CN113186198A - Brown planthopper resistant gene Bph41 and encoding protein and application thereof - Google Patents

Abstract

The invention relates to a brown planthopper resistant gene Bph41, and a coding protein and application thereof. The brown planthopper resistant gene Bph41 comprises a nucleotide sequence shown in any one of the following sequences: i) 1, the nucleotide sequence shown in SEQ ID NO; or ii) the nucleotide sequence shown in SEQ ID No.1 is substituted, deleted and/or added with one or more nucleotides to encode the nucleotide sequence of the same functional protein. The research of the invention discovers a brown planthopper resistant gene Bph41, and the gene is transferred into a common rice variety, so that the resistance of the rice to brown planthopper can be obviously improved, the harm caused by the brown planthopper is reduced, and the purposes of increasing and stabilizing yield are achieved.

Description

Brown planthopper resistant gene Bph41 and encoding protein and application thereof

Technical Field

The invention relates to the field of plant genetic engineering, in particular to a brown planthopper resistant gene Bph41, and a coded protein and application thereof.

Background

Rice is an important food crop, and is staple food for more than half of the world. As the fine genetic map and physical map of rice genome are completed, the transgenic technology is relatively easy and has collinearity with other gramineous crop genome, so that the rice genome is regarded as a model plant. With the completion of genome sequencing of various organisms including rice, humans began to enter the post-genome era. The comprehensive development of functional genome research has become the leading field of life science. Therefore, the research of the functional gene of the rice has great significance for the development of social economy and biological research.

The problem of food safety is a challenge for people all over the world. 50. The dwarfing breeding in the 60 th generation and the two technological revolution of the hybrid rice cultivation in the 70 th generation greatly improve the rice yield. The popularization of dwarf high-yield rice leads to the serious damage of plant hoppers. Especially, the frequency and the scale of the outbreak of the planthopper in the whole Asian rice production area entering the 21 st century have the tendency of gradually expanding, and the outbreak of the planthopper is the biggest threat in rice production at present.

For a long time, the control of brown planthoppers has mainly relied on the application of chemical insecticides. Since outbreaks of brown planthopper occur frequently in the maturing and filling stage of rice, the rice plants grow vigorously, and the operation of applying the insecticide to the base of the rice plants is very difficult. In fact, due to the fact that the chemical insecticide is applied in large quantities in successive years, the drug resistance of the brown planthopper is increased by times, and the prevention and control effect of the chemical insecticide is limited. Meanwhile, the chemical insecticide is used for preventing and controlling the brown planthopper, so that the production cost of farmers is increased, and the chemical insecticide also causes environmental and ecological problems such as poisoning of non-target organisms, pollution to the environment and grains and the like.

The cultivation of the anti-brown planthopper rice variety by using the anti-brown planthopper gene is the most economic and effective method in the comprehensive prevention and control of brown planthopper. The research results of the International Rice Research Institute (IRRI) and the rice production practice in southeast asia show that even rice varieties with only moderate resistance level can sufficiently control the brown planthopper population below the level causing harm, so that the actual harm and yield loss to rice are avoided. Therefore, the mining of the brown planthopper resistance gene of the rice and the application of the gene in a rice breeding project are fundamental measures for preventing and treating the brown planthopper of the rice.

The research of brown planthopper resistant gene of rice begins in the early 70 th century. Up to now, more than thirty major anti-brown planthopper anti-pest genes of rice have been identified and located in the resources of cultivated rice and wild rice (see in particular Du et al, 2020.Current understating of the genetic, and molecular control of infection resistance in rice. mol Breeding2020,40: 24). 8 genes (Bph14, Bph26, Bph3, Bph29, Bph9, Bph18, Bph32 and Bph6) have been cloned, and these genes were obtained by using the map-based cloning method (Du et al, 2009; Tamura et al 2014; Liu et al 2014; Wang et al 2015b; Zhao et al 2016; Ji et al 2016; Ren et al 2016; Guo et al 2018).

