Cryptomeria fortunei CfICE1 gene and application thereof

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: 2020-10-13
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 fo...

Method used

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  • Cryptomeria fortunei CfICE1 gene and application thereof
  • Cryptomeria fortunei CfICE1 gene and application thereof
  • Cryptomeria fortunei CfICE1 gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Cloning of Cryptomeria CfICE1 gene

[0027] Experimental materials: the branches of cedar clones in good condition and free from diseases and insect pests were collected as cuttings. Cuttings of 10-15 cm (containing 2 or 3 buds) were collected by cutting the upper incision horizontally and the lower incision obliquely. After soaking in clean water overnight, add No. 6 ABT rooting powder (0.10mg mL -1 ) to induce rooting for 4h. 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. The 2-year-old seedlings were cultured in an incubator, pre-cooled at 15°C for 1 day, and cultured at 4°C for 3 days. The young leaves were taken in liquid nitrogen and quickly stored in a -80°C ultra-low temperature refrigerator.

[0028] Gene source: According to the high-throughput transcriptome sequencing results of Cryptomeria thaliana, the ICE1 gene (AT...

Embodiment example 2

[0047] Implementation Case 2: Construction of Arabidopsis overexpression vector and functional verification

[0048]Construction of eukaryotic expression vector: Using cedar cDNA as a template, according to the amplified coding region of CfICE1 gene, primers were designed using Oligo7, 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 enzyme-digested products were connected, transformed, and single clones were sequenced with T4 ligase, and the bacterial liquid with correct sequencing results was used to extract and recombine Expression vector plasmid, the recombinant expression vector plasmid was named as PBI121-CfICE1.

[0049] Planting of Arabidopsis:

[0050] 1) In an aseptic operatin...

Embodiment example 3

[0061] Implementation case 3: CfICE1 gene expression in different tissues of Cryptomeria chinensis and different low temperature stress treatments.

[0062] Different tissues of Cedar (root, stem, leaf, cone, stem, young leaf) and lateral branches of Cedar under different low temperature stress treatments. Low temperature treatment: place the tertiary lateral branches of Cedar cedar 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 were first placed at 4°C, and then lowered to the target temperature at 4°C / h for treatment, with 5 branchlets per treatment, and each treatment was repeated 3 times, immediately frozen in liquid nitrogen for 15 minutes, and stored in a -80°C ultra-low temperature refrigerator.

[0063] Phenotype observation: observe the phenotypic changes of different low temperature treatments (such as image 3 Shown), as the temperature decreases, from left to right, the leaves also turn from g...

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Abstract

The present invention discloses a cryptomeria fortunei CfICE1 gene and an application thereof, and belongs to the technical field of genetic engineering. A new ICE gene is cloned from cryptomeria fortunei tissues and named CfICE1. A nucleotide sequence is shown as SEQ ID NO.1. An amino acid sequence of an expressed protein is shown as SEQ ID NO.2. An expression vector PBI-CfICE1 is constructed totransform arabidopsis thaliana for functional verification and the CfICE1 is found to be over-expressed in the arabidopsis thaliana to promote increased cold resistance of the arabidopsis thaliana; and a qRT-PCR technology is used to analyze expression amount of the CfICE1 gene under different cryptomeria fortunei tissues and different low temperature stress, and the results show that the expression amounts of the CfICE1 are the highest in seeds and young leaves of cryptomeria fortunei at 4 DEG C. The above results indicate that the CfICE1 gene participates in growth and development of the cryptomeria fortunei and response to the low temperature stress, can be used in improvement of plant resistance genetic engineering, and has a broad application prospect.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and more specifically relates to a cedar gene CfICE1 and its application. Background technique [0002] Cryptomeria fortunei (Cryptomeria fortunei) is a subtropical coniferous tree species unique 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. It is preferred to be widely cultivated. However, the production of cryptomeria is often severely limited by various biotic and abiotic stresses. Low temperature is one of the important environmental factors affecting its growth, productivity and ecological distribution. Globally, hundreds of billions of dollars are lost each year due to reduced agricultural and forestry production due to low temperatures or cold damage. Therefore, it is of great significance to study the effec...

Claims

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Application Information

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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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