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Tobacco drought response gene NtRHF1 and application of encoding protein thereof

A technology of encoding protein and tobacco, which is applied in the field of plant genetic engineering, can solve problems such as inhibiting root growth, short internodes, and decreased activity of antioxidant protective enzymes, achieving broad application prospects and huge potential for economic benefits

Inactive Publication Date: 2014-04-09
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Antioxidant protective enzymes SOD, POD, CAT and other enzyme activities decreased significantly
Second, the impact of drought on the growth and development of tobacco is mainly manifested in: (1) drought stress reduces the germination of seeds and the survival of seedlings; (2) inhibits the growth of roots, thereby affecting the absorption of mineral nutrients; (3) Drought stress leads to dwarf plants, short internodes, small leaves, and premature senescence
There are few reports on the cloning and functional identification of the gene encoding E3 ligase in tobacco

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Embodiment 1: material drought stress treatment

[0018] The seeds of Yunyan 87 were sterilized by 15% sodium hypochlorite, placed in a petri dish containing distilled water, and cultivated in a light culture room with a temperature of 24-26°C, a humidity of 60%, and a light-dark cycle of 12h / 12h. When the seedlings grow to two leaves and one heart (about 14 days), the distilled water is poured out, and a 20% PEG 6000 solution stress treatment experiment is carried out. The treatment time was 1, 3, 6, 12, and 24 hours, respectively; the stress-treated tobacco was used as the experimenter, and the unstressed tobacco seedlings were used as the control group for the expression analysis of RHF1. The drought treatment of transgenic tobacco plants adopts artificial controlled watering method. When the plants grow to 5 leaves (about 30 days), stop watering for 14 days, and observe the growth of each strain.

Embodiment 2

[0019] Embodiment 2: Cloning of RHF1 (RING-H2 Finger 1) gene

[0020] 1. Synthesis of first-strand cDNA in tobacco leaves

[0021] The isolation and purification of total RNA from tobacco leaves were performed according to the instructions of the TRIZOL kit (Invitrogen, USA). Draw 1-2 μ g of the total RNA of tobacco leaves in a 1.5 ml centrifuge tube, according to the RevertAid of Fermentas company TM First Strand cDNA Synthesis Kit Product Description Perform reverse transcription to obtain the first strand cDNA of tobacco leaves.

[0022] 2. PCR amplification of NtRHF1 gene

[0023] Using the first-strand cDNA as a template, perform PCR amplification of the NtRHF1 gene to obtain a PCR amplification product.

[0024] The full-length cDNA primers of the tobacco NtRHF1 gene were designed according to the tobacco expressed sequence tag (Expressed Sequence Tag, EST) information in the website http: / / www.ncbi.nlm.nih.gov / :

[0025] Upstream primer: 5′-ATGAGTTTTGTTTTCCGAG-3′; ...

Embodiment 3

[0029] Example 3: Expression analysis of NtRHF1 gene

[0030] 1. In this example, the fluorescent quantitative RT-PCR method was used to analyze the expression of NtRHF1 gene under 20% PEG 6000 stress. The experimental samples were the tobacco leaves of the experimenter and the control group described in Example 1. In each sample, according to the method described in Example 2, 5 μg of total RNA was reverse-transcribed into first-strand cDNA as a template.

[0031] According to the full-length cDNA sequence of NtRHF1 gene, specific primers for fluorescent quantitative PCR were designed as follows:

[0032] Upstream primer: 5'-TTGGTGCTCGGTGTCTTCTTTAT-3';

[0033] Downstream primer: 5'-GTGCCCTCAGTGTTTCGTAATCT-3'.

[0034] BioRad iQ5 real-time quantitative PCR instrument was used for PCR amplification, and the amplified product was 244bp.

[0035] 2. For each sample, the upstream primer: 5'-TGGCATCACACTTTCTAAAC-3' and the downstream primer: 5'-CAACGGAATCTCTCAGCCCT-3' were use...

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Abstract

The invention discloses a tobacco drought response gene NtRHF1 and an application of encoding protein thereof. The nucleotide sequence of the tobacco drought response gene NtRHF1 is shown by SEQ ID NO: 1, and the amino acid sequence of the encoding proteins is shown by SEQ ID NO: 2. The adaptability of plants to drought stress can be increased through the over-expression of the gene, and the tobacco drought response gene NtRHF1 has a wide application prospect in the field of tobacco drought.

Description

technical field [0001] The invention relates to the field of plant genetic engineering, in particular to the application of a tobacco drought response gene NtRHF1 and its encoded protein. Background technique [0002] In recent years, with global warming, drought has become a frequent natural disaster in crop production in my country. Tobacco, as an important economic crop in China, has high requirements for water throughout the growth period. Most of China's tobacco areas are in arid and semi-arid environments, and high-quality tobacco areas are mostly located in hilly and mountainous areas. The growth and development of tobacco plants are often affected by soil drought, resulting in a reduction in the yield and quality of tobacco leaves. Drought has become a constraint on the production and quality of tobacco leaves in my country. One of the main limiting factors for quality improvement. Therefore, strengthening the excavation and utilization of tobacco drought-resistant ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/52C12N9/00A01H5/00
Inventor 夏宗良苏新宏张小全朱伟
Owner HENAN AGRICULTURAL UNIVERSITY
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