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Cloning of apple stress-resistant related gene MdSIMYB2 and application of cloning of apple stress-resistant related gene MdSIMYB2

An apple, gene technology, applied in the field of molecular biology and biology, can solve the problem of long cycle of resistant varieties

Active Publication Date: 2014-11-12
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, apple is a perennial woody plant, and it takes a long time to select resistant varieties through conventional breeding.

Method used

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  • Cloning of apple stress-resistant related gene MdSIMYB2 and application of cloning of apple stress-resistant related gene MdSIMYB2
  • Cloning of apple stress-resistant related gene MdSIMYB2 and application of cloning of apple stress-resistant related gene MdSIMYB2
  • Cloning of apple stress-resistant related gene MdSIMYB2 and application of cloning of apple stress-resistant related gene MdSIMYB2

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0126] Example 1: Cloning of apple MdSIMYB2 gene.

[0127] 1. RNA extraction and reverse transcription:

[0128] The leaves of tissue-cultured Gala apple seedlings treated with 200mM NaCl salt for 24h were extracted and their RNA was reverse transcribed:

[0129] 1) Extract total RNA using the Tiangen kit RNAplant Plus Reagment, the specific method is as follows:

[0130] 1) Take 1g of frozen ground apple leaves, add 5ml of extraction reagent (4°C), shake and mix.

[0131] 2) Leave it at room temperature for 5 minutes.

[0132] 3) Centrifuge at 12,000rpm at 4°C for 1 minute, and transfer the supernatant to a new RNase-free centrifuge tube.

[0133] 4) For every 10ml of supernatant, add 2ml of 5M NaCl and mix gently.

[0134] 5) Add 6ml of chloroform to every 10ml of supernatant, and mix well by inverting up and down.

[0135] 6) Centrifuge at 10,000°C for 15 minutes at 4°C, and transfer the upper aqueous phase to a new RNase-free centrifuge tube.

[0136] 7) Add isopropa...

example 2

[0185] Example 2: Apple MdSIMYB2 amino acid sequence analysis

[0186] 1) Amino acid sequence alignment between MdSIMYB2 and AtMYB5, AtMYB17, AtMYB108, and PhPH4

[0187] 1) The coding region of the MdSIMYB2 gene has 1095 bp of nucleotides. Sequence analysis using the software DNASTAR revealed that the gene encodes 364 amino acids, the protein size is 39.10 kD, and the pI is 8.17. Using the analysis software of expasy website (http: / / www.expasy.org / ) and the database of NCBI website (http: / / www.ncbi.nlm.nih.gov / ), the functional domain and conserved domain of MdSIMYB2 protein were analyzed Through comparative analysis, it was found that the MdSIMYB2 predicted protein contains two conserved functional domains R2R3 unique to the MYB family (R2 includes amino acids 50-103, and R3 includes amino acids 108-161). The results are shown in the appendix figure 1 a.

[0188]2) Search out the amino acid sequences of AtMYB5 (NM_112200.2), AtMYB17 (NM_115989.3), AtMYB108 (NM_111525.3), P...

example 3

[0193] Example 3: Response of the MdSIMYB2 gene to abiotic stress

[0194] 1) The apple Gala tissue culture seedlings with the same growth status were subjected to stress treatment (untreated as control), and were treated with drought (PEG2%), NaCl (200mM), low temperature (4°C) and ABA (100μM) for 0h and 6h respectively , 12h, 24h, 48h. The processed materials were fixed in liquid nitrogen and stored at -80°C. Finally, RNA was extracted together and reverse transcribed to obtain cDNA.

[0195] 2) Design specific primers for MdSIMYB2 gene (MdSIMYB2-BDL-R) and apple reference primers (Md18S-F, Md18S-R).

[0196] Md18S-F: 5'-AAACGGCTACCACATCCA-3', its sequence is shown in SEQ.ID.NO.15;

[0197] Md18S-R: 5'-CACCAGACTTGCCCTCCA-3' The sequence is shown in SEQ.ID.NO.16;

[0198] MdSIMYB2-BDL-R: 5'-GATAGTTCATCCAGCGG-3' The sequence is shown in SEQ.ID.NO.17;

[0199] 3) Perform a PCR reaction using Md18S-F and Md18S-R as primers and the cDNA in step 1) of Example 3 as a template, ...

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Abstract

The invention relates to cloning of an apple stress-resistant related gene MdSIMYB2 and an application of the cloning of the apple stress-resistant related gene MdSIMYB2. A homology-based cloning and rapid-amplification of cDNA ends (RACE) technology is utilized to obtain a novel apple stress-resistant related gene MdSIMYB2 from a gala apple, the nucleotide sequence of the MdSIMYB2 is shown in SEQ.ID.NO.1, and the protein amino acid sequence is shown in SEQ.ID.NO.2. The gene is transferred into a tomato, and the stress capabilities of drought resistance, salt resistance, cold resistance and the like of a transgenic tomato strain are improved; and through the overexpressing of the gene MdSIMYB2 in the apple, the expression level of the transgenetic apple can be improved, and moreover, the stress capabilities of drought resistance, salt resistance, cold resistance and the like of a transgenic apple strain are increased.

Description

1. Technical field [0001] The invention relates to the cloning and application of an apple stress-resistance-related gene MdSIMYB2, belonging to the fields of molecular biology and biotechnology. 2. Background technology [0002] Plants are often attacked by various adverse environments and pests throughout their lives. During the long-term adaptation and evolution process, plants have formed a series of defense response mechanisms to resist damage from adverse environments. Among them, environmental stress mainly includes various biotic stress and abiotic stress. Biological stress mainly comes from insects, herbivores, and plant pathogens; there are many types of abiotic stress, including drought, salinity, extreme temperature, chemical pollution, and oxygen damage. In order to adapt to the complex and changeable environment and reduce the damage to the organism caused by the adverse environment, complex processes involving multiple genes, multiple signaling pathways, and ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/84A01H5/00
Inventor 郝玉金由春香曹忠慧王荣凯
Owner SHANDONG AGRICULTURAL UNIVERSITY
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