阅读说明：本技术 木薯抗病相关基因MeAHL17及其应用 (Cassava disease-resistant related gene MeAHL17 and application thereof ) 是由 丁泽红 胡伟 铁韦韦 颜彦 于 2021-05-27 设计创作，主要内容包括：本发明公开了一种木薯抗病相关基因MeAHL17及其应用；所述MeAHL17的CDS核苷酸序列如SEQ ID No.1所示。本发明准确鉴定了MeAHL17在抵抗木薯细菌性枯萎病中的功能,为木薯细菌性枯萎病改良提供了理论依据和关键基因,具有重要的应用价值。(The invention discloses a cassava disease-resistant related gene MeAHL17 and application thereof; the CDS nucleotide sequence of MeAHL17 is shown in SEQ ID No. 1. The invention accurately identifies the function of MeAHL17 in resisting the bacterial wilt disease of cassava, provides theoretical basis and key genes for improving the bacterial wilt disease of cassava, and has important application value.)

1. A cassava disease resistance related gene MeAHL17 is characterized in that: the CDS nucleotide sequence of MeAHL17 is shown in SEQ ID No. 1.

2. The MeAHL17 protein encoded by the cassava disease resistance related gene MeAHL17 in claim 1, wherein the amino acid sequence of the protein is shown as SEQ ID No. 2.

3. A promoter of a cassava disease resistance related gene MeAHL17 has a nucleotide sequence shown in SEQ ID No. 3.

4. Use of the promoter of the cassava disease resistance-related gene MeAHL17 as defined in claim 1 or the promoter of the MeAHL17 as defined in claim 3 for preventing and treating bacterial wilt disease of cassava.

5. Use of the promoter of the cassava disease resistance-related gene MeAHL17 as set forth in claim 1 or the promoter of the MeAHL17 as set forth in claim 3 in breeding new plant varieties.

6. Use according to claim 5, characterized in that: the plant is cassava.

Technical Field

The invention relates to the field of biological breeding, and in particular relates to a cassava disease resistance related gene MeAHL17 and application thereof.

Background

Cassava (Manihot esculenta Crantz) is an important economic crop and grain crop in tropical and subtropical regions, and plays a significant role in agriculture and industrial production in China. Bacterial wilt of Cassava (CBB) is one of the very serious diseases in the current cassava production, which can cause the cassava yield to be greatly reduced and seriously threaten the income increase of farmers and the sustainable development of the cassava industry.

Therefore, how to effectively excavate the cassava bacterial wilt resistance candidate genes and carry out detailed functional research on the cassava bacterial wilt resistance candidate genes so as to provide important gene resources for improving the cassava bacterial wilt, and the method is a difficult problem to be solved urgently in the current cassava biological breeding field.

Bacterial blight of cassava is an important agronomic trait. Because of the high heterozygosity of the cassava genome, the construction of a segregation population through parent hybridization and the positioning of the bacterial blight disease-resistant gene of the cassava by adopting a traditional map-based cloning method are limited to a great extent; meanwhile, the research foundation of cassava is poor, a transgenic system is not mature, the excavation of the resistance gene of the bacterial wilt of cassava is greatly limited, and no available gene is applied to the genetic improvement of the resistance of the cassava.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a cassava disease resistance related gene MeAHL17 and application thereof, which can provide gene resources and important reference basis for improving the cassava bacterial wilt and provide basis for gene improvement and biological breeding.

In order to achieve the aim, the invention provides a cassava disease resistance related gene MeAHL17, wherein the CDS nucleotide sequence of MeAHL17 is shown as SEQ ID No. 1.

The amino acid sequence of the MeAHL17 protein coded by the cassava disease resistance related gene MeAHL17 is shown as SEQ ID No. 2.

The invention also provides a promoter of the cassava disease resistance related gene MeAHL17, and the nucleotide sequence of the promoter is shown in SEQ ID No. 3.

The invention also provides application of the promoter of the cassava disease resistance related gene MeAHL17 or MeAHL17 in preventing and treating bacterial wilt of cassava.

The invention also provides application of the promoter of the cassava disease resistance related gene MeAHL17 or MeAHL17 in breeding new plant varieties, preferably, the plant is cassava.

The invention has the beneficial effects that:

(1) the invention accurately identifies the function of MeAHL17 in resisting the bacterial wilt of cassava, provides theoretical basis and key genes for improving the bacterial wilt of cassava, and has important application value;

(2) the invention shows that the A/G allelic variation of the promoter region of MeAHL17 is related to the resistance of cassava bacterial wilt disease. By detecting the genotype, the early prediction and the rapid screening of the bacterial wilt resistance of cassava are facilitated, and the disease-resistant breeding process of the cassava is accelerated.

