Homeobox gene HTF1 capable of regulating conidium germination of Magnaporthe grisea of and use thereof

A technology of homeobox and conidia, applied in the field of genetic engineering

Inactive Publication Date: 2009-09-16
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For animals, the homeobox gene plays an important role in embryonic development, gene transcription regulation, cell differentiation, and tumorigenesis; for plants, the homeobox gene mainly regulates embryonic development, leaf development, and intercellular communication. Functio

Method used

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  • Homeobox gene HTF1 capable of regulating conidium germination of Magnaporthe grisea of and use thereof
  • Homeobox gene HTF1 capable of regulating conidium germination of Magnaporthe grisea of and use thereof
  • Homeobox gene HTF1 capable of regulating conidium germination of Magnaporthe grisea of and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] Embodiment 1: HTF1 gene cloning and conserved domain analysis

[0013] According to the HTF1 sequence (MGG_00184.5) published in the Magnaporthe grisea genome database, gene-specific primers were designed, and the full-length sequence of HTF1 DNA was obtained by PCR amplification using the genomic DNA of the wild-type strain 70-15 of Magnaporthe grisea as a template. In addition, using 70-15 total RNA as a template, the full-length sequence of HTF1 cDNA was amplified by reverse transcription and cloned into the pGEM-T easy vector of Promega Company. The sequencing results showed that it was consistent with the sequence published in the rice blast fungus genome database. The results of Southern hybridization showed that the gene was a single-copy gene in Magnaporthe grisea. According to bioinformatics prediction, HTF1 possesses the unique domains of all homeobox proteins, that is, it contains a typical three-helix structure, and has a helix-turn-helix spatial structure b...

Embodiment 2

[0014] Example 2: HTF1 gene knockout

[0015] The invention uses a homologous recombination method to knock out the HTF1 gene. First construct the target gene knockout vector. The specific method is as follows: first, using the genomic DNA of strain 70-15 as a template, using primers 1AF: 5'-CGGAGCTCCCCAGCTATGGTCAAATCA-3', 1AR: 5'-CCCAAGCTTCCTTCTATCTCCTTTGGTCGT-3' and 1BF: 5'-CCGCTCGAGCACCACGGGACTACAACAC-3', 1BR: 5 '-GGGGTACCACCAAATGCTCGCTCACTA-3' were respectively amplified to obtain fragments on the left and right sides of the ORF of the HTF1 gene as homologous recombination homologous fragments, the sizes of which were 910bp and 810bp respectively. Fragments A and B obtained by PCR amplification were double-digested with HindIII, SacI, KpnI, and XhoI, respectively, purified and recovered, and used for the construction of a knockout vector. The construction process includes conventional enzyme digestion, ligation, transformation and further PCR and enzyme digestion verific...

Embodiment 3

[0016] Example 3: Functional complementation of HTF1 knockout mutants

[0017] In order to confirm that the abnormal growth and development of conidia of positive knockout transformants is caused by the knockout of HTF1 gene, this study used yeast homologous recombination method to construct the fusion expression vector of HTF1 gene promoter and GFP gene, At the same time, it is also the functional complement carrier of HTF1. The specific method is to first amplify from the promoter to the end of the gene, and the fragment of the terminator must be removed. The promoter is the sequence 1500 bp before the start codon of the gene. The 5' end of the primer is added with a linker more than 20 bp homologous to the front-end sequence of the GFP gene of pKB04, which is required for homologous recombination. The Advantage 2 Polymerase Mix of Clontech Company was used for amplification, and the amplified PCR fragments were gel-recovered and purified. The amplification primers are 1NF...

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Abstract

The invention provides a homeobox gene HTF1 capable of regulating conidium germination of Magnaporthe grisea of and use thereof. Gene clone shows that the homeobox gene HTF1 is same to HTF1 sequence (MGG_00184.5) published by the Magnaporthe grisea genome database and comprises the typical structure domain of homeobox protein. The gene knock-out mutant losses the capability of generating conidium and conidiophore increases, and the rice inoculated by hypha block generates pathogenicity. The gene knock-out mutant restores the capability of generating conidium after function complementation test. The homeobox structure domain sequence is knocked out from the HTF1 gene therefore the Magnaporthe grisea can not generate conidium. The HTF1 gene can be used as the molecule target of the new pesticide design. The HTF1 mutant strain can be produced into engineering bacteria, thereby gradually reducing the stock number of the natural Magnaporthe grisea and playing important role in preventing and treating rice blast.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to knockout of the rice blast fungus homeotype box HTF1 gene, functional complementation and application thereof. Background technique [0002] Rice blast caused by Magnapothe grisea (Herbert) Barr (asexual generation Pyricularia grisea) infecting rice is one of the most important diseases on rice. The sexual generation is a haploid heterothallic ascomycete. The fungus can infect more than 50 kinds of grass plants, including many weeds and important cereal crops such as wheat, barley, corn, etc., and also cause certain economic losses. Due to the importance of the disease and the operability of traditional and molecular inheritance of Magnaporthe grisea, it has become one of the ideal model systems for studying the interaction between plant pathogenic fungi and hosts. However, due to the complex pathogenicity and variability of rice blast fungus, high-resistant rice varieties oft...

Claims

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Application Information

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IPC IPC(8): C12N15/31C07K14/37C12Q1/68C12R1/645
Inventor 王宗华许金荣刘文德谢世勇鲁国东
Owner FUJIAN AGRI & FORESTRY UNIV
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