CN110004155B - Disease-resistant gene and protein for controlling plant aversion and aphid resistance characters and application thereof - Google Patents

Abstract

The invention relates to a disease-resistant gene and protein for controlling plant aversion and aphid resistance characters and application thereof, belonging to the technical field of bioengineering. According to the invention, candidate genes are subjected to extensive prediction according to gene functions and a re-sequencing result, a specific upstream primer is designed by inserting a 20bp sequence at 400bp upstream of an initiation codon of a disease-resistant gene (ppa000596m), a downstream primer is designed by using a conserved region at downstream of a stop codon of a ppa000596m gene, and a disease-resistant gene which does not exist in a peach genome is obtained by amplification. The existence of the gene and the aphid resistance character show coseparation, the expression mode of the new gene is further analyzed, the result shows that the expression of the gene in the population and the aphid resistance of a single plant show consistency characteristics, and the new gene is the new disease-resistant gene for controlling the aphid resistance character of the avoidance of the peach tree. The gene and the protein coded by the gene can be applied to multiple aspects of screening of aphid-resistant single plants, enhancing of aphid-resistant characters of plants, breeding of aphid-resistant peach varieties and the like.

Description

Disease-resistant gene and protein for controlling plant aversion and aphid resistance characters and application thereof

Technical Field

The invention relates to a disease-resistant gene and protein for controlling plant aversion and aphid resistance characters and application thereof, belonging to the technical field of bioengineering.

Background

Peach is an important economic tree species in China, according to statistics of grain and agriculture organizations of the United nations, the planting area of peach in China is 72.84 ten thousand hectares in 2014, the total yield is 1245 ten thousand tons, which respectively account for 48.7 percent and 54.6 percent of the total world amount and are at the top of the world. The aphids are one of main pests influencing the growth of peach trees, and the aphids harmful to the peach trees comprise 3 species of green peach aphids, pink peach aphids and peach aphids, wherein the green peach aphids (green peach aphids) are the most serious, and meanwhile, the green peach aphids are plant piercing-sucking pests widely distributed in the world. The aphids suck juice from young plant tissues by a piercing-sucking mouthpart, so that the loss of plant nutrition is caused, the development is hindered, the plants grow abnormally, are senilism and even die, the aphids are also propagation carriers of a plurality of plant viruses except the direct damage caused by piercing-sucking, and the production loss caused by the aphids is even more serious than the direct damage of the aphids. In agricultural production, aphids are generally prevented and controlled by chemical pesticides, so that the production cost and the risk of pesticide residues on fruits are increased, and natural enemies of pests in the nature can be killed, so that the pollution and the damage to the ecological environment are caused. In addition, the aphids have generated drug resistance to organophosphorus, carbamate and pyrethroid insecticides which are widely applied in recent years, so that the difficulty of prevention and control is gradually increased, the breeding of aphid-resistant varieties of peaches is an important way for preventing the harm of the aphids of the peaches in production, and the aphid-resistant varieties are helpful for fundamentally reducing the adverse effects of the aphids on the peach industry.

The study on the aphid-resistant character of the peach tree at home and abroad mainly focuses on the identification of aphid-resistant germplasm resources, the analysis of genetic resistance rules, the positioning of aphid-resistant genes and the like. 5 seeds of peach of Wangliang and the like are subjected to field identification of aphid resistance characters, and 15 resistant resources are selected: wild peach, birthday star peach and flowering peach. After artificial inoculation, the classification of peach species against aphids requires further discussion (Settling belviour and productive potential of the green aphids on the basis of their individual genetic activities and related complex of green aphids) because it is assumed that the peaches and pecans are antibiotic-resistant, the Amygur species are repellent-resistant and foreign, and different results are obtained based on the law of aphid-specific piercing potential maps, and thus the classification of peach species against aphids is required (transgenic aphid and productive potential of the green aphids on the basis of related complex of the genus Prunus 1998,89(3): 233) 242, Sauge M H, etc.). At the earliest, French develops the research on the aphid resistance of peach trees, and screens three aphid-resistant materials: weeping peach, stock variety 'Rubira' and wild peach 'P1908'.

Genetic analysis shows that the pholiota japonica and the 'Rubira' show monogenic inheritance for the resistance of green peach aphids, the resistance phenotypes are very similar, and the pholiota japonica and the 'Rubira' both have strong aversion type resistance, namely aphids cannot propagate on the tips of the branches, usually leave the hosts within 3 days, and red or yellow allergic necrotic spots appear on piercing and sucking parts. However, unlike the weeping peach, 'Rubira' has a strong inducible resistance characteristic, i.e. the damage of the early aphid inhibits the damage behavior of the later aphid, and it is speculated that the regulation mechanisms of the two materials may not be the same. Thus, the resistance regulatory sites of pholiota indica and 'Rubira' are designated Rm1 and Rm2, respectively. Lambert et al located Rm2 site in a 2.88Mb terminal region of chromosome 1, and in 2016, this team reduced the region to 1.14Mb (identification SNP markers having an associated with a large and large gene in reach [ surrounding per ca (L.) ] Batsch ] using a high-intensity region with an object of marker-associated selection (MAS), Tree Genetics & genes 2016,12(6):1-21, Lambert P, etc.). Different from the 'weeping peach' and the 'Rubira', the resistance of the wild peach to green aphid is quantitative character locus, aphid piercing and sucking potential map shows that the wild peach is antibiotic resistance, the population propagation rate of the green aphid on the wild peach can be obviously inhibited, QTL positioning analysis shows that 7 effective loci influencing green aphid resistance exist in the wild peach, and the main effective locus is positioned on No.3 chromosome.

The natural immune response of plants plays a key role in the process of resisting pathogen infection. The first layer is that plants recognize molecular substances from pathogenic microorganisms through cell surface immune receptors to activate natural immunity, which is called pathogen-associated molecular pattern-triggered immunity (PTI), also called basal resistance; the second layer is pathogenic microorganisms with strong pathogenicity secrete effector proteins to plant cells, the proteins usually modify host proteins through posttranslation to inhibit basic resistance of plants, and the plants recognize the effector proteins by using NBS-LRR type anti-disease proteins (R proteins) to reactivate immune response to limit disease occurrence, namely effector protein-induced immune response (ETI). In the ETI reaction, a protein (R protein) coded by a resistance gene (R gene) can realize the perception of pathogen invasion by recognizing a molecular pattern substance carried by a pathogen, and then starts the downstream defense reaction of a plant body, and the R protein can be used as an alarm recognizer and a defense reaction switch in the process of interaction of the plant and the pathogen.

Natural plant immunity plays an equally important role in protecting plants from phytophagous insects. Similar to the invasion process of pathogens, plant-feeding insect-carried Molecular Pattern Substances (HAMPs) or plant-feeding insect-derived effector proteins (HAEs) can also be recognized by plant cells to initiate a defense response in plants (underlinking plant discovery responses; an infectious first responses the expression of a refractory resistance against diseases, transgenic responses, 2013,22(4): 697-. Researchers have discovered a variety of molecular pattern substances from insect oral secretions that can activate the defense response to PTI. Recent studies have also found that effector proteins capable of interacting with host plants and stimulating host plant defense responses are present in insect saliva. It shows that the molecular pattern substance carried by phytophagous insects triggers immune response (HTI) and ETI play an important role in the process of resisting insect harm of plants.

