Application of clematis terniflora isopentenyl transferase PT1 gene, overexpressed arabidopsis thaliana strain thereof and construction method of overexpressed arabidopsis thaliana strain

A technology of lotus isoprene and a construction method, applied in the field of plant genetic engineering, can solve problems such as insufficient UV stress resistance, and achieve the effect of strong resistance

Pending Publication Date: 2022-01-21
ZHEJIANG SCI-TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the use of gene technology to enhance the ability of plants to resist UV stress is not deep enough.

Method used

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  • Application of clematis terniflora isopentenyl transferase PT1 gene, overexpressed arabidopsis thaliana strain thereof and construction method of overexpressed arabidopsis thaliana strain
  • Application of clematis terniflora isopentenyl transferase PT1 gene, overexpressed arabidopsis thaliana strain thereof and construction method of overexpressed arabidopsis thaliana strain
  • Application of clematis terniflora isopentenyl transferase PT1 gene, overexpressed arabidopsis thaliana strain thereof and construction method of overexpressed arabidopsis thaliana strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] The present embodiment has carried out the cloning and expression of Clematis chinensis isopentenyl transferase PT1 gene, and the specific method is as follows:

[0067] Trizol method was used to extract the clematis cone RNA, reverse transcription to obtain cDNA, and high-fidelity enzyme was used to amplify the PT1 gene sequence (SEQ ID NO. 1) from the cDNA template.

[0068] Forward primer: 5'-ATGATTTCTGCTTTAGCTTCTCCA-3'

[0069] Reverse primer: 5'-TCAACAAACAAAGGGAATAAGCAAA-3'

[0070] The PCR amplification system is: template 1 μL, 2× PCR buffer 10 μL, upstream and downstream primers 0.8 μL each, dNTP 0.4 μL, Polymerase 0.4 μL, supplemented with ddH 2 0 to 50 μL. Denaturation at 95°C for 3 min; 35 cycles of 15 S at 95°C, 30 S at 52°C, 1 min at 72°C; extension at 72°C for 5 min, and storage at 4°C. PCR products were stored at -20°C after electrophoresis, recovery and sequencing.

[0071] Use SteadyPure DNA Gel Recovery Kit (AG Company) to perform gel recovery of t...

Embodiment 2

[0082] In this example, the overexpressed Arabidopsis thaliana lines obtained in Example 1 were used for screening, and four transgenic Arabidopsis thaliana plants were obtained. Studies on changing plant phenotypes are as follows:

[0083]Six overexpressed Arabidopsis thaliana strains and 12 original wild-type Arabidopsis thaliana strains were sown respectively. The specific operation and culture conditions were as follows: Arabidopsis thaliana seeds were sown on the surface of the seedling block, and placed in the light at a temperature of 21±2°C. Grow in an incubator with a light cycle of 16h light / 8h dark, and a light intensity of 8000Lx. After about 15 days, the seedlings were transplanted, and the Arabidopsis thaliana was transferred from the seedling block to a 6cm × 6cm square flowerpot, and the culture conditions remained unchanged.

[0084] Primer validation experiments were performed on each transgenic Arabidopsis, such as figure 1 The gel electrophoresis results s...

Embodiment 3

[0087] This embodiment utilizes the transgenic Arabidopsis thaliana obtained in Example 2 and the original wild-type Arabidopsis thaliana plant to carry out the functional verification of the transgenic plant in improving the plant's resistance to ultraviolet stress, as follows:

[0088] (1) UV irradiation treatment

[0089] The transgenic Arabidopsis thaliana plants and wild-type Arabidopsis thaliana plants were given UV irradiation under the same conditions. The irradiation conditions are as follows: the irradiation time is 72h, and the irradiation intensity is about 14000Lx.

[0090] (2) Phenotypic analysis after UV treatment

[0091] It can be seen that the growth state of transgenic Arabidopsis was significantly better than that of the control after UV treatment for 72 h. The leaves of wild-type Arabidopsis thaliana were severely dehydrated and appeared dry, withered and wilted, while the leaves of transgenic Arabidopsis grew well, only a few of the leaves were wilted, ...

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Abstract

The invention discloses application of a clematis terniflora isopentenyl transferase PT1 gene, an overexpressed arabidopsis strain thereof and a construction method of the overexpressed arabidopsis strain. The nucleotide sequence of the clematis terniflora isopentenyl transferase PT1 gene is as shown in SEQ ID NO. 1. A model plant arabidopsis thaliana is adopted, and a positive strain of arabidopsis thaliana trans-PT1 gene is obtained by constructing a plant PT1 gene overexpression vector and transgenosis operation. The invention proves that the plant morphology of the transgenic arabidopsis thaliana strain has the characteristics of multiple glandular hair on the leaf surface and dark leaf color. After ultraviolet stress, the O2 <-> generation rate and H2O2 content increase in the transgenic arabidopsis thaliana leaves are remarkably lower than those of wild arabidopsis thaliana, the MDA content increase in the leaves is remarkably lower than those of the wild arabidopsis thaliana leaves, and the leaf conductivity increase is remarkably lower than that of the wild arabidopsis thaliana leaves. After extreme ultraviolet stress, the survival rate of the transgenic arabidopsis thaliana is remarkably higher than that of wild arabidopsis thaliana.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and provides the application of Clematis paniculata prenyltransferase PT1 (Prenyltransferase 1, PT1) gene, its overexpressed Arabidopsis strain and its construction method. Background technique [0002] Clematis terniflora DC. is a plant of the genus Clematis in the family Ranunculaceae. It has a long history of application as traditional Chinese medicine and folk herbal medicine. The whole herb is used to treat chronic pharyngitis and chronic prostatitis, etc., and has high medicinal value. [0003] The prenyl transferase in plants or microorganisms can connect the prenyl substituents produced by the mevalonate or methylerythritol phosphate pathway in organisms to different small molecular compounds or different substitution positions of compounds . As an active group, isopentenyl can further undergo oxidation, cyclization, dehydration and rearrangement reactions under the action of mo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N15/82A01H5/12A01H6/20
CPCC12N9/1085C12N15/8271C12N15/8261
Inventor 杨丙贤钟卓珩黄露漫张梦邓霖芳付红伟张琳
Owner ZHEJIANG SCI-TECH UNIV
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