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A kind of Cedar cfice1 gene and its application

A technology of gene and cedar, applied in cedar gene CfICE1 and its application field, can solve problems such as unclear function and achieve the effect of deepening cognition

Active Publication Date: 2022-06-03
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Since Chinnusamy et al. successfully isolated the ICE1 gene from Arabidopsis thaliana, the research on its homology has mainly focused on wheat, tomato, rice and other crops, while its expression in perennial woody plants, especially gymnosperm Role in plants is unclear

Method used

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  • A kind of Cedar cfice1 gene and its application
  • A kind of Cedar cfice1 gene and its application
  • A kind of Cedar cfice1 gene and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: cedar CfICE1 gene cloning

[0027] Experimental materials: The branches of cedar clones in good condition and free of diseases and insect pests were collected as cuttings. Cuttings of 10-15 cm (containing 2 or 3 shoots) were collected by making a flat upper cut and an oblique lower cut. After soaking in water overnight, add No. 6 ABT rooting powder (0.10mg mL -1 ) induced rooting for 4 h. On June 30, 2015, the cuttings were placed in the cutting pool of the Baima Experimental Base of Nanjing Forestry University, and transplanted in the spring of the second year. Two-year-old seedlings were cultured in an incubator, pre-cooled at 15°C for 1 d, and incubated at 4°C for 3 days, and young leaves were taken in liquid nitrogen and quickly stored in a -80°C ultra-low temperature freezer.

[0028] Gene source: According to the high-throughput transcriptome sequencing results of cedar, the ICE1 gene (AT1G12860) sequence was downloaded from the Arabidopsis thaliana ...

Embodiment example 2

[0047] Example 2: Construction of Arabidopsis thaliana overexpression vector and functional verification

[0048]Construction of eukaryotic expression vector: Using cedar cDNA as a template, according to the amplified CfICE1 gene coding region, use Oligo7 to design primers, upstream primer XbaI-ICE-F: 5′-GCTCTAGAATGTTCTCGAGAATGAACACTG-3′, downstream primer BamHI-ICE -R: 5'-CGCGGATCCCATTGCAGAATGACAGCTTGCAG-3', the coding region was amplified using the above primers with restriction sites. At the same time, the PBI121 expression vector was also double digested with BamH I enzyme and XbaI enzyme, and then the two digested products were connected, transformed, and single cloned with T4 ligase. Expression vector plasmid, the recombinant expression vector plasmid was named PBI121-CfICE1.

[0049] Planting of Arabidopsis:

[0050] 1) In a sterile bench, treat Arabidopsis seeds with 75% alcohol for 30s;

[0051] 2) Absorb alcohol with a pipette, add 84 for 2min 30s, and then absorb...

Embodiment example 3

[0061] Example 3: CfICE1 gene expression in different tissues of cedar and under different low temperature stress treatments.

[0062] Different tissues (roots, stems, leaves, cones, stems, young leaves) of cedar and lateral branches of cedar treated with different low temperature stress. Low temperature treatment: The tertiary branches of cedar were placed at room temperature (25°C), 4°C, 0°C, -4°C, -8°C, -12°C, -16°C, -20°C for 12 hours, 4°C and below The lateral branches of the cultivars were first placed at 4 °C, and then lowered to the target temperature at 4 °C / h, with 5 branchlets per treatment, 3 repetitions per treatment, immediately frozen in liquid nitrogen for 15 min, and stored in a -80 °C ultra-low temperature refrigerator.

[0063] Phenotypic observation: observe the phenotypic changes of different low temperature treatments (such as image 3 shown), the leaves also changed from green to yellow from left to right as the temperature decreased.

[0064] Total RN...

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Abstract

The invention discloses a cedar gene CfICE1 and its application, belonging to the technical field of genetic engineering. The present invention clones a new ICE gene from Cedar tissue, which is named as CfICE1, its nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of the expressed protein is shown in SEQ ID NO.2. By constructing the expression vector PBI‑CfICE1 and transfecting Arabidopsis for functional verification, it was found that the overexpression of CfICE1 in Arabidopsis enhanced the cold resistance of Arabidopsis; the expression of CfICE1 gene in different Cedar tissues and different low temperature stresses was detected by qRT‑PCR The expression level was analyzed, and the results showed that the expression level of CfICE1 was the highest in the seeds and young leaves of Cryptomeria lanceolata at 4°C. The above results indicated that the CfICE1 gene was involved in the growth and development of Cryptomeria chinensis and the response to low temperature stress, and it could be used in the genetic engineering improvement of stress resistance of plants with broad application prospects.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and more particularly relates to a cedar gene CfICE1 and its application. Background technique [0002] Chinese cedar (Cryptomeria fortunei) is a subtropical coniferous tree species endemic to China. It is mainly distributed in coastal mountainous areas or high-altitude areas. It has good resistance to sulfur dioxide, chlorine, hydrogen fluoride, etc., because of its precious wood and ornamental value. and is preferentially cultivated widely. However, cedar production is often severely limited by various biotic and abiotic stresses. Low temperature is one of the important environmental factors affecting its growth and development, productivity and ecological distribution. Globally, the loss of agricultural and forestry production due to low temperature or low temperature damage costs hundreds of billions of dollars each year. Therefore, it is of great significance to study the effe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/00A01H6/20
CPCC07K14/415C12N15/8273
Inventor 徐进张莹婷崔洁冰张萌
Owner NANJING FORESTRY UNIV
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