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Alfalfa stress response gene MsNAC3 and application thereof

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

Inactive Publication Date: 2014-04-23
申玉华 +5
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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 stress response gene MsNAC3 and application thereof
  • Alfalfa stress response gene MsNAC3 and application thereof
  • Alfalfa stress response gene MsNAC3 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Acquisition of alfalfa stress response gene MsNAC3

[0037] 1. Gene cloning

[0038] The present invention designs homologous primers based on the alfalfa MsNAC1 (JN099384) sequence:

[0039] Upstream primer: 5'-ATGGAAAGAACTCACTTCAATATC-3' (SEQ ID NO.1)

[0040] Downstream primer: 5'-GAAAAGGTTTTGGGCAAGTAC-3' (SEQ ID NO.2)

[0041] The alfalfa seedlings were placed at 4°C for cold stress 4h before the experiment. Take young alfalfa leaves and extract total RNA. TaKaRa RNAiso Plus is used for total RNA extraction. Then, alfalfa total RNA is used as a template and Oligod (T) is used as a primer for reverse transcription reaction. The reaction system is as follows:

[0042]

[0043] The reaction procedure is: 42°C for 60 minutes, 70°C for 15 minutes, and the reaction product is stored at -20°C for later use.

[0044] Using the above cDNA as a template for PCR, the reaction system is as follows:

[0045]

[0046]

[0047] The PCR reaction program is 94℃3min, (94℃10s, 55℃20s, 7...

Embodiment 2

[0050] Example 2 Real-time fluorescent quantitative PCR to detect the expression characteristics of MsNAC3 gene in alfalfa under stress conditions

[0051] The plump alfalfa seeds were selected and sown on MS solid medium and cultured in a light incubator with a light cycle of 16h / d. Then, the two-week-old seedlings were transferred to each containing 250mmol·L -1 NaCl, 10%PEG6000, 100μmol·L -1 Abscisic acid (ABA) MS liquid medium was subjected to stress treatment, and chilling treatment was carried out in a refrigerator at 4°C. The treatment time of NaCl, PEG6000, ABA and 4℃ was 1, 2, 4, 8, 12 and 24h. The untreated ones were used as controls. After the treatments were completed, the total RNA of the leaves and roots of each treatment was extracted and stored in -70℃ refrigerator for use.

[0052] Design fluorescent quantitative expression primers based on the full-length cDNA sequence of MsNAC3:

[0053] Upstream primer: 5'-TGTTGATGCCACTCTGAACAC-3' (SEQ ID NO.3)

[0054] Downstrea...

Embodiment 3

[0060] Example 3 Subcellular localization of MsNAC3 gene

[0061] In order to further study the mechanism of MsNAC3 protein, a cell location detection vector fused with reporter gene EGFP was constructed, combined with Agrobacterium-mediated transient expression technology and laser confocal detection technology, to clarify the subcellular location of MsNAC3 protein.

[0062] Design primers based on the sequence of the complete open reading frame of MsNAC3:

[0063] Upstream primer: 5'-TCTAGAATGGAAAGAACTCACTTCAATATC-3' (SEQ ID NO.7)

[0064] Downstream primer: 5'-GGTACCGAAAAGGTTTTGGGCAAGTAC-3' (SEQ ID NO.8)

[0065] Using the cDNA reversed from the total RNA of alfalfa leaf as a template, a PCR reaction was performed. The amplified product was recovered and connected to the pMD18-T vector for sequencing. The plasmid was extracted from a single positive colony that was sequenced correctly, and the plasmid was digested with restriction enzymes XbaI and KpnI. The target fragment was reco...

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Abstract

The invention relates to an alfalfa stress response gene MsNAC3 and an application thereof. A novel NAC (N-Acetyl Cysteine) family related gene is cloned and authenticated from an alfalfa genome by using an RT-PCR (Reverse Transcription-Polymerase Chain Reaction) technology and is named as MsNAC3 with a nucleotide sequence as shown in SEQ ID No.11, and a fusion expression vector containing an enhanced green fluorescent protein (EGFP) gene is constructed. The expression pattern of the gene MsNAC3 in alfalfa and the relation between the gene and adversity stress (cold damage, salt damage and drought) are analyzed by using a fluorescent quantitative PCR technology, in such a way, people find that the cold resistance, drought resistance and salt resistance of a transgenic tobacco plant can be improved through the overexpression of the gene in tobacco; the gene can be used for genetically transforming other monocotyledons and dicotyledons and improving the stress resistance of the monocotyledons and the dicotyledons.

Description

Technical field [0001] The invention belongs to the field of genetic engineering, and specifically relates to alfalfa stress response gene MsNAC3 and its application. Background technique [0002] NAC transcription factors (including NAM, ATAF1 / 2 and CUC2) are plant-specific transcription factors with multiple regulatory effects. They are widely found in terrestrial plants. The main structural feature of this type of transcription factor is that the N-terminal 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-terminal is a highly diverse transcriptional activation regulatory region, rich in serine, threonine, and pro. Studies have found that the C-terminus of some NAC proteins has protein binding activity. Studies have shown that some members of the NAC family play a role in adversity response networks. The expression of ANAC019, ANAC055 and ANAC072 in Arabidopsis is indu...

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 申玉华徐振军黄文婕唐立红林景卫李望丰
Owner 申玉华
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