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Method for improving citrus huanglongbing resistance

A technology for citrus huanglongbing and disease resistance, which is applied in the field of molecular biology, can solve the problems of the antibacterial peptide gene huanglongbing resistance mechanism and the strength is unclear, and no new citrus huanglongbing resistance has been obtained, so as to improve the Disease resistance and yield in citrus, and the effect of enhancing resistance levels

Pending Publication Date: 2017-01-18
SOUTHWEST UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, the resistance mechanism and strength of antimicrobial peptide genes to Huanglongbing disease in plants are still unclear, and there are no reports of new strains resistant to citrus Huanglongbing [5,6,11,14]

Method used

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  • Method for improving citrus huanglongbing resistance
  • Method for improving citrus huanglongbing resistance
  • Method for improving citrus huanglongbing resistance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1 Method introduction

[0029] 1. Extraction of DNA

[0030] Select 0.1-1 g of citrus veins, use Aidlab company kit (cat.No.DN15) to extract genomic DNA, and store at -20°C for later use.

[0031] 2. RNA extraction and cDNA synthesis

[0032] Select 0.1 g of citrus leaf veins and use the EASYspin Plant RNA Rapid Extraction Kit (Aidlab, cat. No. RN09) to extract total RNA from leaves, and use non-denaturing agarose gel electrophoresis and UV spectrophotometer scanning to detect the quality of RNA. cDNA first-strand synthesis according to BIO-RAD iScript TMcDNA Synthesis Kit (BIO-RAD, cat. No. 170-8891) instructions. The synthesized cDNA was stored at -20 for future use.

[0033] 3. PCR Amplification of Genomic Sequence

[0034] 10 Ex PCR buffer (Mg 2+ free) 2.5μL, 2.5mmol / L dNTPs 2μL, 25mmol / L MgCl 2 2 μL, primer 1 (5 μmol / L) 1 μL, primer 2 (5 μmol / L) 1 μL, Ex Taq DNA polymerase 1U, genomic DNA about 60ng, add ddH 2 0 to 25 μL. The amplification prog...

Embodiment 2

[0039] Example 2 Vector construction

[0040] 1. Acquisition of a specific promoter

[0041] According to the cowpea phloem-specific promoter GRP (GenBank accession number: AF250148.1), primers (GRP-f and GRP-r, see Table 5) were designed, and a fragment of about 600 bp was obtained by PCR amplification from the cowpea genome. The sequence analysis after cloning the amplified DNA fragment shows that it is the GRP specific promoter of cowpea, see SEQ ID No.4.

[0042] The primer sequence used in the embodiment of table 5

[0043]

[0044]

[0045] 2. Synthesis of antimicrobial peptide gene spCB

[0046] According to the cecropin gene Cecropin B sequence, there are secreted antibacterial peptide genes CB, PR1aCB and PR1aCBer artificially synthesized by Shanghai Bioengineering Co., Ltd. The ATG at the 5' end of PR1aCB and PR1aCBer contains the signal peptide sequence PR1a from the tobacco PR1a gene, and the plant translation enhancer sequence AAGGAGAUAUAACA is added upst...

Embodiment 3

[0050] Example 3 Genetic Transformation

[0051] 1. Introduce the constructed plant expression vector plasmid into Agrobacterium EHA105 by electric shock method.

[0052] Referring to the Bio-RAD MicroPulser user manual, the above-mentioned vectors GRP::CB, GRP::PRlaCB and GRP::PRlaCBer were introduced into Agrobacterium EHA105 by electric shock transformation method.

[0053] 2. The vector specifically expressing the antimicrobial peptide gene was integrated into the citrus genome

[0054] The genetic transformation of citrus was carried out by the method mediated by Agrobacterium tumefaciens, and the medium used is shown in Table 1.

[0055] Table 1 Medium for genetic transformation of citrus mediated by Agrobacterium tumefaciens

[0056]

[0057]

[0058] MS: Murashige & Skoog medium (Murashige & Skoog, 1962); Gelrite: plant gel (Sigma, product number: G1910); BA: 6-benzylaminopurine; IAA; 3-indoleacetic acid; 2,4-D: 2,4- Dichlorophenoxyacetic acid; Cef: cephalospo...

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Abstract

The invention belongs to the technical field of molecular biology, in particular relates to a method for improving citrus huanglongbing resistance, and aims to solve the technical problem of providing a new choice for improving the citrus huanglongbing resistance. The technical scheme of the invention is the method for improving the citrus huanglongbing resistance. The method is particularly as follows: integrating an antibacterial peptide gene into a citrus genome under the regulation and control of a phloem specific promoter, wherein the antibacterial peptide gene is an antibacterial peptide gene PR1aCB, CB or PR1aCBer; the CB has the nucleotide sequence as shown in SEQ ID No.1; the PR1aCB has the nucleotide sequence as shown in SEQ ID No.2; the PR1aCBer has the nucleotide sequence as shown in SEQ ID No.3. The phloem specific promoter guides resistance genes, such as the antibacterial peptide and the like, to be efficiently expressed in the phloem, so that the citrus huanglongbing resistance level is enhanced.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a method for improving resistance to citrus huanglongbing. Background technique [0002] Citrus huanglongbing is one of the most devastating diseases of citrus production in the world. It can harm almost all citrus varieties. According to statistics, citrus huanglongbing has spread in 19 provinces and autonomous regions in my country, the affected area accounts for more than 80% of the total citrus cultivation area, and the output accounts for about 85% of the total output. Citrus Huanglongbing is caused by Gram-negative bacteria of the genus Cadidatus liberobacter, including Cadidatus liberobacterasiatium, Cadidatus liberobacteriafricanum, and Cadidatus liberobacteramericanus [2] . The psyllid is the only insect vector for the pathogen of Huanglongbing to enter the plant host. In addition, the pathogen can also spread long distances between different pla...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/62C12N15/12A01H5/00
CPCC12N15/8223C07K14/43586C07K2319/02C07K2319/04C12N15/8281
Inventor 邹修平许兰珍陈善春彭爱红何永睿雷天刚姚利晓
Owner SOUTHWEST UNIV
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