0002] During the growth and development of plants, plants are often affected by adverse external environments, among which drought has been proven to be the most destructive abiotic stress, which seriously affects the growth, development and yield of plants. However, when plants are stimulated by an unfavorable external environment, they do not accept it passively. In fact, during the long process of evolution, plants have established a set of mechanisms to respond to adversity stress, which involves the plant's perception of adversity signals, the transmission of adversity ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/12C12N15/11A01H5/00
Inventor 刘继红黄小三
Owner HUAZHONG AGRI UNIV
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