The piercing-sucking pests refer to a general term of pests which can absorb juice nutrients from interstitial spaces of host plant tissue cells through mouthparts and maintain self growth, and mainly comprise aphids, whiteflies, plant lice, stinkbugs and the like. When the sucking pests take the food, the sucking pests firstly secrete the sphingomyelin to form a protective layer on the sucking mouth parts, then secrete watery saliva in the sucking process, and specific substances in the saliva participate in the regulation of the metabolic process of host plants so as to maintain the long-term nutrient absorption. Several genes have been found which confer resistance to piercing-sucking pests on plants. The Mi1.2 gene of tomato confers resistance to 3 piercing-sucking pests (potato aphid, whitefly and psyllid) and to 3 root-knot nematodes on tomato. The cloned Vat gene in melon confers resistance to Aphis gossypii. The aphid-resistant regulatory genes which have been cloned in plants are only Mi1.2 and Vat genes, and they are NBS-LRR disease-resistant genes. In addition, there is additional evidence that disease resistance genes are involved in resistance regulation by piercing-sucking pests. For example, silencing a disease-resistant gene in lettuce results in its loss of resistance to the lettuce's Oncorhynchus species. The resistance of the medicago truncatula in leguminous model plants to blue-green aphids, alfalfa aphids and pea aphids is in a monogenic dominant genetic control rule, and the gene positioning result shows that disease-resistant gene clusters exist in the positioning intervals of the resistance sites. Therefore, the plant can mobilize the innate immune system through the disease-resistant genes to resist the parasitic behavior of the piercing-sucking pests, and is the main way for the plant to resist the harm of the piercing-sucking pests.

From the above, the research of the aphid resistance mechanism of peach trees is developed, the aphid resistance character regulation gene is cloned, the genetic basis and the molecular regulation mechanism of aphid resistance formation are clarified on the gene level, the establishment of the aphid resistance character molecular marker auxiliary selection system and the basis of variety directional improvement are provided, the breeding efficiency of new aphid resistance varieties of peaches is improved, and the method has important scientific and practical significance.

Zhengzhou fruit tree institute has been engaged in peach genetic improvement work for a long time, wherein the aphid resistance character is one of important target characters concerned, and long-term breeding work accumulates a plurality of aphid resistance and aphid susceptible strains and aphid resistant and susceptible segregation populations, wherein the earliest aphid resistant excellent strain can be traced back to a hybrid single plant '9651' planted in 1996, the resistance of the hybrid single plant is derived from 'Shouxixing peach', a 6 th generation genetic population is constructed at present, and the fruit quality of part of the excellent line reaches the level of producing main cultivated varieties. FIG. 1 shows typical characteristics of an anti-aphid strain and an aphid susceptible strain after damage of aphids, wherein upper and lower rows of pictures respectively show phenotype changes of the anti-aphid strain and the aphid susceptible strain at 0h, 12h, 3d and 7d after damage of the aphids, as can be seen from FIG. 1, a typical resistance reaction 'red hypersensitive spot' appears on the anti-aphid strain at 3 days, the number of the aphids is reduced, no hypersensitive spot appears on the aphid susceptible strain, and a 'leaf rolling' reaction is caused by mass propagation and feeding of the aphids on the aphid susceptible strain after 7 days of aphid inoculation.

The genetic analysis of the aphid-resistant character of the god of longevity peach source in the prior art finds that: in the aphid resistance segregation population, the resistance is discontinuously distributed and has obvious double peak characteristics; the aphid-resistant individual (RR or RR) is crossed with the aphid-susceptible individual (RR) to give a 1: 0 or 1: 1, the cross of resistant individuals (RR or Rr) with resistant individuals occurs in a ratio of 1: 0 or 3: 1, no resistant individuals are found in the cross between susceptible individuals, no obvious change is found in the 6 th generation of resistance, which indicates that the resistance is controlled by dominant single gene, the gene is located in the same linkage group with yellow/white pulp in linkage analysis, the genetic distance is about 27.5cM, and the Rm2 type resistance is presumed to be one type (see 'the genetic analysis of pink god' on green peach aphid resistance, the fruit tree academic report 2016,33(5):578 584, cow goon, etc.).

At present, genetic analysis is carried out on the aphid resistance character from the god of longevity peach, the position of the aphid resistance gene from the god of longevity peach is preliminarily determined, but the aphid resistance gene is still not accurately positioned, and a disease resistance gene for controlling the aphid resistance character of plant evasion is not found.

Disclosure of Invention

The invention aims to provide a novel disease-resistant gene for controlling plant aversion aphid-resistant character, and the disease-resistant gene regulates the aphid-resistant character of the god of longevity.

The invention also provides the protein coded by the novel disease-resistant gene for controlling the aphid-resistant character of plant evasion, and the protein regulates the aphid-resistant character of the god of longevity.

The invention also provides application of the novel disease-resistant gene for controlling the aphid resistance character of the plant evasion in screening the aphid-resistant single plant, and the aphid-resistant single plant can be accurately screened by taking the disease-resistant gene as a molecular marker.

The invention also provides application of the disease-resistant gene for controlling the aphid resistance character of the aversion of the plant in enhancing the aphid resistance character of the plant, and the existence of the disease-resistant gene can effectively enhance the aphid resistance character of the plant.

The invention also provides application of the disease-resistant gene for controlling plant evasion aphid-resistant characters in breeding of peach aphid-resistant varieties, and the peach aphid-resistant varieties containing the disease-resistant gene can be obtained through cultivation.

The invention also provides application of the protein for controlling the plant aversion resistance aphid-resistant character in screening an aphid-resistant individual plant, and the aphid-resistant individual plant can be accurately screened by taking the protein as a molecular marker.

The invention also provides application of the protein for controlling the aphid resistance of the plant evasion in enhancing the aphid resistance of the plant, and the existence of the protein can effectively enhance the aphid resistance of the plant.

In order to achieve the purpose, the invention adopts the technical scheme that:

a disease-resistant gene for controlling plant aversion and aphid-resistant character has a nucleotide sequence shown in SEQ ID NO. 2.

According to the invention, candidate genes are subjected to a great prediction according to gene functions and a re-sequencing result, a specific upstream primer is designed by inserting a 20bp sequence at 400bp upstream of an initiation codon of a disease-resistant gene (ppa000596m), a downstream primer is designed by using a conserved region at downstream of a stop codon of a ppa000596m gene, and a disease-resistant gene which does not exist in a peach genome is obtained by amplification, wherein the disease-resistant gene is shown as SEQ ID No. 2. The existence of the gene and the aphid resistance character show coseparation, the expression mode of the new gene is further analyzed, the result shows that the expression of the gene in the population and the aphid resistance of a single plant show consistency characteristics, and the new gene is the new disease-resistant gene for controlling the aphid resistance character of the avoidance of the peach tree.

The amino acid sequence of the protein for controlling the aphid resistance character of the avoidance of plants is shown in SEQ ID NO. 3.

According to the nucleotide sequence of the disease-resistant gene for controlling the aphid resistance of the evasion of plants, the amino acid sequence of the protein expressed by the disease-resistant gene is predicted, and the protein is a key protein in the aphid resistance regulation of the god of longevity peach.

The new disease-resistant gene for controlling plant evasion aphid-resistant character is applied to screening aphid-resistant single plants.

In practical application, the disease-resistant gene can be used as a molecular marker to detect whether the disease-resistant gene exists, and the single plant with the disease-resistant gene can be determined to be the aphid-resistant single plant, so that the aphid-resistant single plant can be accurately screened.

The application of the disease-resistant gene for controlling the aphid-resistant character of the plant evasion in enhancing the aphid-resistant character of the plant.

The existence of the disease-resistant gene can effectively enhance the aphid resistance of the plant, and in practical application, the disease-resistant gene can be applied to other susceptible plants in a genetic engineering mode, so that the aphid resistance of the susceptible plants is improved.

