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CN-121801955-B - Method for improving citrus canker resistance by CsENPP gene

CN121801955BCN 121801955 BCN121801955 BCN 121801955BCN-121801955-B

Abstract

The invention belongs to the technical field of molecular biology, and particularly discloses a method for improving citrus canker resistance by utilizing CsENPP gene, wherein the coded protein of CsENPP gene is citrus nucleotide pyrophosphatase/phosphodiesterase 1, the CsENPP1 coded sequence is a nucleotide sequence shown as SEQ ID No.1, and the citrus canker resistance is improved based on overexpression of CsENPP gene in citrus cells. The invention integrates a gene encoding citrus nucleotide pyrophosphatase/phosphodiesterase 1 into citrus through an expression vector, the incidence of canker of the obtained transgenic material can be reduced to 48.9% of the incidence of canker of the prior citrus, the incidence of canker can be obviously reduced, and the area of lesions is reduced.

Inventors

  • LI QIANG
  • CHEN SHUANGLING
  • SONG QINGWEI
  • ZHANG MIAO
  • PENG AIHONG
  • LEI TIANGANG
  • HE YONGRUI

Assignees

  • 西部(重庆)科学城种质创制大科学中心

Dates

Publication Date
20260512
Application Date
20260311

Claims (8)

  1. 1. A method for improving citrus canker resistance by using CsENPP gene, wherein the protein coded by CsENPP gene is citrus nucleotide pyrophosphatase/phosphodiesterase 1, the csenpp1 coding sequence is a nucleotide sequence shown as SEQ ID No. 1, and the citrus canker resistance is improved based on overexpression of CsENPP gene in citrus cells.
  2. 2. A method for increasing citrus canker resistance using CsENPP gene as claimed in claim 1, comprising the steps of: (1) Cloning a citrus CsENPP coding sequence; (2) Constructing CsENPP1 over-expression vectors; (3) CsENPP1 over-expression vector is introduced into citrus leaves and identified to obtain transgenic material with improved canker resistance.
  3. 3. A method for increasing citrus canker resistance by use of CsENPP gene according to claim 2, wherein in step (1), the cloning method of the coding sequence of citrus CsENPP1 is as follows: Extracting total RNA of citrus, reversely transcribing the total RNA into cDNA as a template, carrying out PCR (polymerase chain reaction) amplification by using primers OE-CsENPP-F and OE-CsENPP-R, and recovering a CsENPP coding sequence DNA fragment; The nucleotide sequences of the primers OE-CsENPP-F and OE-CsENPP-R are shown as SEQ ID No. 2 and SEQ ID No.3 respectively.
  4. 4. The method for improving citrus canker resistance by using CsENPP gene as claimed in claim 2, wherein in the step (2), the CsENPP1 over-expression vector is constructed by ligating CsENPP coding sequence DNA fragment recovered by cleavage of KpnI and BamHI to pLGNe vector recovered by cleavage of KpnI and BamHI, and constructing the over-expression vector pLGNe-CsENPP1.
  5. 5. The method for improving canker resistance of citrus using CsENPP gene as claimed in claim 2, wherein in step (3), the method for transforming citrus with CsENPP1 over-expression vector is that the over-expression vector pLGNe-CsENPP1 is transformed with agrobacterium tumefaciens by electric shock method, and then the agrobacterium tumefaciens bacterial liquid carrying over-expression vector is injected into citrus leaves.
  6. 6. The method for improving citrus canker resistance by using CsENPP genes according to claim 5, wherein whether CsENPP genes are successfully introduced into citrus leaves is identified by using PCR primers, and whether CsENPP genes in citrus leaves are overexpressed is identified by using qRT-PCR primers.
  7. 7. The method for improving citrus canker resistance by using CsENPP genes as claimed in claim 6, wherein when qRT-PCR primers are used for identifying whether CsENPP genes in citrus leaves are over-expressed, the qRT-PCR primers are RT-CsENPP1-F and RT-CsENPP1-R, and the nucleotide sequences of the primers RT-CsENPP1-F and RT-CsENPP1-R are shown as SEQ ID No. 4 and SEQ ID No. 5 respectively.
  8. 8. The method for improving citrus canker resistance by using CsENPP gene according to claim 2, wherein after the transgenic material is obtained in the step (3), the transgenic material is subjected to resistance evaluation, and it is judged that CsENPP overexpression improves the citrus canker resistance.

