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Barley leaf rust resistance protein and its coding gene and application

A gene and coding technology, applied in the field of barley leaf rust resistance related protein and its coding gene and application, can solve the problem of short sequence length and so on

Active Publication Date: 2019-01-08
杭州禾骑士未来生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Its accuracy rate can reach up to 99.99%, but the sequence length is short

Method used

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  • Barley leaf rust resistance protein and its coding gene and application
  • Barley leaf rust resistance protein and its coding gene and application
  • Barley leaf rust resistance protein and its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Example 1, Cloning and sequencing of barley leaf rust resistance genes Rlr1 and Rlr2

[0090] The total RNA of barley variety Vada adult plant leaves was extracted with RNAprep Pure Plant Total RNA Extraction Kit (Tiangen), and the total RNA of barley leaves was reverse transcribed into cDNA with reverse transcription kit SuperScriptTMⅢRT (Invitrogen).

[0091] The method for reverse transcription with the reverse transcription kit SuperScriptTMⅢRT (Invitrogen) is as follows:

[0092] (a) Add the following reagents in sequence to the RNA-free PCR tube: Oligo(dT)18 (500μg / ml) 1μl; total RNA 1-5μg; dNTP mix (10mM) 1μl; DEPC H 2 O to make up to 13 μl. Incubate at 65°C for 5 minutes, quickly ice-bath for 2 minutes, and centrifuge slightly.

[0093] (b) Add the following reagents sequentially to the above PCR tube: 4 μl of 5×First-Strand buffer; 1 μl of 0.1M DTT; 1 μl of RNaseOUTTM (40U / μl); 1 μl of SuperScriptTMIII (200U / μl). Reverse transcription at 50°C for 50min.

[...

Embodiment 2

[0102] Expression pattern analysis of embodiment 2, Rlr1 and Rlr2

[0103] The primers for fluorescent quantitative PCR were designed according to the requirement of fluorescent quantitative PCR analysis primers and the CDS sequences of Rlr1 and Rlr2 genes, and the expression of barley ubiquitin conjugating enzyme 18 (Hv-UBC18) gene was used as an internal reference. Primer sequences are shown in Table 1.

[0104] Table 1 Primer sequences used in the analysis of expression patterns of Rlr1 and Rlr2

[0105] Primer name

Primer sequence (5'→3')

Location

Rlr1-QPCR-F

GTACGCTTGCAATACTGACCATC

734-756 of sequence 3

Rlr1-QPCR-R

TCCAAAAGAGACCAGAAGCAATAC

No. 1076-1099 of sequence 3

Rlr2-QPCR-F

ATGCAGCGACGTGGCAC

Positions 1-17 of sequence 4

Rlr2-QPCR-R

GCTCGGAAATTGTCTAGACTG

150-170 of sequence 4

Hv-UBC18-F

CGATGCACCCGCACATTTACAG

Hv-UBC18-R

CGTTGCGGCAGTTCCTAAC

[0106] The barl...

Embodiment 3

[0111] The virus-induced gene silencing (VIGS) experiment of embodiment 3, Rlr1 and Rlr2

[0112] 1. Construction of recombinant vector

[0113] According to the CDS sequences of Rlr1 gene and Rlr2 gene, primers with LIC linkers were designed (see Table 2), and the VIGS fragments of Rlr1 gene and Rlr2 gene were amplified from the leaf cDNA of barley variety Vada, and the sizes were 393bp and 465bp (including LIC Linker sequence) DNA fragments, the band of interest was recovered using Tiangen's gel recovery kit. Simultaneously extract the pCa-γbLIC plasmid (the pCa-γbLIC plasmid contains the DNA sequence corresponding to the γRNA of barley stripe mosaic virus (BMSV), and at the same time introduce the LIC junction site downstream of the γb gene in the plasmid for cloning foreign gene fragments) , and digested with NEB ApaI to linearize the plasmid, and recover the target band (denoted as pCa-γbLIC / ApaI) with Tiangen gel extraction kit.

[0114] Table 2 Primers used in virus-i...

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Abstract

The invention discloses a protein related to barley leaf rust resistance as well as a coding gene and application thereof. The protein provided by the invention is protein A and / or protein B, wherein the protein A is a protein described as the sequence 1 or a leaf rust resistance-related protein of which the amino acid sequence is replaced and / or deleted and / or added through one or more amino acid residues; the protein B is a protein described as the sequence 2 or a leaf rust resistance-related protein of which the amino acid sequence is replaced and / or deleted and / or added through one or more amino acid residues. Experiment results prove that the transgenic barley plant simultaneously expressing Rlr1 gene and Rlr2 gene has obviously enhanced resistance for leaf rust; the barley leaf rust resistant protein has a wide market and application prospect in the industrial fields of genetic improvement of cash crops, particularly barley.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to two proteins related to barley leaf rust resistance, coding genes and applications thereof. Background technique [0002] Leaf rust is a widely distributed disease of wheat crops, found in most wheat and barley production areas in the world. In barley production, barley leaf rust can cause 30% yield reduction in normal years, and up to 60% yield reduction in epidemic years. Research and practice have proved that using disease-resistant genes and cultivating disease-resistant varieties is the most economical, safe, effective and environmentally friendly measure to prevent and control crop leaf rust. [0003] To apply the disease resistance gene, first clone the disease resistance gene. In recent years, the cloning methods of plant disease resistance genes have been continuously developed along with the progress of molecular biology technology and high-throughput sequencing techno...

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
CPCC07K14/415C12N15/8282
Inventor 漆小泉王亚军
Owner 杭州禾骑士未来生物科技有限公司