Genome-wide association analysis (GWAS) was first applied to genetic analysis of human diseases. In recent years, with the rapid development of genome technology, especially the improvement of sequencing technology means, whole genome sequencing of many animals and plants has been completed, and GWAS becomes a powerful tool for researching complex traits of crops. Compared with the traditional map-based clone discovery gene, the correlation analysis method has the advantages of no need of constructing a special mapping population, time saving, realization of fine positioning and the like. Meanwhile, GWAS breaks through the limitation that the map-based cloning method is generally suitable for smaller genome species, and only needs the SNPs markers and phenotypic traits of the whole genome. In recent years, GWAS analysis methods have also been developed rapidly. Research results of scientists and international colleagues in China show that the GWAS method discovers target genes in aspects of crop morphological characters, physiological characters, yield-related characters and the like. For example, LOC _ Os01g62780, LOC _ Os11g08410, NAL1(Yano et al 2016, Genome-side association testing using floor-Genome sequencing along with tillering genes in rice site. Nature Genetics,48: 927-.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a brown planthopper resistant gene Bph41, and a coding protein and application thereof. The gene is transferred into a common rice variety, so that the resistance of the rice to brown planthopper can be obviously improved.

In a first aspect, the invention provides an anti-brown planthopper gene Bph41, which comprises a nucleotide sequence shown in any one of the following:

i) 1, SEQ ID NO; or

ii) the nucleotide sequence shown in SEQ ID No.1 is substituted, deleted and/or added with one or more nucleotides to encode the nucleotide sequence of the same functional protein.

The invention further provides a protein encoded by the brown planthopper resistant gene Bph41, wherein the protein comprises an amino acid sequence shown in any one of the following sequences:

i) an amino acid sequence shown as SEQ ID No. 2; or

ii) the amino acid sequence of the protein with the same function obtained by replacing, inserting or deleting one or more amino acids in the amino acid sequence shown in SEQ ID No. 2.

The invention further provides a primer pair for detecting the molecular marker of the brown planthopper resistant gene Bph41, wherein the primer pair comprises:

forward primer sequence: 5'-ATGGCGACGACGACCGGCAG-3' the flow of the air in the air conditioner,

reverse primer sequence: 5'-TCATAATAATGTACAAGAGAGTATAGC-3' are provided.

Further, the molecular markers are one or more of rs4_11033430, rs4_11033910, and rs4_ 11034040; wherein, the polymorphism of rs 4-11033430 is C/G, rs 4-11033910 and the polymorphism of rs 4-11034040 is T/C.

The invention further provides a biological material containing the brown planthopper resistant gene Bph41, wherein the biological material is one or more of a vector, an expression cassette or a transgenic cell.

The invention further provides application of the brown planthopper resistant gene Bph41 or the coding protein thereof, or the primer pair, or the biological material in identification of the brown planthopper resistance of plants.

The invention further provides the brown planthopper resistant gene Bph41 or a coding protein thereof, or the primer pair, or the application of the biological material in brown planthopper resistant plant breeding.

Further, the application is as follows: and (3) over-expressing the brown planthopper resistant gene Bph41 in the plant, or crossing the plant over-expressing the brown planthopper resistant gene Bph41 with other plants to cultivate a brown planthopper resistant strain.

Further, the plant is rice.

The invention further provides a method for detecting the brown planthopper resistant gene Bph41, which comprises the following steps:

obtaining genomic DNA of a plant to be tested, and amplifying the genomic DNA by the primer pair of claim 3;

if the basic groups of the rs4_11033430, rs4_11033910 and rs4_11034040 in the amplification result are C, T and A respectively, the result shows that the plant to be detected contains the brown planthopper resistant gene Bph 41.

The invention has the following beneficial effects:

(1) the invention successfully clones the brown planthopper resistant gene Bph41 of rice by utilizing the relevance analysis of the whole genome.

(2) The Bph41 gene cloned by the invention has obvious brown planthopper resistance, which is of great significance for comprehensively understanding the diversity of brown planthopper resistance gene types of rice.

(3) The brown planthopper resistant gene Bph41 provided by the invention can improve the brown planthopper resistance of rice, and Bph41 is applied to rice breeding through genetic transformation or hybridization, so that the brown planthopper resistance of rice varieties can be improved, the harm of brown planthopper is reduced, and the purposes of increasing yield and stabilizing yield are achieved.

(4) The piercing-sucking insects are a large class of insect pests in agricultural production, and the brown planthopper resistant gene Bph41 clone and the brown planthopper resistant function prove that the piercing-sucking insects have important reference function for the anti-piercing-sucking insect research of other plants.

Drawings

FIG. 1 shows 1520 parts of rice cultivar cladonides according to example 1 of the present invention.