The disease-resistant gene for controlling plant evasion aphid-resistant character is applied to breeding of peach aphid-resistant varieties.

In practical application, the peach tree line which contains the disease-resistant gene and is stably inherited can be obtained through genetic engineering, traditional crossbreeding or a combination of the two ways, so as to obtain a new peach aphid-resistant variety.

The protein for controlling the aphid resistance of plant evasion is applied to screening of individual aphid resistance plants.

In practical application, the protein can be used as a molecular marker to detect whether the protein exists, and the individual plant with the protein can be determined to be the aphid-resistant individual plant, so that the aphid-resistant individual plant can be accurately screened.

The protein for controlling the aphid resistance character of the plant evasion is applied to the aspect of enhancing the aphid resistance character of the plant.

The existence of the protein can effectively enhance the aphid resistance of the plant, and in practical application, the protein can be applied to other plants in an exogenous input mode so as to improve the aphid resistance of the infected plant.

Drawings

FIG. 1 is a typical representation diagram of a 'powdered god of longevity' source aphid-resistant and aphid-susceptible strain damaged by aphids in the prior art;

FIG. 2 is a diagram showing the results of fine localization of the isolated population of aphids resistant and susceptible to aphids in the present invention;

FIG. 3 is an analysis chart of gene expression patterns of 0-48 h of aphids inoculated on aphid resistant and susceptible varieties in a fine localization interval in the invention;

FIG. 4 is a diagram showing the results of the 20bp insertion site upstream of the start codon of ppa000596m in multiple resistant varieties according to the present invention;

FIG. 5 is a diagram showing the design of a primer amplified to a new disease resistance gene PpRm3 in the present invention;

FIG. 6 is a diagram showing BlastN results of PpRm3 full-length cDNA in NCBI website;

FIG. 7 is a diagram showing the BlastX results of the amino acid sequence encoded by the full-length cDNA of PpRm3 in NCBI website;

FIG. 8 is a diagram showing the co-segregation of PpRm3 gene and aphid resistance trait by specific primer detection in the invention;

FIG. 9 is a diagram showing the expression of the PpRm3 gene in the leaf of an aphid-resistant and aphid-susceptible variety which invades for 0-48 h in the invention;

FIG. 10 is a diagram of the expression of the PpRm3 gene in the leaves of an aphid-resistant and aphid-susceptible segregation population;

FIG. 11 is a graph of the distribution of PpRm3 in aphid-resistant and aphid-susceptible varieties as a result of the re-sequencing in accordance with the present invention;

FIG. 12 is a diagram showing the distribution of PpRm3 similar genes in peach genome;

FIG. 13 is a map of a phylogenetic tree analysis of a PpRm 3-like gene of the present invention and a similar gene in 20 peach genomes;

FIG. 14 is a schematic diagram of the introduction of PpRm3 into cultivars by gene recombination and crossover between Shouxing peach and cultivar peach in the present invention.

Detailed Description

The present invention will be described in further detail with reference to specific examples. The equipment and reagents used in the examples and the experimental examples were commercially available except as specifically indicated. '01-77-3' is obtained by hybridizing '87-71' (aphid resistance) and '92-60' (flat peach), '01-77-03' is derived from '87-71', '87-71' is an ornamental and edible dual-purpose variety cultivated by agroforestrial academy of agriculture and forestry in Beijing, and '87-71' is derived from Shouxing peach (progress of dual-purpose peach breeding research in Beijing area [ J ]. Beijing agriculture, 2000, 18(6):23-25, Liu Jia 26877, Wangyu Ying, Song Jing I.).

Example 1 of a novel disease resistance Gene that controls the aphid resistance trait for plant evasion

The nucleotide sequence of the new disease-resistant gene for controlling the plant aversion aphid-resistant character in the embodiment is shown in SEQ ID NO. 2.

Example 1 protein for controlling plant aphid resistance traits

The amino acid sequence of the protein for controlling the aphid resistance of the plant evasion in the embodiment is shown in SEQ ID No. 3.

Example 1 application of disease-resistant Gene for controlling plant aphid-resistant traits in screening aphid-resistant Individual plants

In the embodiment, the disease-resistant gene is used as a molecular marker to detect whether the disease-resistant gene exists, and the single plant with the disease-resistant gene can be determined to be the aphid-resistant single plant, so that the aphid-resistant single plant can be accurately screened.

The method for detecting the disease-resistant gene comprises the following steps: designing a specific primer for PCR detection, and designing a specific DNA probe for detection.

The PCR detection upstream primer is as follows: 5'-AAAGTACTCAGAATAAGCTTTGACTT-3' (shown in SEQ ID NO. 6); the downstream primer is: 5'-ACTCAATACATCTATTACGTCTTCACC-3' (shown in SEQ ID NO. 7).

Example 1 of disease-resistant Gene controlling plant aphid resistance Property for enhancing plant aphid resistance Property

In the embodiment, the disease-resistant gene is transferred into other susceptible plants in a genetic engineering mode, so that the aphid resistance of the susceptible plants is improved.

Example 1 application of disease-resistant Gene for controlling plant aphid-resistant traits in peach aphid-resistant variety Breeding

In the embodiment, the peach tree line which contains the disease-resistant gene and is stably inherited is obtained by combining genetic engineering and traditional crossbreeding, so that a new peach aphid-resistant variety is obtained.

Example 1 application of proteins controlling plant aversion and aphid resistance traits in screening of aphid-resistant individuals

In the embodiment, the protein is used as a molecular marker to detect whether the protein exists, and the single plant with the protein can be determined to be the aphid-resistant single plant, so that the aphid-resistant single plant can be accurately screened.

The mode for detecting the protein is as follows: antibodies specific for the protein are designed for detection.

Example 1 application of proteins controlling plant aphid resistance traits to enhancement of plant aphid resistance traits

In the embodiment, the protein is applied to other plants in an exogenous input mode so as to improve the aphid resistance of the infected plants.

Test example 1 Fine localization of Rm3 site and screening of candidate genes in the region

At present, the research work on the genetic law of the aphid-resistant character of peaches in China is obviously lagged, but related materials have been applied to new variety breeding. In 1976, in order to cultivate varieties of peaches for both ornamental and fresh food, Beijing academy of agriculture and forestry, the superior line '8771' of peaches for both ornamental and fresh food is obtained by taking 'white phoenix' (aphid susceptible) and 'pink god of longevity' (green aphid resistant to peaches and ornamental varieties) as parents and performing 3-generation hybridization or backcross. After the '8771' major line is introduced into the peach breeding problem group of Zhengzhou fruit tree institute, through years of phenotypic observation and resistance identification, the '8771' is found to retain the resistance of the parent 'pink god of longevity' to green aphid of peach. The '8771' is used as a resistant parent, genetic analysis of a plurality of progeny groups discovers that the resistance of a tree body to green peach aphids is expressed as a genetic rule controlled by a dominant single gene, and the resistance is located in the same linkage group with a gene controlling yellow/white pulp of a No.1 chromosome, and is similar to the phenotype of the green peach aphids of weeping peach blossoms and 'Rubira', the resistance of the aphid resistant plant to the green peach aphids forms red or yellow allergic necrosis spots at the piercing and sucking parts 2-3 days after the green peach aphids pierce and suck, so that the resistance of the green peach blossoms of the long-life peach blossoms and the 'Rubira' is considered as the same type of evasion, and the green peach blossoms of the long-life peach blossoms have no relationship with the green peach blossoms of the weeping peach blossoms and the 'Rubira', and the resistance of the green peach blo. This part of the content can be summarized in Table 1. The above-mentioned aphid-resistant researches at home and abroad mainly focus on the direction of aphid-resistant material screening, genetic rule analysis, aphid-resistant gene positioning, etc., and researches on cloning of aphid-resistant regulatory genes of peach trees and functional verification of related genes, etc. have not been reported yet.

