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Protein coded sequence of protein kinase 1 activated by Brassica napus L. cell mitogen

A cabbage rape, cell division technology, applied in the field of protein coding sequences, can solve the problems of unclear salt tolerance, drought resistance and insect resistance, etc.

Inactive Publication Date: 2005-03-16
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In the analysis of prior art documents, although the journal "The Plant Journal" published the article "Activation of AtMEK1, an Arabidopsis mitogen-activated protein kinase, in vitro and in vivo: analysis in 2002, 29: 637-647 of active mutants expressed in E. coli and generation of the active form in stress response in seedlings (Activation of the Arabidopsis mitogen-activated protein kinase kinase AtMEK1 in vivo and in vitro: Mutant expression analysis of activity in Escherichia coli and seedling stress response in The active form of the gene MKK1 has been fully described under different abiotic stress treatments, but the effect of the gene on other environmental factors such as salt tolerance, drought resistance and insect resistance is still unclear

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0121] Cloning of BnMKK1 Gene in Brassica napus

[0122] 1. Tissue separation (isolation)

[0123] Brassica napus (the variety is "Huyou 15") is purchased from the market, and the Brassica napus is placed at 28°C to germinate for 24 hours, and then sowed in the greenhouse. When the Brassica napus leaves are 3-5 pieces, it is ready to extract DNA or RNA.

[0124] 2. RNA isolation (RNA isolation)

[0125] Take part of the tissue, grind it with a mortar, add it to a 1.5mL EP tube filled with lysate, shake it fully, and then transfer it into a glass homogenizer. After homogenization, transfer to 1.5mL EP tube, and extract total RNA (TRIzol Reagents, GIBCO BRL, USA). The quality of total RNA was identified by formaldehyde denaturing gel electrophoresis, and then the RNA content was determined on a spectrophotometer.

[0126] 3. Cloning of Full-length cDNA

[0127] According to the conserved amino acid sequence of the Arabidopsis AtMKK1 gene, using the principle of homologous g...

Embodiment 2

[0139] Sequence Information and Homology Analysis of BnMKK1 Gene in Brassica napus

[0140] The novel Brassica napus BnMKK11 full-length cDNA of the present invention has a length of 1464bp, and the detailed sequence is shown in SEQID NO.3, wherein the open reading frame is located at 39-1007 nucleotides (1114 nucleotides). The amino acid sequence of Brassica napus BnMKK1 was deduced according to the full-length cDNA, with a total of 370 amino acid residues, a molecular weight of 35708.39 Daltons, and an isoelectric point (pI) of 5.59. See SEQ ID NO.3 for the detailed sequence.

[0141] The full-length cDNA sequence of Brassica napus BnMKK1 and its encoded protein were analyzed by BLAST program in the Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDStranslations+PDB+SwissProt+Superdate+PIR databases for nucleotide and protein Homology search, it was found that it has 90% identity with the Arabidopsis gene AtMKK1 at the nucleotide level (attached table 2); at t...

Embodiment 3

[0143] Eukaryotic expression of Brassica napus gene BnMKK1 protein or polypeptide in Arabidopsis and stress resistance identification of transgenic plants

[0144] Construction of Expression Vector Containing Target Gene (Brassica napus Gene BnMKK1)

[0145] According to the full-length sequence (SEQ ID NO.3) of Brassica napus gene BnMKK1, design primers that amplify the complete coding reading frame, and introduce restriction endonuclease sites on the upstream and downstream primers respectively (this can be selected depending on the vector) in order to construct the expression vector. Using the amplified product obtained in Example 1 as a template, after PCR amplification, the Brassica napus gene BnMKK1 gene cDNA was cloned into an intermediate vector (such as pBluescript), and further cloned into a binary expression vector (such as pBI121 and improved pCAMBIA2300 ), under the premise of ensuring the correct reading frame, the identified expression vector was transformed in...

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Abstract

The invention relates to cabbage type rape mitogen-activated protein kinase 1 protein coding sequence in gene engineering technology field. The separating DNA molecule includes the polypepetide nuleotide sequence possessing cabbage type rape BnMKK1 protein activity, the sequence being 70% homolog with 39-1007 nucleotides of SEQ ID NO.3, or the said nucleotide sequence can hybridized with 39-1007 nucleotides of SEQ ID NO.3. The invention has good resistance to environment stress of draught, plant diseases and insect pests, can apparently reduce the biomass loss of crops in draught, plant diseases and insect pests conditions.

Description

technical field [0001] The present invention relates to a protein coding sequence used in the technical field of genetic engineering, in particular to a protein coding sequence of kinase 1 (BnMKK1) protein kinase activated by mitogen in Brassica napus. Background technique [0002] During the growth of crops, they will encounter various natural disasters, such as drought, waterlogging, pests and diseases, etc., resulting in reduced production. An important breeding method at present is to genetically engineer crops with various stress-resistant genes to obtain new varieties of stress-resistant crops. In order to resist the stress of the adverse external environment, plants have developed a complex signal transmission system to help them overcome the current predicament in the long-term natural evolution, among which the MAP kinase (Mitogen-actived protein kinase) signal transmission system plays a role in the stress resistance process. played an extremely important role. M...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N9/10C12N15/29C12N15/54C12N15/63C12N15/79
Inventor 余舜武张利达唐东芹左开井唐克轩
Owner SHANGHAI JIAO TONG UNIV