FIG. 2 shows the results of identifying 1520 copies of the rice cultivar resistant to brown planthopper according to example 1 of the present invention; in the figure, the abscissa WG represents the weight gain rate of the brown planthopper, and the ordinate represents the number of rice varieties; the insect source is brown planthopper biotype I.

FIG. 3 is a Manhattan diagram of the genome wide association analysis provided in example 1 of the present invention.

FIG. 4 is a graph showing the results of haplotypes A and B resistant to Nilaparvata lugens provided by examples of the present invention.

FIG. 5 shows the result of identifying the brown planthopper resistance of the transgenic rice provided by the basic invention embodiment.

FIG. 6 is an example of functional molecular markers 68-75 developed from the genome sequence of Bph41 according to an embodiment of the present invention.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1 cloning of Bph41 Gene and molecular marker development

1. Whole genome relevance analysis

1520 rice varieties, which are located in 74 countries worldwide, are obtained from the institute of crop science of the Chinese academy of agricultural sciences, and mainly include indica rice and japonica rice, and the evolutionary trees of these rice varieties are shown in fig. 1, in which regions with different gray levels represent different subgroups and different branches represent different types.

The invention carries out brown planthopper resistance measurement on 1520 parts of rice varieties, and the method is to measure the weight of the female brown planthopper adults before and after eating for 48 hours so as to obtain the weight gain rate (WG) of the brown planthopper. The average rate of weight gain of 12 brown planthoppers per rice variety was used to indicate the level of resistance. The results shown in fig. 2, which are plotted as a histogram of the rate of weight gain of the rice variety 1520 hours after feeding by brown planthopper; in the figure, the abscissa WG represents the weight gain rate of the brown planthopper, and the ordinate represents the number of rice varieties; the insect source is brown planthopper biotype I. The results show that: the resistance level of 1520 rice varieties to brown planthopper shows a left-biased distribution, i.e. the pest-susceptible varieties are much more numerous than the pest-resistant varieties.

The present invention further extracts genome-wide SNPs data with minor allele frequency of greater than 0.01 and deletion rate of less than 0.2 from 3K rice genome data (Li, Z, et al, 2014The 3,000rice genes project. GigaScience 3,7) using Plink software. The first 7 principal components (Q) are used as fixed effects, and the genetic matrix (K) between each individual is used as random effect to carry out population structure correction. GWAS analysis was performed using a mixed linear model in EMMAX software. The whole genome significance threshold was determined using the formula p ═ 0.05/N (N is the number of SNPs). Significance p-value threshold of about 1.0 × 10^-8. p value less than threshold (-log)₁₀(p) SNPs exceeding 8) are considered to be significant SNPs. The 150kb regions upstream and downstream of the physical position of the significant SNP are determined as significant regions, and a plurality of overlapping significant regions are combined into one significant region.

2. Determination of candidate genes

The analysis of the genome-wide association analysis yielded the results shown in FIG. 3, FIG. 3 being a Manhattan diagram of the genome-wide association analysis; each dot represents the degree of association of one SNP marker with the resistance to Nilaparvata lugens, the smaller the p value (-log)₁₀(p) greater), the greater the degree of association with brown planthopper resistance; log (log)₁₀(p) sites exceeding threshold 8 are considered significant association sites; chromosome 6, 0.8-1.57Mb, is the most highly correlated whole genomeRegion, -log₁₀(p) far above threshold 8; the model used was a mixed linear model of the EMMAX software. The results show that: a significant region of 10.48-12.47Mbp exists in chromosome 4, which indicates that the region contains the brown planthopper resistant gene and is used as a candidate region. One significant SNP rs4_11033430 within the candidate region is located in the exon region of LOC _ Os04g19750 and is predicted to cause amino acid changes. LOC _ Os04g19750 encodes an F-box-LRR protein, in combination with Nipponbare reference genome Gene functional annotation information (http:// rice. plant biology. msu. ed u /). Cpr30 in Arabidopsis encodes the F-box protein and remains resistant to Pseudomonas syringae (Gou et al,2019), and LOC _ Os04g19750 is a homologous protein of cpr30 in rice as a candidate gene. 1520 parts of material were divided into haplotype a and haplotype B based on the different base pattern of the significant SNP rs4_11033430 in 1520 parts of material, with 1470 parts of material for haplotype a and 36 parts for haplotype B. Haplotype B material was significantly more resistant to brown planthopper than haplotype a material (as shown in figure 4, haplotype a had a significantly higher brown planthopper weight gain rate (WG) than haplotype B). G68 in haplotype B was selected as the material for amplifying LOC _ Os04G 19750.

