Application of wheat stripe rust pstg_06371 gene in stripe rust control and breeding method of stripe rust resistant wheat

A technology for wheat stripe rust and wheat stripe rust, applied in the field of genetic engineering and crop molecular breeding, can solve the problems of wheat-stripe rust genetics research difficulties, small number of key pathogenic genes, and difficult genetic transformation, etc. Effects of wheat stripe rust resistance

Active Publication Date: 2020-07-14
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Due to its biotrophic and obligate parasitic characteristics, genetic transformation of wheat stripe rust is difficult; and wheat itself is also difficult to carry out genetic transformation, so the genetic research of wheat-stripe rust interaction is very difficult and the progress is slow
At present, the number of infection or pathogenic key genes identified in wheat stripe rust is relatively small, and no genes have been effectively used in wheat disease resistance breeding

Method used

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  • Application of wheat stripe rust pstg_06371 gene in stripe rust control and breeding method of stripe rust resistant wheat
  • Application of wheat stripe rust pstg_06371 gene in stripe rust control and breeding method of stripe rust resistant wheat
  • Application of wheat stripe rust pstg_06371 gene in stripe rust control and breeding method of stripe rust resistant wheat

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] (1) PCR amplification to obtain the PSTG_06371 gene fragment

[0045]Taking the complete genome sequence of wheat stripe rust PST-78 as a reference (GenBank accession number AJIL00000000.1 or BROAD download link ftp: / / ftp.broadinstitute.org / pub / annotation / fungi / puccinia / genomes / pucciniastriifor misspst- 78 / ), using the sequence of hypothetical protein gene PSTG_06371 (GenBank: KNF00443.1) as a template to design PCR primers. The sequence of the forward primer CQM06371-F1 is shown in Seq ID No.3: 5`-CTTCTTTTCCCTGATATTCCTACCA-3`; the sequence of the reverse primer CQM06371-R1 is shown in Seq ID No.4: 5`-CAAATTGGAAAAGATGAGGTGT-3`. CQM06371-F1 / R1 amplifies 43 bp downstream of the gene start codon (ATG) to the 3' non-coding region of the gene, and the length of the amplified fragment in the reference genome (physiological race PST-78) is 1074 bp.

[0046] Compared with the gene structure PSTG_06371 annotated in the reference genome PST-78, the transcript amplified in CRY29 ...

Embodiment 2

[0063] Embodiment 2 Anti-stripe rust resistance identification of wheat stripe rust

[0064] Propagation of fresh spores of wheat stripe rust: plant the susceptible variety Huixianhong in the greenhouse, inject the spore aqueous solution with a syringe during the one-leaf-one-heart stage, then spray water with a watering can and cover with a plastic film to keep it moist. Inoculation can be repeated 2-3 times to ensure full onset, with an interval of 1 week between each time. Preparation of the spore aqueous solution: Take the dried and frozen (-80°C) spores, suspend them with an appropriate amount of tap water until they are light orange, place them on a shaker at room temperature and shake them for 30 minutes (180rpm), and then inoculate them by injection. Sporadic disease can be seen about 20 days after inoculation, and a large number of leaves can be seen after about 30 days. A large number of fresh spores can be collected for inoculation identification of transgenic mater...

Embodiment 3

[0069] Example 3 Identification of PSTG_06371 Gene Expression in Stripe Rust-resistant Wheat

[0070] Twelve days after inoculation of wheat seedling leaves, the surface of wild-type CB037 leaves was covered with a large number of spore piles. About 100 mg of leaves of CB037 and transgenic materials were taken at different periods after wheat was inoculated with stripe rust, RNA was extracted by Trizol method, and cDNA was prepared for real-time fluorescent quantitative PCR. The primer sequences are CQM06371-F3 and CQM06371-R3; the specific sequences are shown in the sequence table Seq ID No.10 and Seq ID No.11. Taking the α-tubulin gene of wheat stripe rust as an internal reference (Huang Xueling et al., Journal of Agricultural Biotechnology, 2012, 20(2): 181-187), the primer TUBA-F / R sequences are shown in the sequence table Seq ID No.12, Seq ID No.13 shown.

[0071] PCR system: 5 μl 2×SYBR GreenMaster, 1 μl forward and reverse primers (10 μM), 1 μl cDNA, 2 μl ddH 2 O, th...

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Abstract

The invention provides application of a puccinia striiformis PSTG_06371 gene to prevention and control of wheat stripe rust. The sequence of the PSTG_06371 gene is shown as SEQ ID No. 5; the PSTG_06371 gene can be used for effectively regulating and controlling growth and proliferation of puccinia striiformis; the PSTG_06371 gene is used as a molecular target for transcriptional regulation or a gene target for inhibiting a protein function; the effect of inhibiting the growth and proliferation of the puccinia striiformis is realized through silencing the PSTG_06371 gene. Wheat obtained by themethod for culturing puccinia striiformis resisting wheat by utilizing the gene has remarkable puccinia striiformis resistance.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and crop molecular breeding, and in particular relates to the application of wheat stripe rust PSTG_06371 gene in the prevention and treatment of stripe rust and the cultivation method of stripe rust resistant wheat. Background technique [0002] Wheat is the second largest grain crop in my country, with an average annual sowing area of ​​about 360 million mu. The stability and improvement of wheat production are related to the national economy and people's livelihood. However, for many years, fungal diseases have posed a serious threat to wheat production, such as rust, powdery mildew, scab, sheath blight and other diseases, seriously affecting the yield and quality of wheat. Among them, wheat stripe rust, commonly known as "jaundice", is caused by obligate parasitic stripe rust (Puccinia striiformis Westend.f.sp.tritici). In my country, the incidence of wheat stripe rust is rampant...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/31C12N15/82A01H5/00A01H6/46
CPCC07K14/37C12N15/8218C12N15/8282
Inventor 吴佳洁张会飞黄德华刘强倪飞付道林
Owner SHANDONG AGRICULTURAL UNIVERSITY
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