Alfalfa draught resistant gene MsTHI1 and encoded protein and application thereof

A kind of technology of alfalfa and gene, applied in the direction of application, genetic engineering, plant genetic improvement, etc., can solve the problem of weak drought resistance and achieve the effect of improving drought resistance

Active Publication Date: 2019-08-09
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most cultivated alfalfa has weak drought resistance, and can only maintain good quality and high yield in soil with certain irrigation conditions

Method used

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  • Alfalfa draught resistant gene MsTHI1 and encoded protein and application thereof
  • Alfalfa draught resistant gene MsTHI1 and encoded protein and application thereof
  • Alfalfa draught resistant gene MsTHI1 and encoded protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 Acquisition of MsTHI1 gene from alfalfa

[0069] (1) Extraction of total RNA and its reverse transcription

[0070] Plant alfalfa No. Longmu 801 in vermiculite, extract total RNA from alfalfa seedlings with a plant total RNA extraction kit purchased from Kangwei Century Biotechnology Co., Ltd., detect its integrity with agarose gel, and measure it with a micro-spectrophotometer concentration. The reverse transcription of RNA was carried out with HiScript II Q Select RT SuperMix for qPCR (+gDNAwiper) kit (Vazyme, China), and the quality of cDNA was identified by PCR with Taq enzyme (Kangwei Century). The reaction system is shown in Table 2.

[0071] Table 2 Reverse transcription reaction system

[0072]

[0073] Reverse transcription primer sequence:

[0074] Upstream primer: 5'-TTTGAGACTTTCAATGTGCCCGCC-3' (Sequence Listing ID NO.3)

[0075] Downstream primer 5'-TAGCATGTGGGAGTGCATAACCCT-3' (Sequence Listing ID NO.4)

[0076] The reverse transcription re...

Embodiment 2

[0092] Example 2 Real-time fluorescent quantitative PCR detection gene expression characteristics in alfalfa under adversity stress conditions

[0093] Select alfalfa seeds with full grains, cultivate them in vermiculite, and water them with 1 / 10 Hoagland nutrient solution. When the seeds were 4 weeks old, the plants were treated with 15% PEG-6000, 150mmol / L NaCl, 150mmol / L NaHCO 3 , 4°C stress treatment, the treatment time was 0h (CK), 3h, 6h, 12h, 24h, 48h, and each treatment set 3 repetitions. The leaves, stems and roots of the plants were taken, frozen in liquid nitrogen, and stored at -80°C. Design fluorescent quantitative expression primers based on the full-length cDNA sequence:

[0094] Upstream primer 5'-TGTGGCCATGATGGTCCTTT-3' (Sequence Listing ID NO.7)

[0095] Downstream primer 5'-TCCTCGGCAGTGTTCATGTC-3' (Sequence Listing ID NO.8)

[0096] Internal reference gene primers:

[0097] Upstream primer: 5'-GGCTGCCATCAAGGAGGAAT-3' (Sequence Listing ID NO.9)

[0098]...

Embodiment 3

[0110] The subcellular localization of embodiment 3MsTHI1 gene

[0111] By constructing a tobacco transient expression vector, use GV3101 Agrobacterium (purchased from Beijing Bomaide Biotechnology Co., Ltd.) to inject into Nicotiana tabacum L (provided by Plant Genetic Engineering and Molecular Biology Experiment, College of Biological Sciences, Northeast Agricultural University) In , transient expression was carried out, and the expression position of MsTHI1 gene was observed under confocal electron microscopy.

[0112] The construction method of tobacco transient expression vector is:

[0113] Primers were designed based on the complete open reading frame of the MsTHI1 gene:

[0114] Upstream primers:

[0115] 5'-CGGGGGACTCTTGACGAGCTCATGACGACGTCGTTCGCC-3' (Sequence Listing ID NO.11)

[0116] Downstream primers:

[0117] 5'-CATGTCGACTCTAGAGGATCCAAATTCACCTTTTCCATTTTGGTT-3' (Sequence Listing ID NO.12)

[0118] The PCR amplification product in Example 1 was ligated with th...

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Abstract

The invention belongs to the technical field of plant genetic engineering, in particular to an alfalfa drought resistance gene MsTHI1 and encoded protein and application thereof. The drought resistantgene MsTHI1 is cloned from an alfalfa genome through a PCR technology, the nucleotide sequence of the drought resistant gene MsTHI1 is shown in the sequence table ID NO.1, and a tobacco transient expression vector, a tobacco overexpression vector and an alfalfa overexpression vector are constructed. Accordingly, an expression pattern of the MsTHI1 gene in alfalfa is analyzed by applying a fluorescence quantitative PCR technology and the relationship between the gene and adversity stress. It is found that the overexpression of MsTHI1 gene in tobacco and alfalfa can improve the drought resistance of tobacco plants, and the gene can be used for plant genetic transformation to improve the drought resistance of plants.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to the alfalfa drought-resistant gene MsTHI1 and its encoded protein and application. Background technique [0002] Drought, as a worldwide problem restricting agricultural production, has been concerned by the world in recent years. Due to climate warming, water scarcity, environmental degradation and many other reasons, the frequency and intensity of extreme climate events have shown a significant increase trend. The loss caused by drought accounts for more than 50% of meteorological disasters, which has become the main limiting condition affecting crop yield and quality. [0003] The effects of drought on plants are extensive and far-reaching. Drought mainly inhibits leaf extension, causes leaf stomata to close, reduces the activity of various enzymes in the process of photosynthesis, and affects the absorption and fixation of carbon dioxide. At t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/82C12N15/66A01H5/00A01H6/82A01H6/54
CPCC07K14/415C12N15/66C12N15/8273
Inventor 张攀尹航柴华张亚玲袁玉莹项继红胡国富崔国文
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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