3. Obtaining the full-length DNA of Bph41

Using brown planthopper resistant parent G68 leaf DNA as a template, designing a Primer according to a Primer-BLAST on NCBI of a Nipponbare reference genome sequence, amplifying a plurality of overlapped DNA sequences covering candidate genes, and splicing a genome sequence covering the genes. And (3) re-synthesizing a primer according to the spliced genome sequence, and amplifying to obtain the full-length DNA of the Bph41, wherein the nucleotide sequence of the full-length DNA is shown as the sequence table SEQ ID NO. 1.

4. Molecular marker of brown planthopper resistant gene Bph41

Compared with the inductive material, 3 SNPs exist in the Bph41 exon region, and the physical positions of the 3 SNPs are rs4_11033430, rs4_11033910 and rs4_11034040 respectively. In the resistant parent, the bases of rs4_11033430, rs4_11033910 and rs4_11034040 are C, T and a, respectively, i.e. haplotype G68; in the susceptible parent, the bases of the 3 SNPs are G, C and C, respectively, i.e., the haplotype Nipponbare. Thus, molecular labeling was performed using:

68-75 labeled primers:

a forward primer sequence ATGGCGACGACGACCGGCAG, shown as SEQ ID NO. 3;

a reverse primer sequence TCATAATAATGTACAAGAGAGTATAGC, shown as SEQ ID NO. 4;

the DNA of the rice brown planthopper resistant variety or breeding material is amplified, if primers 68-75 are used, an amplified fragment of haplotype G68 can be amplified, and the existence of a brown planthopper resistant gene Bph41 of rice is marked. Therefore, the molecular marking method provided by the invention has very high efficiency in identifying the existence of the Bph41 resistance gene, can predict the brown planthopper resistance of rice plants, and accelerates the breeding process of brown planthopper-resistant rice varieties.

Example 2 functional verification and application of Bph41 Gene

1. Construction of genetic transformation vectors

And (3) constructing a Bph41 gene overexpression vector. The vector used was pCXUN (provided by professor Wangganggu of Ohio State University, USA), and pCXUN vector was digested with XcmI, and the foreign fragment was added with A and ligated directly.

The Bph41 gene is directly amplified by adopting a PCR method, and is connected with a vector after being added with A. After sequencing verification, the obtained vector is the Bph41 gene overexpression vector, and the vector is transferred into agrobacterium tumefaciens EHA 105. Selecting monoclonal for amplification culture, performing PCR verification, adding equal volume of 50% glycerol, mixing, and storing at-70 deg.C.

2. Genetic transformation

The above-mentioned Bph41 gene overexpression vector was introduced into brown planthopper-sensitive rice cultivar Kasalath by the Agrobacterium EHA 105-mediated genetic transformation method (Hiei et al, 1994, effective transformation of rice (Oryza sativa L.), mediated by Agrobacterium and sequence analysis of the nucleic acids of the T-DNA. plant Journal 6: 271-282).

Verification of transgenic function of Bph41 Gene

The Bph41 gene over-expression transformation vector obtains 20 positive transgenic plants. After harvesting the seeds, using T₂The Bph41 transgenic plant is used for insect resistance identification by adopting a seedling stage group method. As shown in FIG. 5, the transgenic positive lines 8-2, 11-12 and 8-9 were identified for resistance to brown planthopper. Identification resultsAs shown in figure 5, after 7 days of brown planthopper inoculation, the whole plant of the pest-susceptible control variety Kasalath died, the transgenic positive plant grew healthily without leaf damage, the pest resistance level was 2-4, and the Bph41 gene was confirmed to have the function of brown planthopper resistance. Therefore, the brown planthopper resistant gene Bph41 can be applied to rice and rice seeds to cultivate rice varieties with brown planthopper resistant performance.

Example 3 validation of molecular markers

1. Materials and methods

1.1 materials: brown planthopper resistant parent G68 (containing brown planthopper resistant gene Bph41), brown planthopper sensitive rice Nipponbare (Nipponbare) and 549 parts of 3K rice.

Molecular marker primer: 68-75, the nucleotide sequences of which are respectively shown as SEQ ID No. 3-4.