TABLE 1 overview of the major aphid-resistant material in peach trees

In the invention, Rm3 locus is finely positioned, and the test material used in the research process is a progeny segregation population of nectarine No. 13 '(Rr) in' 01-77-3 '(Rr) ×', and total number of 147 individuals are obtained. Firstly, re-sequencing two parents, analyzing a sequencing file by using integrated Genomics Viewer software, wherein the analysis result is shown as figure 2, the figure shows that exchange conditions of an exchange single plant and a positioning interval are displayed, polymorphic sites are searched in a forebody positioning interval (a rectangular frame region, No.1 chromosomes 45.08-46.23 Mb), the polymorphic sites of a population are analyzed and found to be 13 exchange single plants in total in the interval, the Rm3 interval can be narrowed to about 300kb (45.55-45.84 Mb), but the abnormal phenomenon of the exchange single plant exists in the 45.85-45.95 Mb interval of the population, the abnormal phenomenon is shown as that the exchange single plant is too many (6 single plants exist in 0.1 Mb), and the two exchange events are both carried out; thus, it is believed that the reference genome has a large segment of genome splice error at that site, or that the reference genome has a large segment of sequence difference from the genome at that site of the aphid-resistant cultivar.

Then, screening candidate regulatory genes for the genes in the positioning interval, and discovering that 33 genes are in total in a 300kb positioning segment through peach genome analysis, and discovering that the 33 genes have no obvious change in the expression quantity of 0-48 h of aphid invasion of the aphid resistant and aphid susceptible varieties through fluorescent quantitative PCR analysis (as shown in figure 3); through the analysis of the re-sequencing data of a plurality of aphid-resistant and aphid-susceptible varieties, the gene structure changes such as frame shift mutation, insertion deletion and the like which can cause the function change of the gene do not exist in the 300kb positioning segment, so that 33 genes in the fine positioning segment are determined not to comprise Rm3 candidate regulatory genes.

Test example 2 discovery of candidate regulatory gene (New R Gene) of Rm3

From the point of gene function analysis, among 33 genes in the fine localization interval, BRI1 related receptor kinase (Prupe.1G558900) and disease-resistant gene (ppa000596m) are very likely to be candidate genes for aphid resistance regulation, so more attention is paid to the two genes in the screening process. During the analysis of the results of the resequencing of the disease resistance gene (ppa000596m), it was found that: a20 bp sequence insertion (the sequence is 5'-GCTATACACTTGACTTGGTA-3', and the first 20bp of the sequence shown in SEQ ID NO. 4) is arranged at the upstream 400bp of the initiation codon of the gene, which indicates that the gene has two genotypes in a disease-resistant variety, and the re-sequencing results of a plurality of infection-resistant varieties show that the sequence insertion and the aphid-resistant character occur simultaneously (as shown in figure 4).

In the case where no good candidate regulatory gene could be found, it was attempted to amplify both genotypes of ppa000596m by inserting the 20bp sequence into the designed specific primers and other sequences nearby.

The designed PpRm3 gene amplification primers are as follows:

upstream primer containing 20bp insertion sequence: 5'-GCTATACACTTGACTTGGTAGCAAG-3' (shown in SEQ ID NO. 4); a downstream primer: 5'-CAGTGACAACAATTTCTTGAAATG-3' (shown in SEQ ID NO. 5).

DNA of '01-77-3' is used as a PCR amplification template, a specific upstream primer is designed by using a 20bp insertion sequence, a downstream primer is designed by using a conserved region downstream of a ppa000596m gene stop codon (as shown in figure 5), and a genome full-length sequence of a brand-new disease-resistant gene (later called PpRm3) which does not exist in a peach reference genome is amplified by PCR. PCR amplification using Baobao organisms

GXL DNA Polymerase (cat # R050A), amplification reaction system as shown in Table 2, and amplification reaction program as shown in Table 3.

TABLE 2 PCR amplification reaction System

TABLE 3 PCR amplification reaction procedure

The full-length sequence of PpRm3 gene is shown below (underlined part indicates exon part, total 6 exons, shown in seq.id no 1):

