Application of wheat transcription factor wrky70 gene in regulating plant growth and development

A transcription factor and plant growth technology, applied in the field of genetic engineering, to increase the number of spikes, reduce the number of tillers, and increase the yield

Active Publication Date: 2022-06-03
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 regulating plant growth and development
  • Application of wheat transcription factor wrky70 gene in regulating plant growth and development
  • Application of wheat transcription factor wrky70 gene in regulating plant growth and development

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Acquisition of gene sequence of wheat transcription factor WRKY70 and construction of plant expression vector.

[0022] Log on 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 in SEQID NO.1 ), and PCR amplification primers were designed according to the CDS of the WRKY70 gene:

[0023] WRKY70-F: 5'-ATGGCGGCACTTGTCACTCC-3' (SEQ ID NO. 2)

[0024] WRKY70-R: 5'-CTACTTATCGTCGTCATCCTTGT-3' (SEQ ID NO. 3)

[0025] The wheat variety Xiaoyan 6 was cultivated, and 14 days after germination, the leaves of the first leaf were inoculated with the leaves of the physiological race of stripe rust No. 32, and the total RNA of the plant was extracted by the TRIzol method, and the cDNA was obtained by reverse transcription PCR. Amplify the target sequence using cDNA as template.

[0026] The specific steps o...

Embodiment 2

[0043] Example 2 Acquisition of Transgenic Wheat Plants

[0044] pWMB006 (Ampicillin resistance, see figure 1 ) plasmid intron Osintron, ligated into the full-length coding sequence of the WRKY70 gene. After the ligation is successful and the sequencing is correct (see Appendix 1 for sequencing primers), the large fragment is recovered by double-cutting with HindIII and EcoRI, and then ligated to pCAMBIA3301 (Kanamycin resistance, see the double-cut with HindIII and EcoRI). figure 2 ) vector, and after the successful construction, Agrobacterium EHA105 was used to transfer into the immature wheat embryos.

[0045] Agrobacterium EHA105 infection: Agrobacterium was shaken at 160 rpm at 28°C the day before the test. Take 1ml of bacterial liquid into a 1.5ml centrifuge tube, add 1.4ul of acetosyringone (0.1M) and mix. Take the ears of wheat (Fielder, purchased from Zhongji High-Tech (Beijing) Biotechnology Co., Ltd.) pollinated for about 15 days, take the seeds and strip the em...

Embodiment 3

[0048] Example 3 Verification of Transgenic Wheat

[0049] The genomic DNA was extracted from the leaves of the transgenic plants and verified by PCR to confirm that the WRKY70 DNA fragment was inserted into the wheat genome.

[0050] Extraction of genomic DNA:

[0051]Take 50 mg of normal growing wheat leaves to be tested and grind them, add 400 μL of plant genomic DNA extract (200 mM Tris-Cl pH7.4, 250 mM NaCl, 25 mM EDTA, 1% SDS), vortex and mix, and centrifuge at high speed for 5 min. Add an equal volume of isopropanol, precipitate at room temperature for 30 min, discard the supernatant after high-speed centrifugation for 5 min, wash the precipitate twice with 70% ethanol, and dissolve the precipitate in 20 μL of nuclease-free ddH 2 O.

[0052] PCR amplification using specific primers of the pWMB006 vector:

[0053] PCR was performed using the genomic DNA extracted above as a template. The system included 4 μL of 5* buffer, 1 μL of upstream primer and 1 μL of downstream...

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Abstract

The application of the wheat transcription factor WRKY70 gene in regulating plant growth and development, by constructing the CDS sequence of the wheat WRKY70 transcription factor gene to the upstream of the overexpression vector with the FLAG protein tag through enzyme-cut ligation technology, and transforming wheat plants, screening and identification , and finally obtain transgenic plants. The analysis of the screened plants found that it has the phenotype of increasing the number of wheat spikes, reducing the plant height, and reducing the effective tillering, that is, the gene affects the yield of the plant. Homologous genes in crops may have the same or similar functions, so they are of great significance in production.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular 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 crops in the world, and its 17% planting area provides more food than 30% of the global population and 20% of energy consumption. In my country, wheat is not only one of the most important carbohydrate sources for people, but also plays a pivotal role in food security. With the increase in 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, heredity, which is the most complex yield factor of wheat, has increasingly become the main bottleneck restricting the improvement of wheat yield and has not been effectively solved. Therefore, it has become the consensus of everyone to promote the stability of gra...

Claims

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Application Information

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Patent Type & Authority Patents(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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