Wheat drought-sensitive gene TaANTHSYS1 and application thereof

A gene and wheat technology, applied to the wheat drought-sensitive gene TaANTHSYS1 and its application fields, can solve the problems of few reports on the role of flavonoid synthesis-related genes and changing the drought resistance of plants.

Pending Publication Date: 2020-12-22
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The search shows that some genes that can significantly change the drought resistance of plants have been found, but there are few reports on the r

Method used

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  • Wheat drought-sensitive gene TaANTHSYS1 and application thereof
  • Wheat drought-sensitive gene TaANTHSYS1 and application thereof
  • Wheat drought-sensitive gene TaANTHSYS1 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026]Example 1. Cloning and expression analysis of TaANTHSYS1

[0027]1.1 Extract Wheat Total RNA

[0028]1. Put the wheat tissue material into a mortar pre-cooled with liquid nitrogen and grind it into powder in liquid nitrogen;

[0029]2. After the liquid nitrogen evaporates and dry, immediately transfer it to a 2ml centrifuge tube. Add 1ml of Invitrogen's TRIzol extract for every 100mg of material. After thawing, use the sample gun to repeatedly suck and blow, vigorously shake and mix the sample. Fully lyse, leave it at room temperature for 5 minutes;

[0030]3. Add 0.2ml of chloroform, vigorously shake and mix for 15 seconds, and place at room temperature for 10 minutes;

[0031]4. Centrifuge at 12000rpm for 15 minutes at 4°C;

[0032]5. Use a pipette to carefully aspirate the upper aqueous phase, add to a new 1.5ml centrifuge tube, add 500μl of isopropanol (1:1 volume), mix well, -20℃, precipitation for 30min or overnight;

[0033]6. Centrifuge at 4°C, 12000rpm for 10min, and discard the supernata...

Embodiment 2

[0071]Example 2. Construction of plant expression vector

[0072]Use plant expression vector p3426, select XbaI and BamHI to double digest p3426 and Blunt vector containing the target gene respectively; recover the large vector fragment and small target gene fragment, use T4 After DNA ligase ligation, the competent cells of Escherichia coli DH10B are transformed, and the plant expression vector with the target gene is obtained after the recombinant is identified.

[0073]1. Double enzyme digestion, taking p3426 empty vector and Blunt as examples

[0074]The p3426 empty vector and Blunt-T plasmid were extracted by alkaline lysis, and 10μg of each was digested. The digestion system is as follows:

[0075]

[0076]Enzyme digestion in a constant temperature water bath at 30°C for more than 2 hours. After double digestion, 1×TAE was used as the electrophoresis buffer, and the digested product was subjected to 0.8% agarose gel electrophoresis. The large fragment of p3426 vector and the target gene band ...

Embodiment 3

[0086]Example 3. Preparation and transformation of Agrobacterium competence

[0087]3.1 Preparation of Agrobacterium AGL1 / EHA105 Competent

[0088]1. Pick a single colony of Agrobacterium tumefaciens from a YEP plate (containing 50μg / ml rifampicin), and inoculate it in YEP liquid medium (containing 50μg / ml rifampicin) at 200 rpm / min and culture overnight at 28°C.

[0089]2. Inoculate 2ml overnight culture medium in 50ml YEP liquid medium containing the same antibiotics and cultivate to OD under the same conditions600Up to 0.5.

[0090]3. Bacterial liquid is ice-bathed for 30 minutes, centrifuged at 4°C, 5000 rpm for 10 minutes, and the bacteria are collected.

[0091]4. Suspend the bacteria body in 10ml of 0.15mol / L NaCl in an ice bath, and collect the bacteria by centrifugation.

[0092]5. Resuspend in 1ml 20mmol / L ice pre-cooled CaCl2In the solution, aliquot 200μl / tube into 1.5ml Eppendorf tubes, quickly freeze in liquid nitrogen for 1 min, and store at -70°C for later use.

[0093]3.2 Transformation ...

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Abstract

The invention discloses a wheat drought-sensitive gene TaANTHSYS1, a plant gene editing related vector containing the gene and an application of the gene to cultivation of drought-tolerant plants. Experiments prove that when the gene is transferred into wheat by an agrobacterium tumefaciens mediated method, the drought resistance of an obtained transgenic plant is obviously reduced, it is proved that the provided gene can be used as a gene editing target, then drought-resistant wheat is obtained by a gene editing method, and wide application prospects are achieved.

Description

Technical field[0001]The invention belongs to the technical field of biological genetic engineering, relates to wheat flavonoid synthesis-related genes and their effects in plant drought processes, and particularly relates to a wheat drought sensitive gene TaANTHSYS1 and applications thereof.Background technique[0002]Drought severely affects crop yields, especially with the development of industry, the shortage of fresh water resources has become more and more serious, making drought stress a social issue of global concern. my country has a large population, and the drought disaster is more serious, which has become an important factor restricting my country's economic and social development. Therefore, cultivating new varieties of drought-resistant crops has become a very urgent task.[0003]The use of transgenic technology to improve plants and transfer new traits into high-biomass plants to develop highly efficient new varieties of transgenic plants is a technology with broad appli...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/84A01H5/00A01H6/46
CPCC07K14/415C12N15/8273
Inventor 王勐骋夏光敏
Owner SHANDONG UNIV
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