TGTATAGCATATAAAAGAACTCCAATACAACTAGAATACTCATCGTGGGGTATTCTTCCAATTCTACTTCTTATTCTTCTCTTACCTCTCTCAATTTCTCTACAATTTGCATAAGGTTTTTTTTAAAAAAAAAATTTATAAATGAAATCATCAATTTTTGTACAAATAATGAACGAAAATGAATTTTGTGAAAACAAACTGAAACAAGACTTGTGCTTTTCTTCTTTTTTTTTCTTTTTCCCTTTTAATTGGACATTGTGCTCGCCTCTTCCCTCTCTTTTCATGCCTCTTTCCCCTAAGCTTCCTTGCATTTTCCTGCCCTTCAATTTTAGCCGTCCAGTAGATCGCGTAGCCAAAAAGATATTATCTTTTCACTGTTCTTCCCACGTGGGAACTTTTGCTTTTGTCGACAGACACGGTTGAGGACCTTGACTATGCGTATTTCGAATTTATTCATCTGCGCTGCAACACATAAACCACTGCCCAGAAAAAATAATTGATTATGAAATAACTTCTTCTGCTCGAGTCGGATCGGAAGGCCAACTTGTGCATAATTTGTAGTTGGTCTTATCTGAACCAGTATCAATTAATGGATGCCAGGAAAGCCC ATAAAGCCTCCTCTTCATCCTCACCATCCTCTTCATCAAAACGTCGGGAGTACCAAGTGTTCTTGAGCTTCAGAGGT GAAGACACACGCAAGGGCTTCACAGGCCACCTCCACGCCGCATTATCTGGTGACGGATTCCGCGCCTTTCTTGATGA CAACGAGCTAAAAAGGGCGGAATTTATAAAAACCCAACTGGAGCAGGCAATCGACGGGTCCATGATCTCCATAATTG TCTTCTCCAAGAGGTATGCCGATTCCAGTTGGTGTCTTGACGAGCTGGTGAAGATCATGGAGTGCAGAGAAAGGCAA CAGGTTTTCCCATTGTTCTATAATGTTGACGCTTCAGATGTCCGGAAACAAACTGGTAGTTTTGCACAAGCATTTGA GAAACATGAAGCGGGCATCTGTGAAGGTAAACATGAGAAAGAAAAGGTACAGCGGTGGAGAAATGCTCTCACTCAAG CTGCAGATTTGTGTGGGGAAGATCTCAAAAATGCTGATGGGTAATTACTAATTTCTTGTTCCTTGGATTATCATCTCCTTACTTGACAACCATATGTTTTCTGTCCTTTTTGCTTTTGTTTTTGTTTTTCCAACCATAATGTTAAATCCCAATCTTAATTGTTAATTATATCTTAGTATAAAAAAAGAAAATAATTTGCTCATTCTAAATTATTAAGTTAGGGATTCAATTTAACCTTGTAGTTTGCTCTGCTTCTGTGTTTAATTGCCAGGCATGAAGCAAAGTTTATCAAGAAAATTCTTGGGAAG GTTAATAACTTGGTGAACAGCAAATACCAATTAGACACCGAAGACCTTGTTGGAATTACTTCTCGGGTGAACGATGT TGTTCGCATGATTGGTATTGAAAATTCAGGTTCTAAGGATGTTGTTCGCATGATTGGTATTTTGGGGATGGGCGGCA TTGGAAAAACAACGCTTGCCAAAACCATTTATAACAAATTTGGACCTATCTTTGAAGGTAGGAGTTTCCTTGCAGAC GTGAGGGAAGTATTTGCAAACCAACGCAGTAATGGTCTGGTTGGTTTGCAAGAACAACTTCTAAATGATATCTTGAA AAACGAGGGCATAAAGGTTGGATCTGTTGCTAAAGGGATCGATATGATAAGAGAAAGACTTTGCTGTAAAAGAGCAC TTGTCATAATTGACGATGCAGATGATCTACAGCAACTAAAAGCAATAGCTAGAGCTCGTGATTGGTTTGGTCCTGGA AGTAGAATTGTTATAACAACAAGAAATCAACATTTGCTAGACCAAGTTGGAGTGGATAGCACATATATGGCTCAAGC AATGGACGAGGAAGAAGCTCTAGAGCTCTTTAGTTGGCATGCCTTTGAAATTGGTTATCCTGATCAAGAATATCTTA ACCTCTCAAAACGTGTAATTCGTTACTGTCAAGGCTTGCCACTAGCACTTCGAGTTGTAGGGTCTTTTCTGATTAAA AGATCCATAGTTGAGTGGGAAAGCCATTTGGAGAAATTGGAAAGGAGTCCTCCTGATGGAGAAATTCAAAAAGTACT CAGAATAAGCTTTGACTTGCTACCTGATCAGGAAAAGAGAGAGATATTCCTTGATATATCTTGTTTCTTTATAGGAA TGGACAAGGACTACGTAACACAAATATTAAAGGGATGTGACTTTTCTGCAACGATAGGAATCAGTGTCCTCATTGAG CGGTGCCTTGTAACTGTTAGTGAGGAAAAGCTGAGGATGCATGATTTGCTTCGAGACATGGGAAGAGAGATCGTTCG TGAAAAGTCCACCGGCCGTGCTGAAAAATTTAGTAGATTGTGGAAAGGTGAAGACGTAATAGATGTATTGAGTGATG AATCTGTAAGTATTTTCCCAGTAAAGTTTTAGAATGCGTCATGCAAGAGAAGCATATGTATCCCATGTCTATAATAAATTAAAATGTCTAAACCAAAGTAAAAGAGGGGGTTCTCACACATTCACACTACCAAGGTGCCATATGAATTTGAACTTGAGACCTTAGTCTACAAGTTTAGATCATTTTTCACTTCACTAGACCCCGTTGATCAAAGCTCAATTAGGCATAACCAACAAAAAATATGTCCCTTGATTGCAAACATAAACTTATAGGGCGTAACATGGTCCAATACTATTCATGTTTGTAAATCATTTTTCTAACTCGGTATCTTTTGTTAATAGGGAACTAAAAAAATTGGAGGAGTTGCTCTAGATTCGGATCTAGA TTTTATTAGCTTCCGTGCACAAGCATTTACCAACATGAAAAAACTGAGGTTACTCCACCTCAGCGGAGTGGAGCTCA CTGGAGAGTACAAAGATTTTCCCCAAAATTTAATATGGTTGAGCTGGCTTCGATTCCCTTTAGAGTCCATACCAGAT GACTTTCCTGTGCAACCAAAACTAGTTGCTTTAGACCTGCAGTGTAGCGAACTCAAAATAGTTTGGAAGGATAGCAA GGTATAATAATCTCAACCTAGTTGTTGTTGGTTCTTTCCTTGTGGTTCTATACATATATTATACTTCACCTTCTCTTCATTTTGGTTTAATTCCTGTATCAGAGACCTTCCTTTTGGATTTTAATTTCCATTCTAATTTTGTTTTTGTGTAACAGTTGCATCAGAATTTGAAAATCCTTAATCTCAGTGGTTCCTATAAGCTAACAAAATCACCAGACTTTTCAAAACTCC CAAATCTGGAGGAATTGATATTGGAAGACTGTGAGAGTTTGTCTGAGGTTCACTCATCCATCGGGGATCTTGGAAGA CTTTCTTTGGTAAATCTTAAAGGCTGCATAATGCTAAAGGATCTCCCACTGAATTTCTATAAATCCAAGTCTATTGA AACTCTTATACTGAATAAGTGTAGGAGTTTTGTAAAGTTGGCTGAGGGCTTAGGGGACATGGTATCATTGACAACTC TGAAAGCGGATGAGACAGCCATAAGACAAATACCATCTTCCATATTAAAATTGAAGAAACTGAAAGTTTTATCATTA TGTAATGTGAATGGGTCGCCATCAACAAATCTTTTGCCTCCTTCGTTGCAAAGTTTAAGCTCTTTAAGAGAATTAGC TCTTGCAGACTGGAGTTTAACTGATGATGCATTCCCCAAGGATCTCGGTAGCCTAATTTCCTTAGAAAATTTAGATC TTGCAGGAAATGATTTTTGCAGCCTACCAAGCTTCAGTCGTCTTTCAAAGCTTCATGATTTGTCTTTAAGTAAGTGC AAAAATCTTCGTGCAATCCCAGATTTACCAACAAATTTGAAAGTCTTAAAAGCAGAATATTGCTTTGAATTGGAAAA AATGCCAGATTTTTCAGAAATGTCAAATATAAAAGAATTGTATCTAAGTGGTTCGGACAAAGTCACTGAGATTCCAG GCCTGGATAAGTCATTAAACTCCATGACAATGATTTCTATGGATGGCTGCACTAATCTCACTGCTGATTTTAGGAAG AACATCCTACAGGTCAATCCTTCTCTCTCTCTCTCAAACACACACATACATCTTTCTCTTTCTATCACATACAACGCATATGTACACATGCAGGCATAAGAATGTATTTCTTATAGAAATTTGGCTCTACATGGTATAGGGATGGACTTCTTGC GGATATGGTGGCATTTTTCTCAGTGGAAATGATATTCCTGATTGGTTCGACTGCGTCCATGACGACGATATTGTGTA TTTCACTGTGCCTCGAAGTGTTGGTCGTAATTTTAAAGGGTTAACTTTGTCCTTCGTTTCCTCTCCAGGCTTTTTAA GTCGTCCTATTAGCATTAGCATTAAAAACATGACCAAGGGGGCTGAGCTTGAAGCCAGGATCATACCCGATTGTCCA ATTGACCAAGGGGGCTGGCTCATTTACAATCCAGTTTCTTATCTTTGGCAGGGACAGTTATCAAATGACGAGCTCAA ATTGCAAGACGGTGATAAAGTCTTGATTGAAATAATAGTGGAGGATTATTATAGGGTGAAGGTCAAGGTGAAGAAAA CAGGTGTTAGTCTGGTATGGGACAAATTTATGAACGAAAATATGATTGATTACCATCTTTGTGCGTATGAACGACGC CCATCTCAAAATCTGGTCAATGATGATGACATCATTCATGTCGAAGATGATAATCACATAACAAAATCACCAGACTT TTCAAAATTCCCAAATCTCGAGAAGTTGATATTGAAAGGTTGTAAGAAGTTGATTAAGGTTCACTCATACATCGGGG ATCTTGGAAGACTTTCTTTGGTAAATCTTGAAGACTGCGAAATGCTAAGGGATCTCCCACTGAATTTCTATAAATCC AAGTCTATTGAAACTCTTATACTGAATGGTTGTTCAAGATTTGAAGACTTGGCTGATGGCTTAGGGGACATGGTATC ATTAACAGTTTTGGAAGCAAATAAGACAGCCATCAGACGAATTCCATCTTCCATAGTAAAATTGAAGAACTTAGAAC ATCTGCTTCTTGCAAACAATTACTTTCGTAGCCTACCAAGTCTCGCTGGTCTTTCAAAGCTCAAGGTCTTGTCTTTA AATGCATGCAGAGAACTTCGTGCAATCCCAGATTTACCAACCAATTTGTATGTTCTGAAAGCAAATGGCTGCCCAAA TTTGGAAACAATTCCAGATTTTTCAAAAATGTCGAATATGAGAAAATTGTATCTCCGTGATTCGGTCAAACTCACTG AGGTTCCAGGCTTGGATAAGTCGTTAAACTCCATGACAAGGATTCGTATGGAAGGCTGCACCAATCTGACTGCCGAT TTTAGGAACAACATCCAACAGGTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCACACACACACACACACACAGAGGCACAGCTGCAGATGCGGCCCATGTAAATATGCATATGCATGTATAAGAATGTGTTGCTTCTGGTGTGCAGAGATGGACTTCTTGCGGATTTGGTGGAATTTATTTGAATGGA ATTTATGATATTCCTGAGTGGTTCAAAATCCTCAATGATGTGGGCAATATCGTCTTCTTTGAAGTTCCTCAAAGAAT CATGGGTCGTGATTTAAAAGGGTTGACTATATGCTTCGTTTACTCTTTTGTTGTTTTTGGCCGAGAACTTGAAGGTC CTATTGGCATTATCGTTAGAAATCTTACCAAACAAACTGCTTTGCACACCAAGATAGCATTTGCCAGATGCGGAAGA CCAGAACCGGATTTGCTTATTTGGAGACTATTGTCAACCGGACTTGAAGACCGTTATCTTTGGCAGGGACAATTGTC AAACGATGTGCTCTGTTTGCAAGCCGGGGACCAAGTCTCCATTCTTGTAAGGCCTCTAGTTGATTTTGTGATAGTGA AGAAGACAGGGGTTCATCTAGAATGGGACAAAGTCATGAAGGAGAATATGGATAATCTGGATCCTCATTTGTATGAT TGGAAAACGAATCGGGATTTTTGATGGGGAGTTGATGAATCCAT

