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Application of wheat transcription factor WRKY70 gene in regulation and control of plant growth and development

A transcription factor, plant growth technology, applied in the field of genetic engineering, to achieve the effect of reducing plant height, increasing the number of grains per ear, and increasing yield

Active Publication Date: 2021-06-25
XUZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, the inheritance of the yield factor, which is the most complex trait of wheat, has increasingly become the main bottleneck limiting the improvement of wheat yield and has not been effectively solved.

Method used

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  • Application of wheat transcription factor WRKY70 gene in regulation and control of plant growth and development
  • Application of wheat transcription factor WRKY70 gene in regulation and control of plant growth and development
  • Application of wheat transcription factor WRKY70 gene in regulation and control of plant growth and development

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Obtaining the gene sequence of wheat transcription factor WRKY70 and constructing a plant expression vector.

[0022] Log in to the NCBI Information Resource Database website https: / / www.ncbi.nlm.nih.gov / nuccore / KY784578.1 to find the sequence of the transcription factor WRKY70 gene (GenBank No: KY784578.1, the nucleic acid sequence is shown as SEQID NO.

[0023] 1), design PCR amplification primers according to the CDS of WRKY70 gene:

[0024] WRKY70-F:5'-ATGGCGGCACTTGTCACTCC-3' (SEQ ID NO.2)

[0025] WRKY70-R:5'-CTACTTATCGTCGTCATCCTTGT-3' (SEQ ID NO.3)

[0026] Cultivate Xiaoyan No. 6 wheat variety, 14 days after germination, inoculate the leaves of Physiological Race 32 on the first leaf as material, extract the total RNA of the plant by TRIzol method, obtain cDNA by reverse transcription PCR, and then The target sequence is amplified using cDNA as a template.

[0027] The specific steps for extracting RNA are as follows: refer to Plant RNA Reagent ins...

Embodiment 2

[0045] The acquisition of embodiment 2 transgenic wheat plants

[0046] Cut out pWMB006 (amphiliptin resistance, see figure 1 ) The intron Osintron of the plasmid is connected into the full-length coding sequence of the WRKY70 gene. After the connection was successful and the sequencing was correct (see Appendix 1 for the sequencing primers), the large fragment was recovered by double cutting with HindIII and EcoRI, and then connected to pCAMBIA3301 (kanamycin resistance, see figure 2 ) vector, after the successful construction, use Agrobacterium EHA105 to transfer into young wheat embryos.

[0047] Infection of Agrobacterium EHA105: the day before the test, the Agrobacterium was shaken at 160 rpm at 28°C. Take 1ml of bacterial solution in a 1.5ml centrifuge tube, add 1.4ul of acetosyringone (0.1M) and mix well. The ears of wheat (Fielder, purchased from Zhongji High-tech (Beijing) Biotechnology Co., Ltd.) about 15 days after pollination were taken, the grains were removed...

Embodiment 3

[0050] Verification of embodiment 3 transgenic wheat

[0051] Genomic DNA was extracted from the leaves of transgenic plants and verified by PCR to confirm that the WRKY70 DNA fragment was inserted into the wheat genome.

[0052] Genomic DNA extraction:

[0053]Take 50 mg of normal growing wheat leaves to be tested, grind them, add 400 μL of plant genomic DNA extraction solution (200 mM Tris-Cl pH7.4, 250 mM NaCl, 25 mM EDTA, 1% SDS), vortex and mix well, centrifuge at high speed for 5 min, and pipette Then add an equal volume of isopropanol, precipitate at room temperature for 30 minutes, discard the supernatant after high-speed centrifugation for 5 minutes, wash the precipitate twice with 70% ethanol, and dissolve the precipitate in 20 μL of nuclease-free ddH 2 O.

[0054] Utilize the specific primer of pWMB006 carrier to carry out PCR amplification:

[0055] PCR was performed using the above-mentioned extracted genomic DNA as a template, and the system included 4 μL of 5...

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Abstract

The invention provides application of the wheat transcription factor WRKY70 gene in regulation and control of plant growth and development, a CDS sequence of the wheat transcription factor WRKY70 gene is constructed to the upstream of an overexpression vector with an FLAG protein tag through an enzyme digestion connection technology, a wheat plant is transformed, screened and identified, and finally a transgenic plant is obtained. The screened plants are analyzed and found to have phenotypes of increasing the grain number per ear of wheat, reducing the plant height and reducing effective tillering, that is, the gene affects the yield of the plants. Homologous genes in crops may have the same or similar functions, so that the genes have important significance in production.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to the application of wheat transcription factor WRKY70 gene in cultivating high-yield plants. Background technique [0002] Wheat is one of the three most important staple food crops in the world. Its 17% planting area provides more food than 30% of the global population supply and 20% of energy consumption. In my country, wheat is not only one of the most important sources of carbohydrates, but also plays a pivotal role in food security. With the increase of the global population, the demand for wheat is increasing, and the contradiction between supply and demand is becoming more and more prominent. At the same time, the inheritance of yield factors, which is the most complex trait of wheat, has increasingly become the main bottleneck limiting the improvement of wheat yield and has not been effectively resolved. Therefore, it has become everyone's consensus to prom...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00A01H6/46
CPCC07K14/415C12N15/8261Y02A40/10
Inventor 王军娟徐明真陈童童徐靓蒋继宏
Owner XUZHOU NORMAL UNIVERSITY
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