Drawings

FIG. 1 is a diagram of the gene mapping and functional SNP analysis of MeAHL 17;

in the figure, FIG. 1A is a view showing a bacterial wilt disease GWAS analysis of cassava, and a candidate gene is located in the interval of 11.65-11.70Mb of chromosome 2;

FIG. 1B is a comparative analysis chart of candidate gene transcriptome;

FIG. 1C is the MeAHL17 gene model, with the red dashed line indicating the location of the functional SNP in the promoter region of MeAHL 17;

FIG. 1D is a graph comparing the activity of the A-containing and G-containing MeAHL17 promoter regions (300 bp and 600bp upstream of the transcription start site) in a dual luciferase assay, each sample comprising 8 biological replicates;

FIG. 1E is a graph comparing the resistance of cassava to bacterial wilt based on the AA and AG MeAHL17 alleles;

FIG. 2 is a graph of analysis of the disease resistance function of MeAHL 17;

in the figure, figure 2A is a graph of the difference in the expression levels of MeAHL17 in cassava with AG alleles (Yunnan8 and ZM9781) and AA alleles (ZM95308 and ruishi x3) measured by qRT-PCR for 3 biological replicates per sample, data expressed as mean ± standard deviation,. indicates P <0.01, at 0 and 2 days after Xam infection;

FIG. 2B is a graph showing the difference in expression level of MeAHL17 in cassava leaves transformed with pCAMBIA1304 (vector control, VC1), pCAMBIA1304:: MeAHL17 (overexpression, OE), pTRV (vector control, VC2) or pTRV:: MeAHL17(RNA silencing, RNAi) in the above four varieties (wherein ZM95308 and Ruishi X3 both carry AA alleles; Yunnan8 and ZM9781 both carry AG alleles), respectively; expression was measured by qRT-PCR, 3 biological replicates per sample were assayed, and data are expressed as mean ± standard deviation, P <0.05, P < 0.01;

FIG. 2C is a graph of the number of bacteria in cassava leaves transformed with pCAMBIA1304 (vector control, VC1), pCAMBIA1304:: MeAHL17 (overexpression, OE), pTRV (vector control, VC2) or pTRV:: MeAHL17(RNA silencing, RNAi) at 0 and 6 days after infection with Xam; data are expressed as mean ± standard deviation, P <0.05, P <0.01, and 4 biological replicates were determined for each sample.

Detailed Description

The present invention is described in further detail below with reference to specific examples so as to be understood by those skilled in the art.

Example 1 cassava disease resistance-related Gene MeAHL17 screening

1. Whole genome correlation analysis is carried out based on cassava germplasm natural population, and gene intervals related to cassava bacterial wilt disease are rapidly locked

The cassava germplasm natural population comes from an agricultural delirium cassava germplasm resource garden. 299 parts of cassava material was arbitrarily selected, fresh young leaves were frozen in liquid nitrogen, and genomic DNA was extracted using DNeasy Plant Mini kit (Qiagen, Beijing). Each sample was used to construct a pair-end library with an insert size of 500bp using 5. mu.g of genomic DNA. The 150bp paired end reads for each sample were sequenced using the Illumina X-Ten platform. Sequencing reads for each sample were aligned to the cassava SC205 reference genome using the BWA mem v0.7.17 program. The alignment results were ranked and repeatedly labeled using Samtools v1.9 and Picard v 1.94.

After deleting low-quality reads, the reads on the single-ended and double-ended comparison are used for detecting SNP by adopting a GATK toolkit v3.5 process. The HaplotypeCaller module is used to create an original genotype file containing SNPs and indels, and further filter them using the following parameters: "QUAL <2.0| | | QD <2.0| | | MQ <40.0| FS >60.0| | MQRankSum < -12.5| | ReadPosRankSum < -8.0-clusterSize 2-clusterWindowSize 5" and "QD <2.0| FS >200.0| ReadPosRankSum < -20.0". The identified SNPs and indels were annotated using SnpEff v3.6c software. Low quality SNPs were filtered using "Major Allele Frequency (MAF) >0.05 and missing data < 20%", yielding a total of 1,313,775 high quality SNPs.

The bacterial wilt of cassava germplasm is evaluated according to the technical specification (NY/T3005-2016) of the bacterial wilt of cassava in China. The average of three biological replicates was used to determine the level of resistance. Then, the cassava bacterial wilt is taken as a phenotypic character, and genome-wide association analysis (GWAS) is carried out. The AQ matrix generated by ADMIXTURE v1.3.0 was considered to be a fixed effect, and the relatives (K) matrix was constructed using SPAGeDi v1.3a. The corresponding P value threshold of 0.000001 is set to control the whole genome type I error rate.

The results show that: the interval 11.65-11.70Mb of chromosome 2 was significantly associated with cassava bacterial wilt disease (FIG. 1A).

