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Chimonanthus praecox CpVIN3 gene and application thereof

A technology of wintersweet and genes, applied in the field of genetic engineering, to achieve high species-specific effects

Active Publication Date: 2019-06-11
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the current research, people have comprehensively understood the function and mechanism of VIN3 in Arabidopsis, cabbage and other model plants, but the relevant reports of CpVIN3 in Wintersweet have not yet appeared, and there are many conjectures about its function and role , a large number of experiments are needed to confirm the

Method used

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  • Chimonanthus praecox CpVIN3 gene and application thereof
  • Chimonanthus praecox CpVIN3 gene and application thereof
  • Chimonanthus praecox CpVIN3 gene and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Cloning of Example 1 Wintersweet CpVIN3 Gene

[0030] The CpVIN3 gene in the transcriptome sequencing of the wax plum was cloned, and the obtained sequence fragments were passed through the BlastN (Nucleotide-nucleotide BLAST) and BlastX (Translated query vs. protein database) for comparative analysis to obtain target clones.

[0031] According to the obtained cDNA sequence of the CpVIN3 gene of Wintersweet, use the software Primer primer 5.0 to design a pair of specific primers spanning the largest ORF frame for PCR amplification. The primer sequences are as follows:

[0032] CpVIN3-F: '5-GCCTCACTCTTTCTGTACCCT-3'

[0033] CpVIN3-R: '5-TTAGTTGGTTACCCCATCAT-3'

[0034] Wintersweet cDNA was used as a template to amplify the CpVIN3 gene of Wintersweet. The PCR reaction system and reaction conditions were as follows:

[0035]

[0036] The PCR amplification conditions are:

[0037]

[0038] The product obtained by PCR amplification is detected with 1% agarose gel, ...

Embodiment 2

[0042] The construction of embodiment 2 Wintersweet CpVIN3 gene plant expression vector

[0043] Combining the distribution characteristics of the restriction sites of the CpVIN3 gene ORF box sequence and the multi-cloning site characteristics of the plant overexpression vector pCAMBIA-1300 used, a pair of gene-specific primers were designed, and restriction sites were added to the upstream and downstream of the primers KpnI and SalI and the corresponding protection bases are used to amplify the CpVIN3 gene coding region carrying a suitable restriction site and clone it into the multiple cloning site of the plant expression vector. The primer names and sequences are as follows:

[0044] p-CpVIN3-F( The box part is the Kpn I restriction site, and the underline is the protected base);

[0045] p-CpVIN3-R( The box part is the Sal I restriction site, and the underline is the protection base).

[0046] Using the bacterium liquid obtained by cloning in Example 1 as a template,...

Embodiment 3

[0048] Example 3 Phenotype observation of CpVIN3 transformed Arabidopsis and memory Arabidopsis

[0049] Inflorescence infection method to transform Arabidopsis, the obtained transgenic Arabidopsis T 0 Hygromycin resistance screening was carried out on the generation seeds, and the positive plants grew well on the resistant medium, while the non-transgenic plants were dwarfed and yellowed on the medium containing hygromycin resistance, such as Image 6 shown. Genomic DNA was extracted from leaves of four transgenic Arabidopsis lines and wild-type Arabidopsis plants, and identified by PCR with specific primers for CpVIN3 gene. the result shows( Figure 7 ), the obtained transgenic Arabidopsis lines could amplify the target band with the same size as the positive control, while the wild-type Arabidopsis did not amplify the target band, indicating that the target gene CpVIN3 had been successfully inserted into Arabidopsis in the genome.

[0050] In order to further verify the...

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Abstract

The invention provides a chimonanthus praecox CpVIN3 gene and application of the chimonanthus praecox CpVIN3 gene in flowering phase regulation. A gene sequence of the chimonanthus praecox CpVIN3 provided by the invention is shown as SEQ ID No.1. The chimonanthus praecox CpVIN3 gene is obtained through cloning for the first time, and the function of the gene is verified in plant arabidopsis thaliana through a transgenic technology. The method comprises the following steps: transferring a CpVIN3 gene into arabidopsis thaliana; the phenotype of the gene is observed to preliminarily verify the function of the gene; compared with the phenotype of transgenic arabidopsis thaliana and the phenotype of wild arabidopsis thaliana, the flowering time of the transgenic arabidopsis thaliana is earlierthan that of the wild arabidopsis thaliana under the condition of 4 DEG C vernalization treatment, and the number of rosette leaves is reduced compared with that of the wild arabidopsis thaliana, which indicates that the gene regulates plant nutrition growth to reproductive growth in the vernalization process.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to the cloning and application of the C. chinensis CpVIN3 gene. Background technique [0002] Wintersweet (Chimonanthus praecox (L.) Link) is a kind of winter flowering perennial shrub unique to my country. It is famous for its color like beeswax and fragrant fragrance. The blooming characteristics of Aoxue make it a popular garden Ornamental plants, in the long history of thousands of years in our country, the cultivation and planting of wintersweet in nurseries and bonsai is unique. In addition, wintersweet is a Quaternary glacier relict plant, which occupies an important position in the ancient evolutionary system [2] . For hundreds of years, people's research on wintersweet has mainly focused on germplasm resources, variety categories and cultivation. In recent years, with the vigorous development of molecular biology, people's understanding of wintersweet has gradually shift...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/02A01H6/20
Inventor 李志能张弈眭顺照李名扬李先源
Owner SOUTHWEST UNIVERSITY
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