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Cold sensitivity related OfHSF11 gene of cinnamomum rixiangxiense as well as encoding protein and application of cold sensitivity related OfHSF11 gene

A gene coding and gene technology, applied in the cold-sensitive related OfHSF11 gene and its encoded protein and application field, can solve the problem that the HSF transcription factor has not been explored

Pending Publication Date: 2022-04-12
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing studies have shown that HSF transcription factors are related to the stress response of plants, but HSF transcription factors in Osmanthus fragrans have not been explored

Method used

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  • Cold sensitivity related OfHSF11 gene of cinnamomum rixiangxiense as well as encoding protein and application of cold sensitivity related OfHSF11 gene
  • Cold sensitivity related OfHSF11 gene of cinnamomum rixiangxiense as well as encoding protein and application of cold sensitivity related OfHSF11 gene
  • Cold sensitivity related OfHSF11 gene of cinnamomum rixiangxiense as well as encoding protein and application of cold sensitivity related OfHSF11 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Constructing the Overexpression Vector of the Japanese Fragrant Osmanthus OfHSF11 Gene

[0029] (1) Obtain the target gene

[0030] According to the Osmanthus fragrans genome database published by Wang Lianggui's research group at Nanjing Forestry University ( http: / / 117.78.20.255 / ), screened all members of the HSF gene family, and named one of the gene sequences OfHSF11.

[0031] (2) Design primers

[0032] The full-length nucleotide sequence of the gene was analyzed by BioXM software, and Sma I and SpeI enzymes were selected as the two restriction endonucleases. Primers were designed using CE design software. Fill in the relevant information as required, specifically including the sequence near the restriction site on the carrier, the full length of the target gene, and fill in the 2 restriction sites (5' end and 3' end) in order to obtain amplification primers. The designed sequence was synthesized by Jierui Biotechnology Co., Ltd., as follows:

...

Embodiment 2

[0057] Example 2 Transformation of Agrobacterium GV3101

[0058] (1) Take out the competent GV3101 stored in a -80°C ultra-low temperature refrigerator and put it on ice to thaw. Add 1 μL of plasmid per 33 μL of competent cells, pipette and mix well, then ice bath for 20 minutes, liquid ammonia quick freezing for 5 minutes, 37°C water bath for 5 minutes, and ice bath for 5 minutes;

[0059] (2) Add 500 μL non-resistant LB liquid medium, culture at 28°C, 200rppm shaker for 1h;

[0060] (3) After the cultivation is completed, centrifuge the bacterial solution at 6000r for 1min, discard part of the supernatant, leave 100μL and spread it evenly on the LB solid medium (containing 50mg / L Kana), seal it with a parafilm, and incubate at 28°C. Cultivate in the box for 40-48h;

[0061] (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 backup plate into L...

Embodiment 3

[0062] Example 3 Infect Nicotiana benthamiana and carry out malondialdehyde (MDA) index determination

[0063] (1) Shaking bacteria: Take out the super1300 empty load, the P19 auxiliary expression vector and the GFP::Super1300-OfHSF11 target gene fusion expression vector transformed into Agrobacterium bacteria at -80°C, take it out at room temperature and melt it to a mixed state of ice and water, and then insert it Thaw in ice, add 300 μL of bacterial solution to 30 mL of LB liquid medium (containing Kana 10 μg·mL -1 ), 28°C, 200rpm in the dark, shake culture, until the bacterial solution OD 600 = Between 0.6-0.8;

[0064] (2) Prepare the mixed bacterial solution: weigh 0.0196g of acetosyringone (AS) powder, use dimethyl sulfoxide to aid dissolution (operate in a fume hood), then add an appropriate amount of sterile water to make the volume to 100ml, and obtain the mother solution. Take 15ml of mother liquor and add 85ml of sterile water to obtain 150μmol L -1 AS; Weigh 0....

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Abstract

The invention discloses a osmanthus fragrans cold sensitivity related OfHSF11 gene as well as an encoding protein and application thereof, and belongs to the field of plant molecular biology. A transcription factor which is separated and cloned from osmanthus fragrans based on a plant gene cloning technology is named OfHSF11, and the nucleotide sequence of the transcription factor is shown as SEQ ID NO.1. On the basis, an over-expression vector is constructed, instantaneous transformation of nicotiana benthamiana is carried out, and phenotypic analysis before and after low-temperature treatment shows that the transcription factor is a transcription factor which is named as OfHSF11 and is named as OfHSF11. The cold sensitivity of the OfHSF11 transfected plant is obviously higher than that of the non-OfHSF11 transfected plant, which indicates that the OfHSF11 gene is a potential cold resistance indicator gene, and a useful gene resource is provided for cold resistance genetic improvement breeding of sweet osmanthus.

Description

technical field [0001] The invention belongs to the field of plant molecular biology, and in particular relates to a cold-sensitivity-related OfHSF11 gene and its encoded protein and application. Background technique [0002] Osmanthus fragrans (Osmanthus fragrans) is an evergreen woody tree species widely used in landscaping in the south of the Yangtze River in my country. It has high ornamental and economic value, but is sensitive to low temperature stress, and its cultivation and application in high latitude areas are limited. Therefore, discovering cold-resistant genes in osmanthus fragrans is of great significance for improving the cold resistance of osmanthus fragrans. [0003] Heat shock transcription factor (HSF), as one of the key components of plant heat stress response, plays an important role in plant heat stress memory. Existing studies have shown that HSF transcription factors are related to the stress response of plants, but HSF transcription factors in Osman...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/84C07K14/415A01H5/00A01H6/82A01H6/00
Inventor 岳远征谭宏岩杨勇宾婧杨秀莲王良桂
Owner NANJING FORESTRY UNIV
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