Description

Method for improving citrus canker resistance by CsENPP gene Technical Field The invention relates to the technical field of molecular biology, in particular to a method for improving citrus canker resistance by utilizing CsENPP gene. Background Citrus is the first fruit in the south of China and is also a global important cash crop. However, citrus canker severely hampers the healthy development of the citrus industry (Hu Junhua et al, 2015, fruit tree theory). Citrus canker is caused by a pathogenic variant of xanthomonas citri (Xanthomonas citri subsp. Citri, xcc), originating from india, java, etc. (Hu Junhua, et al, 2015, fruit tree theory). The major citrus producing areas of Fujian, hunan, guangdong and the like in China are seriously affected by citrus canker. The canker pathogen mainly infects citrus leaves, branch tips, fruits and the like, wherein the damage of seedlings and saplings is serious (He Xiuling and the like, 2007, chinese agronomic report). The disease tree can have the phenomena of fallen leaves, dead tips, weak tree vigor, fallen fruits and the like, and the yield and quality of citrus are seriously affected. There are tens of Rutaceae plants that are compromised by citrus canker, most of which are economic cultivars. The most susceptible to sweet orange has been found by research, followed by lime and pomelos (Yuan Chengdong et al, 1997, phytosanitary; li Min et al, 2013, southern China fruit tree). At present, the control of the hazard of citrus canker generally adopts an integrated control strategy, mainly chemical control and secondarily biological control. However, chemical prevention and control measures are not friendly to the environment, are extremely easy to pollute the environment, require a large amount of manpower and material resources, have poor biological prevention and control effects and high cost, so that the loss caused by canker is urgently needed to be reduced by cultivating new disease-resistant varieties (Zhu Xuemei and the like, 2017, modern gardening). The long period of crossbreeding makes the breeding efficiency low. With the rise of molecular biology, research on disease-resistant defense reactions of pathogenic bacteria and plants is started from the direction of disease-resistant genetic engineering. Genetic engineering approaches have also been tried in anti-ulcer studies (Duan Minjie et al, 2016, university of southwest, university of teachers; gu Ruirui et al, 2017, china agricultural science) to obtain transgenic materials resistant to ulcer disease. For example Chen Shanchun et al obtained the tussah antimicrobial peptide D gene-resistant strain of brochure, neo-orange, navel orange (Chen Shanchun et al, 1996, chinese agricultural science), the exogenous genes NLS, chit, xa21 and PthA were resistant to citrus canker after transfer to the crystal sugar orange, ponkan and sweet orange (Mendeset al.,2010,Plant Pathology;Yanget al, 2011,Plant molecular biology), csBZIP40 was an important transcription factor responsive to citrus canker infection, and it was hypothesized to affect resistance of citrus varieties via the SA pathway (Liet al.,2019, ploS one), the CsLOB1 gene was the target protein of canker pathogen PthA, rendering citrus more susceptible to canker (Liet al.,2014,Proceedings of the National Academy of Sciences), and CRISPR/Cas9 targeted knockdown of the citrus canker gene CsLOB1 promoter resulted in plants with increased resistance to citrus canker (Penget al.,2017,Plant biotechnology journal). Nucleotide pyrophosphatase/phosphodiesterase (NPPs) is an N-glycosylase present in monocotyledonous and dicotyledonous plants and is capable of catalyzing hydrolytic cleavage of the pyrophosphate and phosphodiester bonds of a variety of nucleotides and nucleotide sugars (Rodr ı 'guez-Lo' pez et al, 2000,Proceedings of the National Academy of Sciences;Nanjo et al, 2006,The Plant Cell). Although nucleotide pyrophosphatase/phosphodiesterase 1 regulation has been partially employed in plant disease resistance, there has been no study in the field of citrus canker. In view of this, the present application has been made. Disclosure of Invention The invention provides a method for improving the resistance of citrus canker by utilizing CsENPP genes, in particular to a method for integrating a citrus nucleotide pyrophosphatase/phosphodiesterase 1 coding gene into citrus through an expression vector, so that the resistance of citrus to canker is effectively improved, and the method has great application value for citrus canker-resistant breeding. The invention adopts the following technical scheme: The invention aims to provide a method for improving citrus canker resistance by utilizing CsENPP1 genes, wherein the coded protein of the CsENPP genes is citrus nucleotide pyrophosphatase/phosphodiesterase 1, the CsENPP1 coded sequence is a nucleotide sequence shown as SEQ ID No. 1, and the citrus canker resistance is improved based on the overexpression of CsENP