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Salt-resistant related gene of osmanthus fragrans and encoding protein and application of salt-resistant related gene

A technology of gene and cinnamon, applied in the salt-resistance-related gene of cinnamon and its encoded protein and application fields, can solve problems such as unexplored

Active Publication Date: 2022-08-02
NANJING FORESTRY UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Trihelix transcription factors are a class of transcription factors that have been widely concerned in recent years and play an important role in regulating plant stress response and development, but Trihelix transcription factors in Osmanthus fragrans have not been explored

Method used

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  • Salt-resistant related gene of osmanthus fragrans and encoding protein and application of salt-resistant related gene
  • Salt-resistant related gene of osmanthus fragrans and encoding protein and application of salt-resistant related gene
  • Salt-resistant related gene of osmanthus fragrans and encoding protein and application of salt-resistant related gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Construction of the overexpression vector of the OfGT3 / OfGT42 / OfGT46 gene

[0026] (1) Obtain the target gene

[0027] According to the published whole genome database of Osmanthus fragrans, all members of the Trihelx gene family were screened, and the three gene sequences were named OfGT3, OfGT42 and OfGT46 respectively.

[0028] (2) Design primers

[0029] The full-length nucleotide sequences of the above genes were analyzed by using BioXM software, and Sma I and Spe I enzymes were selected as two restriction endonucleases. Primers were designed using CE design software. Fill in the relevant information as required, including the sequence near the restriction site on the vector, the full length of the target gene, and fill in the two restriction sites (5' end and 3' end) in sequence, and then the amplification can be obtained. primers. The designed sequence is sent to Jerui Biotechnology Co., Ltd. for synthesis.

[0030] OfGT3F: aagcttctgcaggggccccgggAT...

Embodiment 2

[0051] Example 2 Transformation of Agrobacterium GV3101

[0052] (1) Take out the GV3101 competent cells stored in the -80°C ultra-salt freezer and melt them on ice. Add 1 μL of plasmid per 33 μL of competent cells, mix by pipetting, ice bath for 20 minutes, liquid ammonia quick-freeze for 5 minutes, 37°C water bath for 5 minutes, and ice bath for 5 minutes;

[0053] (2) Add 500 μL of non-resistant LB liquid medium, and cultivate for 1 hour at 28°C on a shaker at 200 rpm;

[0054] (3) After the cultivation is completed, centrifuge the bacterial liquid at 6000 rpm for 1 min, discard part of the supernatant, leave 100 μL to spread evenly on LB solid medium (containing 50 mg / L Kana), seal with parafilm, and place it upside down at 28°C Culture in incubator for 40-48h;

[0055] (4) Bacterial inspection and backup: the target band in the bacterial inspection is correct and the brightness is consistent ( Figure 4 ), then pick the corresponding colony in the backed up plate into ...

Embodiment 3

[0056] Embodiment 3 Infect Nicotiana benthamiana and carry out MDA index measurement

[0057] (1) Shake bacteria: Super1300 empty, P19 auxiliary expression vector and GFP::Super1300-OfGT3 / GFP::Super1300-OfG42 / GFP::Super1300-OfG46 target gene fusion expression vector that has been transformed into Agrobacterium. Take it out at -80°C and thaw it to a mixed state of ice and water, then insert it into ice to melt, and add 300 μL of bacterial solution to 30 mL of LB liquid medium (containing 10 μg·mL of Kana). -1 ), 28 ℃, 200 rpm shaking culture in the dark, until the bacterial liquid OD 600 = Between 0.6-0.8;

[0058] (2) Preparation of mixed bacterial solution: Weigh 0.0196g of AS powder, use dimethyl sulfoxide to assist dissolution (operate in a fume hood), then add an appropriate amount of sterile water to make up to 100ml to obtain a mother liquor, take 15ml of the mother liquor and add 85ml of sterile water to obtain 150 μmol·L-1 of acetosyringone (AS); weigh 0.2035 g of Mg...

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Abstract

The invention discloses a salt-resistant related gene of osmanthus fragrans and an encoding protein and application of the salt-resistant related gene, and belongs to the field of plant molecular biology. Three Trihelx transcription factors separated and cloned from osmanthus fragrans based on a plant gene cloning technology are respectively named as OfGT3, OfGT42 and OfGT46 genes, nucleotide sequences of the three Trihelx transcription factors are sequentially shown as SEQ ID NO.1-3, corresponding amino acid sequences of the three Trihelx transcription factors are sequentially shown as SEQ ID NO.4-6, the nucleotide sequences of the three Trihelx transcription factors are sequentially shown as SEQ ID NO.3, and the nucleotide sequences of the three Trihelx transcription factors are sequentially shown as SEQ ID NO.4-6. On the basis, an over-expression vector is constructed, the gene is introduced into the Bensi tobacco by using an agrobacterium floc soaking method to obtain a transgenic plant, membrane lipid peroxidation index analysis before and after treatment of 250mM NaCl shows that the salt tolerance of a non-transgenic plant is obviously lower than that of a transgenic plant, and the salt tolerance of the non-transgenic plant is obviously lower than that of the transgenic plant. Therefore, the gene is a potential salt tolerance indicator gene.

Description

technical field [0001] The invention belongs to the field of plant molecular biology, and in particular relates to a salt-resistance-related gene of cinnamon and cinnamon as well as its encoded protein and application. Background technique [0002] Rixianggui (Osmanthus fragrans cv.'rixianggui') is a horticultural variety of sweet-scented osmanthus, named for its continuous flowering and strong aroma. It is an excellent garden tree species that integrates greening, beautification, and fragrance, both ornamental and practical, with high ornamental and economic value. However, it is sensitive to salt stress, and its cultivation and application in high latitude regions are limited. Therefore, it is of great significance to discover salt resistance genes in osmanthus fragrans for the improvement of salt resistance of osmanthus fragrans. Trihelix transcription factors are a class of transcription factors that have received extensive attention in recent years and play an importan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/84A01H5/00A01H6/82
CPCC07K14/415C12N15/8273C12N15/8205
Inventor 王良桂曾贵敏朱美林刘芳伊李晓芹杨秀莲银征何方
Owner NANJING FORESTRY UNIV
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