Calprotectin gene of Meloidogyne graminicola and application of calprotectin gene

A root-knot nematode and calreticulin technology, which can be used in applications, nematicides, genetic engineering, etc., can solve problems such as large side effects, economic losses, and agricultural production hazards

Active Publication Date: 2020-05-22
SUZHOU CITY INVASIVE PEST PREVENTION & CONTROL TECH CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Root-knot nematodes have a wide range of hosts and strong adaptability, causing serious damage to agricultural production and causing huge economic losses
Crop rotation, biological fumigation, biological control and chemical pesticides are the main methods to control nematodes, but there are problems such as poor control effect and environmental pollution
There are outstanding problems such as poor specificity, large side effects, and limited control effect

Method used

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  • Calprotectin gene of Meloidogyne graminicola and application of calprotectin gene
  • Calprotectin gene of Meloidogyne graminicola and application of calprotectin gene
  • Calprotectin gene of Meloidogyne graminicola and application of calprotectin gene

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1: DNA extraction of root-knot nematode Pseudomonas graminearum

[0030] 1. Extraction of total RNA

[0031] Use TRIZOL Reagent to extract total RNA, the steps are as follows:

[0032] 1) ddH used in the RNA extraction process 2 O, centrifuge tubes and tips, etc. are treated with 0.1% DEPC.

[0033] 2) Collect root-knot nematode Pseudomonas graminearum in a 1.5mL centrifuge tube, freeze quickly in liquid nitrogen, add 200 μL Trizol, grind it thoroughly with a micro tissue homogenizer, then add 800 μL Trizol, mix well, and let stand at room temperature for 5 minutes;

[0034] 3) Add 200 μL of chloroform, shake fully, place at room temperature for 3 minutes, and centrifuge at 12,000 rpm at 4°C for 15 minutes;

[0035] 4) Pipette the supernatant into another 1.5mL centrifuge tube, add 550μL isopropanol, mix well, let stand at room temperature for 10min, and centrifuge at 12000rpm at 4°C for 10min;

[0036] 5) Discard the supernatant, add 1mL 75% ethanol to e...

Embodiment 2

[0046] Embodiment 2: the cloning of calreticulin gene (MgCRT) of root-knot nematode calreticulin (MgCRT)

[0047]The M. graminearum transcriptome data were spliced ​​and compared one by one, and a 1712bp sequence was obtained, which was highly similar to the M. incognita CTR sequence, and the sequence already contained the complete 5' end and ORF of the CRT gene. Sequence, the 3' sequence is incomplete, according to this sequence, design and verify primers, see Table 1, use the cDNA obtained in Example 1 to perform PCR amplification.

[0048] Table 1 Primer Sequence

[0049] Primer sequence Mgcrt-F-57 5′-ACGAAGATTAAGAATGGCCG-3′ Mgcrt-R-656 5′-CTTTCTGCCTTTTCGCCATC-3′ Mgcrt-ORF-F 5′-TCTAAACATAAGAGCGACTACGG-3′ Mgcrt-ORF-R 5′-GTGCGGTATTTGGTAGGGAG-3′

[0050] PCR reaction system: 1 μL cDNA, 1 μL primer F (10 μM), 1 μL primer R (10 μM), 2.5 μL 10×Buffer, 2 μL dNTP (2.5 mM), 0.2 μL Taq, 16.3 μL dd H 2 O.

[0051] PCR reaction conditions:...

Embodiment 3

[0056] Embodiment 3: RACE technology clones the full-length sequence of MgCRT gene

[0057] According to the obtained calreticulin gene sequence of root-knot nematode graminearum, the specific primer Mgcrt-R3' (5'-ACATTTGCGGACCTGGAAC-3') and the 3' adapter primer were designed for nested PCR. reaction to amplify the 3' end sequence of the target gene. According to 3′-Full RACE Core Set with PrimeScript TM RTase (TAKARA) operating instructions, use 3'RACE Adapter primers for reverse transcription reaction, synthesize 1st Strand cDNA; use specific primers and 3'RACE Outer Primer for PCR reaction to obtain target fragments for sequencing, and compare the obtained sequences with known sequences The full-length sequence of calreticulin (MgCRT) cDNA was obtained by splicing. The full length of MgCRT gene cDNA is 1715bp, and its nucleotide sequence is shown in SEQ ID NO.1, which contains an open reading frame (ORF) of 1248bp. The cDNA open reading frame encodes 415 amino acids, an...

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Abstract

The invention discloses a calprotectin gene of Meloidogyne graminicola and application of the calprotectin gene, and belongs to the technical field of biology. The calprotectin gene is a calprotectingene MgCRT, disclosed for the first time, of the Meloidogyne graminicola, a nucleotide sequence of the calprotectin gene MgCRT is represented by SEQ ID NO.1, and an amino acid sequence of an expression protein of the calprotectin gene MgCRT is represented by SEQ ID NO.2. Experiments prove that the gene is expressed in subabdominal esophageal glands of the Meloidogyne graminicola, dsRNA of the geneis designed and synthesized, Meloidogyne graminicola larvae are steeped in a dsRNA solution, are washed and are inoculated to rice, and after the gene MgCRT of the Meloidogyne graminicola is silencedby virtue of a method, nematodes invading rice are remarkably decreased, so that the calprotectin gene MgCRT of the Meloidogyne graminicola is relevant with invasion hosts of the Meloidogyne graminicola, and the gene MgCRT can be utilized as a target for preventing and controlling the invasion of the Meloidogyne graminicola on crops by virtue of an RNA interference technique.

Description

technical field [0001] The invention belongs to the field of biological technology, and more specifically relates to a calreticulin gene of root-knot nematode graminae and its application. Background technique [0002] Root-knot nematodes (Meloidogyne spp.) are a class of sessile endoparasitic plant pathogenic nematodes. Root-knot nematode has a wide range of hosts and strong adaptability, which seriously harms agricultural production and causes huge economic losses. Crop rotation, biological fumigation, biological control and chemical pesticides are the main methods to control nematodes, but there are problems such as poor control effect and environmental pollution. There are outstanding problems such as poor specificity, large side effects, and limited control effect. [0003] Calreticulin has been found in humans and various animal parasitic nematodes and has been confirmed to be related to nematode parasitism and human diseases. In particular, it has been found that in...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/113C12N15/11C07K14/435A01N57/16A01P5/00
CPCC07K14/4354C12N15/113A01N57/16C12N2310/14
Inventor 邓艳凤田忠玲
Owner SUZHOU CITY INVASIVE PEST PREVENTION & CONTROL TECH CENT
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