1.2 methods

Extracting the genome DNA of the rice sample by a CTAB extraction method. Sample DNA was amplified using primers 68-75. 50 μ l system. A50. mu.l reaction system included: 2X Phanta Max Buffer, 25.0. mu.l; 10mM dNTP, 1.0. mu.l; ddH₂O, 9.0. mu.l; 10. mu.M primer, 2.0. mu.l; phanta Max Super-F DNA Polymerase, 1.0. mu.l, and 50ng of DNA template. The amplification reaction was performed on a Bioer PCR instrument: 3min at 95 ℃; 15s at 95 ℃, 60s at 72 ℃ and 30 cycles; 5min at 72 ℃. 68-75 the amplification products were tested by Sanger sequencing for the base forms of G68 and Nipponbare at rs4_11033430, rs4_11033910 and rs4_ 11034040. 549 cultivars of the 3K rice were extracted from the SNPs dataset of 3K rice at bases rs4_11033430, rs4_11033910 and rs4_11034040 by software Plink.

2. As a result: the above-mentioned method is used to respectively amplify rice variety G68 and Nipponbare and extract 549 SNPs of variety. The result shows that C, T and A varieties can be amplified by using 68-75 molecular marker primers in rs 4-11033430, rs 4-11033910 and rs 4-11034040, and C, T and A varieties (haplotype G68) extracted from 549 parts of rice show insect resistance to brown planthopper. While the varieties from which the above bases could not be amplified or extracted (haplotype Nipponbare) exhibited pest-sensitivity to Nilaparvata lugens (FIG. 6).

Therefore, the molecular marking method provided by the invention can accurately screen the gene Bph41 containing the brown planthopper resistance gene, thereby greatly improving the breeding efficiency.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

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Claims (10)

1. An anti-brown planthopper gene Bph41, which is characterized by comprising a nucleotide sequence shown in any one of the following:

i) 1, SEQ ID NO; or

ii) the nucleotide sequence shown in SEQ ID No.1 is substituted, deleted and/or added with one or more nucleotides to encode the nucleotide sequence of the same functional protein.

2. A protein encoded by the brown planthopper-resistant gene Bph41 according to claim 1; the protein comprises an amino acid sequence shown in any one of the following:

i) an amino acid sequence shown as SEQ ID No. 2; or

ii) the amino acid sequence of the protein with the same function obtained by replacing, inserting or deleting one or more amino acids in the amino acid sequence shown in SEQ ID No. 2.

3. A primer pair, which is used for detecting the molecular marker of the brown planthopper resistant gene Bph41 of claim 1; the primer pair comprises:

forward primer sequence: 5'-ATGGCGACGACGACCGGCAG-3' the flow of the air in the air conditioner,

reverse primer sequence: 5'-TCATAATAATGTACAAGAGAGTATAGC-3' are provided.

4. The primer pair of claim 3, wherein the molecular marker is one or more of rs4_11033430, rs4_11033910, and rs4_ 11034040; wherein, the polymorphism of rs 4-11033430 is C/G, rs 4-11033910 and the polymorphism of rs 4-11034040 is T/C.

5. A biomaterial comprising the brown planthopper resistant gene Bph41 of claim 1; the biological material is one or more of a vector, an expression cassette or a transgenic cell.

6. Use of the brown planthopper resistant gene Bph41 or its encoded protein according to claim 1, or the primer pair according to claim 3 or 4, or the biological material according to claim 5 for identifying brown planthopper resistance in plants.

7. Use of the brown planthopper resistant gene Bph41 or its encoded protein according to claim 1, or the primer pair according to claim 3 or 4, or the biological material according to claim 5 in breeding brown planthopper resistant plants.

8. The application according to claim 7, wherein the application is: and (3) over-expressing the brown planthopper resistant gene Bph41 in the plant, or crossing the plant over-expressing the brown planthopper resistant gene Bph41 with other plants to cultivate a brown planthopper resistant strain.

9. Use according to any one of claims 6 to 8, wherein the plant is rice.

10. A method for detecting the brown planthopper resistance gene Bph41 according to claim 1, comprising:

obtaining genomic DNA of a plant to be tested, and amplifying the genomic DNA by the primer pair of claim 3;

if the basic groups of the rs4_11033430, rs4_11033910 and rs4_11034040 in the amplification result are C, T and A respectively, the result shows that the plant to be detected contains the brown planthopper resistant gene Bph 41.

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