2. Further screening for candidate genes based on comparative transcriptome analysis

Transcriptome data were obtained from the NCBI-SRA database under accession number SRP 045199. After removal of the linker sequence and low quality reads, clean reads were aligned to the SC205 reference genome by HISAT2 v 2.0.4. Gene expression levels were calculated by StringTie v1.3.4d using default parameters. Gene expression levels were normalized using FPKM. The differentially expressed genes were identified using DESeq2 software. Differentially expressed genes are defined as FDR <0.01 and fold change > 2.

Based on the cassava genome annotation, there are 6 candidate genes in the 11.65-11.70Mb interval on chromosome 2. Interestingly, only Sc02g014000 expression was significantly induced by the pathogen under the cassava bacterial wilt disease treatment conditions (fig. 1B).

Therefore, it was concluded that Sc02g014000 is an important candidate gene for regulating bacterial blight of cassava, which encodes a nuclear localization protein containing an AT-hook domain, which was designated MeAHL17 based on gene homology, and the CDS nucleotide sequence of MeAHL17 is shown in SEQ ID No. 1; the amino acid sequence is shown as SEQ ID No. 2; the nucleotide sequence of the promoter of MeAHL17 is shown in SEQ ID No. 3.

Example 2

1. Based on comparative genome analysis, SNP (single nucleotide polymorphism) sites significantly related to gene expression are excavated

Based on genome re-sequencing data, one SNP variation was found in the MeAHL17 promoter region (at-53 bp upstream of the start codon, i.e., 948 site of the nucleotide sequence of the promoter of MeAHL 17) (A/G, FIG. 1C).

To explore the effect of a/G allelic variation on the activity of the MeAHL17 promoter, we performed a dual luciferase assay in cassava protoplasts. MeAHL17 promoter sequences (-1 to-300 bp and-1 to-600 bp) with A or G were cloned and inserted into pGreenII0800-LUC vector and transformed into cassava protoplasts. Relative luciferase activity was determined using a dual luciferase reporter assay kit (RG027, Beyotime, shanghai). The results show that: the G-containing MeAHL17 promoter was more active than the a-containing MeAHL17 promoter (fig. 1D), indicating that a/G allelic variation affects expression of MeAHL 17.

2. A large amount of cassava germplasm analysis finds that A/G variation is obviously associated with cassava bacterial wilt resistance

By screening a large number of cassava germplasm we found that the germplasm carrying the AA allele by MeAHL17 was more sensitive to bacterial blight of cassava than the germplasm carrying the AG allele (fig. 1E). It is deduced from this that allelic variation of A/G in the promoter region regulates the expression of MeAHL17, which in turn influences the resistance of bacterial blight of cassava.

Example 3 overexpression and validation of RNAi function indicate that MeAHL17 plays an important role in the resistance of bacterial blight of cassava

To examine the expression profile of MeAHL17 in response to bacterial wilt (Xam), we collected 4 representative cassava varieties (of which ZM95308 and Ruishi X3 carry AA alleles; Yunnan8 and Yunnan 3) at 0 and 2 days after Xam infestationZM9781 carries an AG allele). Total RNA was isolated using cDNA synthesis kit (K1622, USA) and reverse transcribed. Then, using MeEF1a as internal reference and 2^-ΔΔCtThe method identified the relative expression level of MeAHL17 by quantitative real-time PCR (qRT-PCR).

The results show that: the expression level of MeAHL17 carrying the AA allele induced less after Xam treatment compared to the AG allele (fig. 2A).

Subsequently, overexpression and RNAi functional verification were performed simultaneously in the above 4 cassava varieties. To overexpress MeAHL17, the coding sequence of MeAHL17 was first amplified and inserted into the pCAMBIA1304 vector, followed by injection of the agrobacterium strain (GV3101) with recombinant vector or pCAMBIA1304 (control) into cassava leaves. To silence MeAHL17, a MeAHL17 specific region was amplified and cloned into pTRV2 vector, followed by injection of agrobacterium strain (GV3101) with recombinant vector or pTRV2 (control) into cassava leaves along with pTRV 1. After two days of cultivation of the plants transformed with pCAMBIA1304:: MeAHL17 and pCAMBIA1304 or two weeks of cultivation of the plants transformed with pTRV:: MeAHL17 and pTRV, Xam was inoculated into the cassava leaves, and the number of bacteria and the gene expression level were checked after 6 days.

The results show that: xam the number of bacterial populations in the different varieties after overexpression of MeAHL17 was significantly reduced compared to the control, whereas the number of bacterial populations in the MeAHL17 silenced varieties (Yunnan8 and ZM9781) was significantly increased compared to the control (FIGS. 2B-C). These results indicate that MeAHL17 plays an important role in combating bacterial blight of cassava, and that allelic variation of A/G in its promoter region is associated with resistance to bacterial blight.

Other parts not described in detail are prior art. Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.

Sequence listing

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Sanya Research Institute, Chinese Academy of Tropical Agricultural Sciences

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