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Wheat leaf developmental gene TaWRKY76 and application thereof

A gene and leaf technology is applied to the leaf development gene-WRKY transcription factor TaWRKY76 and its application field, which can solve the problem that the function of WRKY gene has not been reported.

Inactive Publication Date: 2013-06-26
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The WRKY gene family is a plant-specific gene family that plays an important role in resistance to diseases and insect pests, but the role of WRKY genes in leaf development has not been reported

Method used

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  • Wheat leaf developmental gene TaWRKY76 and application thereof
  • Wheat leaf developmental gene TaWRKY76 and application thereof
  • Wheat leaf developmental gene TaWRKY76 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1: Cloning of TaWRKY76 gene

[0029] 1.1 Wheat cultivation: germinate Shanrong 3 and Jinan 177 wheat seeds, use Hoagland culture medium, 21 ℃, long-day sunshine (light 16h / darkness 8h), culture to 2 leaves 1 heart stage, and extract RNA;

[0030] 1.2 Extraction of total RNA:

[0031] Reagents and utensils: RNAiso (TaKaRa), chloroform (Chloroform), isopropyl alcohol (Isopropyl alcohol), absolute ethanol (Ethanol), DEPC water, all plastic consumables are treated with DEPC water for 24 hours, 123 ° C, 40 min high pressure damp heat Bacteria, and then dried in an oven at 65°C (about 2 days), glassware and metal utensils were treated at 180°C for 6 hours.

[0032] 1) Material collection: 0.05g of leaves and 0.07g of roots (absorbed with absorbent paper before material collection) were put into a 2ml plastic centrifuge tube, a steel column was added, quick-frozen in liquid nitrogen, and oscillated and ground by an oscillator;

[0033] 2) Add 1.5ml Trizol extract,...

Embodiment 2

[0118] Example 2: Expression profile analysis of TaWRKY76 gene

[0119] 2.1 Material collection: Cultivate Wheat Shanrong 3 and Jinan 177 to the stage of two leaves and one heart according to the method in 1.1, and extract the total RNA of leaves and roots respectively;

[0120] 2.2 Extraction of total RNA, digestion of DNA and reverse transcription of RNA: see 1.2-1.4 for the method

[0121] 2.3 Adjustment of internal reference for semi-quantitative analysis:

[0122] Using cDNA as a template, carry out PCR reaction, the system is slightly modified from 1.5. Primers are as follows

[0123] TaAct-S: 5'-GTTCCAATCTATGAGGGATACACGC-3'

[0124] TaAct-A:5'-GAACCTCCACTGAGAACAACATTACC-3'

[0125] Adjust the internal reference of all samples to a consistent level;

[0126] 2.4 TaWRKY76 gene expression profile analysis: use the above cDNA as a template, and use TaWRKY76 semi-quantitative primers for PCR amplification, and the system is slightly modified from 2.3;

[0127] 2.5 Elec...

Embodiment 3

[0128] Example 3: Subcellular Subcellularization of the TaWRKY76 Gene

[0129] 3.1 Biolistic transformation of onion epidermal cells

[0130] 3.1.1 Onion skin preparation

[0131] Buy fresh onions from the market, culture them in water for two days until they grow new roots, cut off the inner epidermis (3×3cm) with tweezers and a blade in an ultra-clean bench, spread the inner layer on 1 / 2MS with the inner layer facing down, try not to leave with bubbles,

[0132] 3.1.2 Preparation of particle bullets

[0133] 1) Sterilize twice with 70% alcohol lotion tungsten powder, add 50% sterilized glycerin to make the concentration reach 60mg / ml;

[0134] 2) Mix the tungsten powder particle solution well before use, take 25μl into a 1.5ml centrifuge tube, and then add

[0135] 10 μl plasmid DNA (2 μg / μl),

[0136] 100μl 2.5mol / L CaCl 2 ,

[0137] 40μl 0.1mol / L spermidine, shake while adding;

[0138] 3) The mixture was vortexed for 3 minutes;

[0139] 4) Stand still for 1min;

...

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Abstract

The invention discloses a wheat leaf developmental gene TaWRKY76 and an application thereof. The nucleotide sequence of the gene complementary deoxyribonucleic acid (cDNA) is shown in SEQ ID No.1. The invention further discloses an application of the gene TaWRKY76 in culture of plants with curled leaves. Experiments prove that the wheat TaWRKY76 can be introduced into plant cells, so that the plants can obtain a phenotype of curled leaves, advantages are provided for improving photosynthesis, and the gene can be widely applied to culture of high-yield crops varieties.

Description

technical field [0001] The invention belongs to the technical field of biological gene engineering, and in particular relates to a leaf development gene—WRKY transcription factor TaWRKY76 and its application. Background technique [0002] Leaf shape is an important trait closely related to crop yield. The three-dimensional structure of leaves can affect light absorption, carbon fixation and gas exchange for photosynthesis. Properly curled leaves are an important feature of super rice. Proper leaf curling improves photosynthetic efficiency, enhances dry matter accumulation, increases yield, reduces solar damage to leaves, and reduces respiration under drought conditions. Therefore, revealing the mechanism controlling leaf curling is of great significance for crop breeding and stress tolerance. [0003] Leaf curling is regulated by hormones and non-hormonal factors. Existing studies have shown that the auxin methyltransferase AtIAMT1 is closely related to leaf curling, but ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/84A01H5/00
Inventor 夏光敏秦桢
Owner SHANDONG UNIV
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