by searching the cDNA sequence of the PpRm3 gene (shown as SEQ ID NO. 2) in the NCBI website BlastN program for similarity with the nucleic acid sequences of all the existing species, several nucleic acid sequences with the highest similarity are shown in peach, and the similarity is about 90% (shown in FIG. 6). Then searching the similarity of the cDNA coding amino acid sequence of the PpRm3 gene and the amino acid sequences of all the existing species by a BlastX program, and indicating that one amino acid sequence with the highest similarity exists in the sweet cherries, and the similarity is 81 percent (as shown in FIG. 7). The results of fig. 6 and 7 indicate that PpRm3 gene is an unreported novel gene encoding a disease resistance protein (R protein). The 20bp detectable insertion site primer is used for amplifying an aphid-resistant segregation population containing 147 single plants, and the existence of the gene and the aphid-resistant character are found to be co-segregation (as shown in figure 8), so that the new gene is used as a candidate gene for aphid-resistant regulation.

Test example 3 analysis of PpRm3 Gene expression Pattern

In order to further determine whether the PpRm3 gene is involved in the aphid resistance regulation process of peach trees, the PpRm3 gene expression conditions of the gene before and after peach aphid inoculation and in a single plant of a flu-resistant segregation population are detected.

The primers used for detection were: an upstream primer: 5'-AAAGTACTCAGAATAAGCTTTGACTT-3' (shown in SEQ ID NO. 6); a downstream primer: 5'-ACTCAATACATCTATTACGTCTTCACC-3' (shown in SEQ ID NO. 7). Amplification was performed using a Roche LightCycler 480SYBR Green I Master (cat. 4887352001) using the PCR reaction system shown in Table 4 and the PCR reaction program shown in Table 5.

TABLE 4 PCR amplification reaction System

TABLE 5 PCR amplification reaction procedure

The detection result is shown in FIG. 9, and the PpRm3 gene is found to have no expression before and after the aphid susceptible variety is inoculated with aphids, has a certain expression level in the aphid resistant variety and shows response to aphid invasion.

In addition, the expression of the PpRm3 gene in the segregating population is also detected, the detection method is the same as the detection of the single plant, the detection result of the expression quantity is shown in FIG. 10, the result shows that the PpRm3 gene is not expressed in the aphid susceptible single plant at all, the gene is expressed in the aphid resistant single plant, the expression of the gene in the population and the aphid resistance of the single plant present perfect consistency characteristics, and the gene and the aphid resistance character have very close connection.

Test example 4PpRm3 was a novel gene introduced into a cultivar by crossing with Shouxiang peach

At present, the DNA fragment containing the full length of the PpRm3 gene is amplified to about 5.5kb (shown as SEQ ID NO. 1), local BlastN comparison is carried out on a 150bp DNA sequence library generated by resequencing an aphid resistant variety and an aphid susceptible variety by using the fragment, and the result shows that hundreds of gene fragments with 100% similarity can be searched in the aphid resistant variety, while DNA fragments with 100% similarity are completely searched in a resequencing library of the aphid susceptible variety (shown in FIG. 11), which indicates that the gene only exists in the aphid resistant variety, but completely does not exist in the aphid susceptible variety, but does not exist in a genotype of the aphid with the gene without normal function.

The peach genome was subjected to homologous gene search using the full length of the PpRm3 gene, and found that there were 20 genes with high similarity among them, 19.6Mb (2), 35.9Mb (1), 44.1Mb (2), and 45.00-46.03 Mb (14) distributed on chromosome 1, and 25.5Mb (1) on chromosome 6, respectively (as shown in fig. 12). FIG. 13 is a phylogenetic tree analysis of the PpRm3 similar genes, each represented by gene number, chromosome physical location, and the 20 peach genomes similar genes, the post-gene shape of which corresponds to its shape above the gene cluster of FIG. 12. It is very obvious that a gene cluster of PpRm3 similar genes (disease-resistant genes) is in a 45.00-46.03 Mb region of the No.1 chromosome, and the PpRm3 has the highest similarity with the gene at 45.73Mb and the gene at 46.03Mb in the gene cluster, which is the basis of a genome sequence of chromosome recombination at the site in the crossing process of the Sasa chinensis and the cultivated species.

In conclusion, the applicant cultured the peach aphid-resistant superior line in the prophase of the subject group, the resistance is derived from peach closely related species, namely the Erythroculter persicum, and has obvious resistance to peach green aphid, and the resistance is regulated and controlled by the Rm3 locus of the No.1 chromosome. The applicant finds the aphid-resistant candidate regulatory gene PpRm3 by combining fine localization with candidate gene screening, and the functional annotation, expression analysis and linkage analysis of the gene support the aphid-resistant candidate regulatory gene; PpRm3 is a disease-resistant gene which does not exist in a peach reference genome, and the gene only appears in an aphid-resistant peach variety; a disease-resistant gene cluster exists at the Rm3 locus of chromosome 1, and the disease-resistant gene with the highest similarity to the PpRm3 gene in the peach genome is positioned near a fine positioning interval. Therefore, it is presumed that the aphid resistance of the peach tree Rm3 is regulated by a disease resistance gene, which is derived from wild peach kindred species, of the tamarind, and is absent but present in the cultivar, and the cultivar is crossed with the tamarind to exchange or insert the gene into the disease resistance gene cluster of chromosome 1, 45.00-46.03 Mb, resulting in the cultivar having resistance to green aphid of peach (as shown in fig. 14).

