Taraxacum kok-saghyz inulin degrading enzyme gene Tk1-FEH and application thereof

A technology for degrading enzymes and inulin, applied in the field of genetic engineering, can solve the problems of short domestication time, high cost, low gum content and low biomass, etc.

Inactive Publication Date: 2020-01-07
RUBBER RES INST CHINESE ACADEMY OF TROPICAL AGRI SCI +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current germplasm of rubber grass is basically wild germplasm, the domestication time is short, there are problems such as low rubber content and biomass, and poor stress resistance. The cost of extracting natural rubber from rubber grass is still high. Therefore, creating high rubber content The new germplasm with high biomass and strong stress resistance is of great significance to the industrialization of rubber grass

Method used

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  • Taraxacum kok-saghyz inulin degrading enzyme gene Tk1-FEH and application thereof
  • Taraxacum kok-saghyz inulin degrading enzyme gene Tk1-FEH and application thereof
  • Taraxacum kok-saghyz inulin degrading enzyme gene Tk1-FEH and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Cloning of rubber grass inulin degrading enzyme gene Tk1-FEH

[0043] The present invention utilizes RT-PCR technology to clone the rubber grass inulin degrading enzyme gene Tk1-FEH from the leaves of rubber grass high rubber content strain 2162, and the specific method is as follows:

[0044] 1. Extract the total RNA in rubber tree leaves, and reverse transcribe to obtain the first strand of cDNA.

[0045]2. Using the first strand of cDNA as a template and Tk1-FEH-F1 and Tk1-FEH-R1 as primers, the cDNA sequence of Tk1-FEH gene was obtained by PCR amplification reaction. The primer sequences are as follows:

[0046] Tk1-FEH-F1: 5'-ATGAGCAAGCCTCTTTCCTCC-3';

[0047] Tk1-FEH-R1: 5'-TTAAACTGTGCTTTTTACAGT-3'.

[0048] Using Premix Taq TM (TaKaRa) for PCR amplification, the reaction system is: cDNA template 1 μL, primers Tk1-FEH-F1 and Tk1-FEH-R1 (10 μmol / L) each 1 μL, 2×PCR Mix 10 μL, add ddH 2 0 to 20 μL.

[0049] The PCR amplification program was: denatura...

Embodiment 2

[0052] Example 2 Expression analysis of rubber grass inulin degrading enzyme gene Tk1-FEH under drought and other stresses

[0053] In this example, the fluorescent quantitative PCR method was used to detect the four tissues of rubber grass leaves, flowers, pedicels and roots, and the expression of Tk1-FEH gene in rubber grass leaves after being treated with drought, abscisic acid, PEG6000, ethephon and methyl jasmonate.

[0054] Drought treatment: First, water the rubber grass tissue culture seedlings transplanted for 3 months to saturation, and then put them in the light incubator for water cut-off treatment. 3-day-old (F3d) leaves were used as material.

[0055] Chemical agent treatment: take rubber grass tissue culture seedlings cultivated in hydroponic culture for one month as materials, soak the roots of the plants in 10 μM abscisic acid, 15% (w / v) PEG6000, 1% (v / v) ethephon and 200 μM jasmine acid methyl ester, leaf samples were taken at 0, 6, 12, 24, 48 and 72 hours, ...

Embodiment 3

[0065] Example 3: Obtaining Transgenic Tk1-FEH Gene Rubber Grass Plants and Identification of Root Biomass and Drought Resistance

[0066] This embodiment provides a method for constructing transgenic Tk1-FEH gene rubber grass plants, the specific steps are as follows:

[0067] (1) Using the Tk1-FEH-19T plasmid prepared in Example 1 as a template, Tk1-FEH-EcoRI-F and Tk1-FEH-XbaI-R as primers to amplify the ORF of the Tk1-FEH gene, the amplified product was subjected to EcoRI and After double digestion with XbaI, it was connected to the expression vector pCAMMBIA1300 after the same digestion, and the constructed recombinant plasmid was named Tk1-FEH-OE-1300.

[0068] The sequences of Tk1-FEH-EcoRI-F and Tk1-FEH-XbaI-R are as follows:

[0069] Tk1-FEH-EcoRI-F:5′-CG GAATTC ATGAGCAAGCCTCTTTCCT-3′ (the underline is the EcoRI restriction site)

[0070] Tk1-FEH-XbaI-R:5′-GC TCTAGA TTAAACTGTGCTTTTTACAGT-3' (the underline is the XbaI restriction site).

[0071] (2) The recombin...

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Abstract

The invention relates to a Taraxacum kok-saghyz inulin degrading enzyme gene Tk1-FEH and application thereof. The sequence of the Taraxacum kok-saghyz inulin degrading enzyme Tk1-FEH is shown in SEQ ID NO: 1. According to the invention, Tk1-FEH gene is overexpressed and the function of the gene is studied. The results show that endurance of Taraxacum kok-saghyz genetically modified by the Tk1-FEHgene to drought stress is improved significantly, after 38 days of water cut-off, the Taraxacum kok-saghyz can still maintain a good growth state, and the root biomass increases significantly under normal growth state, with the dry root weight increasing by more than 170%, which indicates that the gene can be used in the research of drought resistance and root biomass improvement of Taraxacum kok-saghyz and other crops. The Taraxacum kok-saghyz inulin degrading enzyme gene Tk1-FEH comes from Taraxacum kok-saghyz, does not produce gene drift, has little impact on the environment, and provides new gene resources for improving drought resistance and root biomass of Taraxacum kok-saghyz and other dandelion plants.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a rubber grass inulin degrading enzyme gene Tk1-FEH and its application. Background technique [0002] Rubber grass (Taraxacum kok-saghyz) is a perennial herbaceous plant of the genus Dandelion in the Compositae family. It is native to Kazakhstan and Xinjiang, China. Its roots can synthesize high-quality natural rubber, and its structure and properties are similar to those produced by the Brasilian rubber tree. . Moreover, rubber grass has the advantages of wide adaptability, short growth and harvest period, rotation or intercropping, suitable for mechanized planting and harvesting, etc., making it a rubber-producing crop with great development prospects. However, the current germplasm of rubber grass is basically wild germplasm, the domestication time is short, there are problems such as low rubber content and biomass, and poor stress resistance. The cost of extract...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/24C12N15/11C12N15/82A01H5/00A01H6/14C12Q1/6895
CPCC12N9/2402C12N15/8261C12N15/8273C12Q1/6895C12Q2600/13C12Q2600/158C12Y302/0108
Inventor 覃碧王锋张立群刘实忠聂秋海张继川王肖肖杨玉双甘霖
Owner RUBBER RES INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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