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Alfalfa adversity stress responsive gene MsNAC2 and application thereof

A kind of alfalfa, gene technology, applied in the field of genetic engineering, can solve the problems of slow growth and low yield, and achieve the effect of improving cold resistance and stress resistance

Inactive Publication Date: 2014-04-09
申玉华 +4
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although rice overexpressing the OsNAC6 gene shows slow growth and low yield, it shows strong resistance to drought, high salinity and Fusarium wilt (Nakashima et al., 2007)

Method used

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  • Alfalfa adversity stress responsive gene MsNAC2 and application thereof
  • Alfalfa adversity stress responsive gene MsNAC2 and application thereof
  • Alfalfa adversity stress responsive gene MsNAC2 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Acquisition of alfalfa stress response gene MsNAC2

[0042] First, based on the sequences of legume peanut NAC transcription factor genes AhNAC2 and AhNAC3 ​​(EU755023 and EU755022), alfalfa NAC gene homologous primers were designed. The upstream primer sequence was 5′-ACCAAACGGTTCAAG GCCGAACC-′3 (SEQ ID NO.1), and the downstream sequence is 5'-CGATACTCGTGCATGATCCAATTG-'3 (SEQ ID NO. 2). Then, using RT-PCR technology, the sequence of the conserved region of NAC was first amplified from the genome of Medicago sativa.

[0043] 1. Extraction of total RNA from alfalfa: Total RNA was extracted using TaKaRa RNAiso Plus.

[0044] 2. Synthesis of the first strand of cDNA:

[0045] The reaction system is as follows:

[0046]

[0047]The reverse transcription program is: 42°C for 60 minutes, 70°C for 15 minutes.

[0048] 3. PCR reaction:

[0049]

[0050]

[0051] The PCR reaction program was: 94°C pre-denaturation for 5 minutes, (94°C for 10s, 55°C for 20...

Embodiment 2

[0059] Example 2 Real-time fluorescent quantitative PCR detection of expression characteristics of MsNAC2 gene in alfalfa under adversity stress conditions

[0060] The two-week-old alfalfa seedlings were transferred to 250mmol·L -1 NaCl, 10%PEG6000, 100μmol L -1 The stress treatment was carried out in abscisic acid (ABA) MS liquid medium, and the chilling injury treatment was carried out in a 4°C refrigerator. The treatment times of PEG6000, ABA and 4°C were 1, 2, 4, 8, 12 and 24 hours, and the treatment times of NaCl were 1, 2, 4, 8, 12, 24 and 48 hours. After the completion, the total RNA of leaves and roots was extracted respectively, and fluorescent quantitative expression primers were designed according to the full-length cDNA sequence of MsNAC2. The upstream primer sequence was 5-TGGTGAAGATGACCCTTTTGC-3′ (SEQ ID NO: 5), and the downstream primer sequence was 5′-AAGCTCCACTTGCAGTTGCAG -3' (SEQ ID NO: 6). The alfalfa Actin gene (EU664318) was used as the internal refere...

Embodiment 3

[0061] The subcellular localization of embodiment 3MsNAC2 gene

[0062] The MsNAC2 gene was constructed into the fusion expression vector pBIGFP containing the eGFP gene to construct the fusion expression vector pBIGFP-MsNAC2 (see Example 4 for details). Then combined with Agrobacterium-mediated transient expression technology and laser confocal detection technology, the expression vector pBIGFP-MsNAC2 was successfully transformed into onion epidermis. Then the expression site of the gene was observed under a fluorescent microscope, and the carrier pBIGFP without MsNAC2 was used as a control, the results are shown in Figure 4 , where, in the onion epidermal cells transfected with pBIGFP-MsNAC2, only the GFP green fluorescence signal can be observed in the nucleus under ultraviolet light ( Figure 4 in Figure B), Figure 4 Figure A in the figure is a photo of onion epidermal cells transfected with pBIGFP-MsNAC2 under white light; while in the control group, clear green fluor...

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Abstract

The invention relates to an alfalfa adversity stress responsive gene MsNAC2 and an application thereof. An NAC-family related gene is cloned from an alfalfa genome by applying RT-PCR (Reverse Transcription-Polymerase Chain Reaction) and an RACE (Rapid-Amplification of cDNA Ends) technology, and named as MsNAC2 with a nucleotide sequence as shown in SEQ ID No.11; and a fusion expression vector with an enhanced type green fluorescent protein (EGFP) gene is constructed. According to the invention, a fluorescent quantitative PCR (Polymerase Chain Reaction) technology is applied to analyze an expression mode of the gene in the alfalfa as well as a relationship between the gene and adversity stress (cold damage, salt damage and drought), so that cold resistance, drought resistance and salt resistance of a transgenic tobacco plant can be found to be improved by over-expressing the gene in tobaccos; and the gene can be applied to genetic transformation of other monocotyledons and dicotyledons, so that stress resistance of the monocotyledons and dicotyledons is improved.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to the alfalfa adversity response gene MsNAC2 and its application. Background technique [0002] NAC transcription factor (including NAM, ATAF1 / 2 and CUC2) is a plant-specific transcription factor with multiple regulatory functions, which widely exists in terrestrial plants. The main structural feature of this type of transcription factor is that the N-terminus is conserved The NAC domain of about 150 amino acid residues can bind DNA and other proteins, and can be divided into 5 conserved regions from A to E; the C-terminus is a highly diverse transcriptional activation regulatory region, rich in serine, threonine, proline Amino acid, glutamic acid, etc., studies have found that the C-terminus of some NAC proteins has protein binding activity. Current studies have shown that some members of the NAC family play a role in the adversity response network. ANAC019, ANAC05...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/10C12N15/84C12N5/10A01H5/00
Inventor 申玉华徐振军杨晓坡林景卫李望丰
Owner 申玉华
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