<110> Zhengzhou fruit tree institute of Chinese academy of agricultural sciences

<120> disease-resistant gene and protein for controlling plant aversion and aphid resistance characters and application thereof

<160>7

<170>SIPOSequenceListing 1.0

<211>6119

<212>DNA

<213> birthday star peach

<221>PpRm3Full-length sequence of gene

<400>1

tgtatagcat ataaaagaac tccaatacaa ctagaatact catcgtgggg tattcttcca 60

attctacttc ttattcttct cttacctctc tcaatttctc tacaatttgc ataaggtttt 120

ttttaaaaaa aaaatttata aatgaaatca tcaatttttg tacaaataat gaacgaaaat 180

gaattttgtg aaaacaaact gaaacaagac ttgtgctttt cttctttttt tttctttttc 240

ccttttaatt ggacattgtg ctcgcctctt ccctctcttt tcatgcctct ttcccctaag 300

cttccttgca ttttcctgcc cttcaatttt agccgtccag tagatcgcgt agccaaaaag 360

atattatctt ttcactgttc ttcccacgtg ggaacttttg cttttgtcga cagacacggt 420

tgaggacctt gactatgcgt atttcgaatt tattcatctg cgctgcaaca cataaaccac 480

tgcccagaaa aaataattga ttatgaaata acttcttctg ctcgagtcgg atcggaaggc 540

caacttgtgc ataatttgta gttggtctta tctgaaccag tatcaattaa tggatgccag 600

gaaagcccat aaagcctcct cttcatcctc accatcctct tcatcaaaac gtcgggagta 660

ccaagtgttc ttgagcttca gaggtgaaga cacacgcaag ggcttcacag gccacctcca 720

cgccgcatta tctggtgacg gattccgcgc ctttcttgat gacaacgagc taaaaagggc 780

ggaatttata aaaacccaac tggagcaggc aatcgacggg tccatgatct ccataattgt 840

cttctccaag aggtatgccg attccagttg gtgtcttgac gagctggtga agatcatgga 900

gtgcagagaa aggcaacagg ttttcccatt gttctataat gttgacgctt cagatgtccg 960

gaaacaaact ggtagttttg cacaagcatt tgagaaacat gaagcgggca tctgtgaagg 1020

taaacatgag aaagaaaagg tacagcggtg gagaaatgct ctcactcaag ctgcagattt 1080

gtgtggggaa gatctcaaaa atgctgatgg gtaattacta atttcttgtt ccttggatta 1140

tcatctcctt acttgacaac catatgtttt ctgtcctttt tgcttttgtt tttgtttttc 1200

caaccataat gttaaatccc aatcttaatt gttaattata tcttagtata aaaaaagaaa 1260

ataatttgct cattctaaat tattaagtta gggattcaat ttaaccttgt agtttgctct 1320

gcttctgtgt ttaattgcca ggcatgaagc aaagtttatc aagaaaattc ttgggaaggt 1380

taataacttg gtgaacagca aataccaatt agacaccgaa gaccttgttg gaattacttc 1440

tcgggtgaac gatgttgttc gcatgattgg tattgaaaat tcaggttcta aggatgttgt 1500

tcgcatgatt ggtattttgg ggatgggcgg cattggaaaa acaacgcttg ccaaaaccat 1560

ttataacaaa tttggaccta tctttgaagg taggagtttc cttgcagacg tgagggaagt 1620

atttgcaaac caacgcagta atggtctggt tggtttgcaa gaacaacttc taaatgatat 1680

cttgaaaaac gagggcataa aggttggatc tgttgctaaa gggatcgata tgataagaga 1740

aagactttgc tgtaaaagag cacttgtcat aattgacgat gcagatgatc tacagcaact 1800

aaaagcaata gctagagctc gtgattggtt tggtcctgga agtagaattg ttataacaac 1860

aagaaatcaa catttgctag accaagttgg agtggatagc acatatatgg ctcaagcaat 1920

ggacgaggaa gaagctctag agctctttag ttggcatgcc tttgaaattg gttatcctga 1980

tcaagaatat cttaacctct caaaacgtgt aattcgttac tgtcaaggct tgccactagc 2040

acttcgagtt gtagggtctt ttctgattaa aagatccata gttgagtggg aaagccattt 2100

ggagaaattg gaaaggagtc ctcctgatgg agaaattcaa aaagtactca gaataagctt 2160

tgacttgcta cctgatcagg aaaagagaga gatattcctt gatatatctt gtttctttat 2220

aggaatggac aaggactacg taacacaaat attaaaggga tgtgactttt ctgcaacgat 2280

aggaatcagt gtcctcattg agcggtgcct tgtaactgtt agtgaggaaa agctgaggat 2340

gcatgatttg cttcgagaca tgggaagaga gatcgttcgt gaaaagtcca ccggccgtgc 2400

tgaaaaattt agtagattgt ggaaaggtga agacgtaata gatgtattga gtgatgaatc 2460

tgtaagtatt ttcccagtaa agttttagaa tgcgtcatgc aagagaagca tatgtatccc 2520

atgtctataa taaattaaaa tgtctaaacc aaagtaaaag agggggttct cacacattca 2580

cactaccaag gtgccatatg aatttgaact tgagacctta gtctacaagt ttagatcatt 2640

tttcacttca ctagaccccg ttgatcaaag ctcaattagg cataaccaac aaaaaatatg 2700

tcccttgatt gcaaacataa acttataggg cgtaacatgg tccaatacta ttcatgtttg 2760

taaatcattt ttctaactcg gtatcttttg ttaataggga actaaaaaaa ttggaggagt 2820

tgctctagat tcggatctag attttattag cttccgtgca caagcattta ccaacatgaa 2880

aaaactgagg ttactccacc tcagcggagt ggagctcact ggagagtaca aagattttcc 2940

ccaaaattta atatggttga gctggcttcg attcccttta gagtccatac cagatgactt 3000

tcctgtgcaa ccaaaactag ttgctttaga cctgcagtgt agcgaactca aaatagtttg 3060

gaaggatagc aaggtataat aatctcaacc tagttgttgt tggttctttc cttgtggttc 3120

tatacatata ttatacttca ccttctcttc attttggttt aattcctgta tcagagacct 3180

tccttttgga ttttaatttc cattctaatt ttgtttttgt gtaacagttg catcagaatt 3240

tgaaaatcct taatctcagt ggttcctata agctaacaaa atcaccagac ttttcaaaac 3300

tcccaaatct ggaggaattg atattggaag actgtgagag tttgtctgag gttcactcat 3360

ccatcgggga tcttggaaga ctttctttggtaaatcttaa aggctgcata atgctaaagg 3420

atctcccact gaatttctat aaatccaagt ctattgaaac tcttatactg aataagtgta 3480

ggagttttgt aaagttggct gagggcttag gggacatggt atcattgaca actctgaaag 3540

cggatgagac agccataaga caaataccat cttccatatt aaaattgaag aaactgaaag 3600

ttttatcatt atgtaatgtg aatgggtcgc catcaacaaa tcttttgcct ccttcgttgc 3660

aaagtttaag ctctttaaga gaattagctc ttgcagactg gagtttaact gatgatgcat 3720

tccccaagga tctcggtagc ctaatttcct tagaaaattt agatcttgca ggaaatgatt 3780

tttgcagcct accaagcttc agtcgtcttt caaagcttca tgatttgtct ttaagtaagt 3840

gcaaaaatct tcgtgcaatc ccagatttac caacaaattt gaaagtctta aaagcagaat 3900

attgctttga attggaaaaa atgccagatt tttcagaaat gtcaaatata aaagaattgt 3960

atctaagtgg ttcggacaaa gtcactgaga ttccaggcct ggataagtca ttaaactcca 4020

tgacaatgat ttctatggat ggctgcacta atctcactgc tgattttagg aagaacatcc 4080

tacaggtcaa tccttctctc tctctctcaa acacacacat acatctttct ctttctatca 4140

catacaacgc atatgtacac atgcaggcat aagaatgtat ttcttataga aatttggctc 4200

tacatggtat agggatggac ttcttgcgga tatggtggca tttttctcag tggaaatgat 4260

attcctgatt ggttcgactg cgtccatgac gacgatattg tgtatttcac tgtgcctcga 4320

agtgttggtc gtaattttaa agggttaact ttgtccttcg tttcctctcc aggcttttta 4380

agtcgtccta ttagcattag cattaaaaac atgaccaagg gggctgagct tgaagccagg 4440

atcatacccg attgtccaat tgaccaaggg ggctggctca tttacaatcc agtttcttat 4500

ctttggcagg gacagttatc aaatgacgag ctcaaattgc aagacggtga taaagtcttg 4560

attgaaataa tagtggagga ttattatagg gtgaaggtca aggtgaagaa aacaggtgtt 4620

agtctggtat gggacaaatt tatgaacgaa aatatgattg attaccatct ttgtgcgtat 4680

gaacgacgcc catctcaaaa tctggtcaat gatgatgaca tcattcatgt cgaagatgat 4740

aatcacataa caaaatcacc agacttttca aaattcccaa atctcgagaa gttgatattg 4800

aaaggttgta agaagttgat taaggttcac tcatacatcg gggatcttgg aagactttct 4860

ttggtaaatc ttgaagactg cgaaatgcta agggatctcc cactgaattt ctataaatcc 4920

aagtctattg aaactcttat actgaatggt tgttcaagat ttgaagactt ggctgatggc 4980

ttaggggaca tggtatcatt aacagttttg gaagcaaata agacagccat cagacgaatt 5040

ccatcttcca tagtaaaatt gaagaactta gaacatctgc ttcttgcaaa caattacttt 5100

cgtagcctac caagtctcgc tggtctttca aagctcaagg tcttgtcttt aaatgcatgc 5160

agagaacttc gtgcaatccc agatttacca accaatttgt atgttctgaa agcaaatggc 5220

tgcccaaatt tggaaacaat tccagatttt tcaaaaatgt cgaatatgag aaaattgtat 5280

ctccgtgatt cggtcaaact cactgaggtt ccaggcttgg ataagtcgtt aaactccatg 5340

acaaggattc gtatggaagg ctgcaccaat ctgactgccg attttaggaa caacatccaa 5400

caggtctctc tctctctctc tctctctctc tctctctctc tctctctctc tctctctctc 5460

tctctctctc tctctctcac acacacacac acacacagag gcacagctgc agatgcggcc 5520

catgtaaata tgcatatgca tgtataagaa tgtgttgctt ctggtgtgca gagatggact 5580

tcttgcggat ttggtggaat ttatttgaat ggaatttatg atattcctga gtggttcaaa 5640

atcctcaatg atgtgggcaa tatcgtcttc tttgaagttc ctcaaagaat catgggtcgt 5700

gatttaaaag ggttgactat atgcttcgtt tactcttttg ttgtttttgg ccgagaactt 5760

gaaggtccta ttggcattat cgttagaaat cttaccaaac aaactgcttt gcacaccaag 5820

atagcatttg ccagatgcgg aagaccagaa ccggatttgc ttatttggag actattgtca 5880

accggacttg aagaccgtta tctttggcag ggacaattgt caaacgatgt gctctgtttg 5940

caagccgggg accaagtctc cattcttgta aggcctctag ttgattttgt gatagtgaag 6000

aagacagggg ttcatctaga atgggacaaa gtcatgaagg agaatatgga taatctggat 6060

cctcatttgt atgattggaa aacgaatcgg gatttttgat ggggagttga tgaatccat 6119

<211>4494

<212>DNA

<213> birthday star peach

<221>PpRm3Gene cDNA sequence

<400>2

atggatgcca ggaaagccca taaagcctcc tcttcatcct caccatcctc ttcatcaaaa 60

cgtcgggagt accaagtgtt cttgagcttc agaggtgaag acacacgcaa gggcttcaca 120

ggccacctcc acgccgcatt atctggtgac ggattccgcg cctttcttga tgacaacgag 180

ctaaaaaggg cggaatttat aaaaacccaa ctggagcagg caatcgacgg gtccatgatc 240

tccataattg tcttctccaa gaggtatgcc gattccagtt ggtgtcttga cgagctggtg 300

aagatcatgg agtgcagaga aaggcaacag gttttcccat tgttctataa tgttgacgct 360

tcagatgtcc ggaaacaaac tggtagtttt gcacaagcat ttgagaaaca tgaagcgggc 420

atctgtgaag gtaaacatga gaaagaaaag gtacagcggt ggagaaatgc tctcactcaa 480

gctgcagatt tgtgtgggga agatctcaaa aatgctgatg ggcatgaagc aaagtttatc 540

aagaaaattc ttgggaaggt taataacttg gtgaacagca aataccaatt agacaccgaa 600

gaccttgttg gaattacttc tcgggtgaac gatgttgttc gcatgattgg tattgaaaat 660

tcaggttcta aggatgttgt tcgcatgatt ggtattttgg ggatgggcgg cattggaaaa 720

acaacgcttg ccaaaaccat ttataacaaa tttggaccta tctttgaagg taggagtttc 780

cttgcagacg tgagggaagt atttgcaaac caacgcagta atggtctggt tggtttgcaa 840

gaacaacttc taaatgatat cttgaaaaac gagggcataa aggttggatc tgttgctaaa 900

gggatcgata tgataagaga aagactttgc tgtaaaagag cacttgtcat aattgacgat 960

gcagatgatc tacagcaact aaaagcaata gctagagctc gtgattggtt tggtcctgga 1020

agtagaattg ttataacaac aagaaatcaa catttgctag accaagttgg agtggatagc 1080

acatatatgg ctcaagcaat ggacgaggaa gaagctctag agctctttag ttggcatgcc 1140

tttgaaattg gttatcctga tcaagaatat cttaacctct caaaacgtgt aattcgttac 1200

tgtcaaggct tgccactagc acttcgagtt gtagggtctt ttctgattaa aagatccata 1260

gttgagtggg aaagccattt ggagaaattg gaaaggagtc ctcctgatgg agaaattcaa 1320

aaagtactca gaataagctt tgacttgcta cctgatcagg aaaagagaga gatattcctt 1380

gatatatctt gtttctttat aggaatggac aaggactacg taacacaaat attaaaggga 1440

tgtgactttt ctgcaacgat aggaatcagt gtcctcattg agcggtgcct tgtaactgtt 1500

agtgaggaaa agctgaggat gcatgatttg cttcgagaca tgggaagaga gatcgttcgt 1560

gaaaagtcca ccggccgtgc tgaaaaattt agtagattgt ggaaaggtga agacgtaata 1620

gatgtattga gtgatgaatc tggaactaaa aaaattggag gagttgctct agattcggat 1680

ctagatttta ttagcttccg tgcacaagca tttaccaaca tgaaaaaact gaggttactc 1740

cacctcagcg gagtggagct cactggagag tacaaagatt ttccccaaaa tttaatatgg 1800

ttgagctggc ttcgattccc tttagagtcc ataccagatg actttcctgt gcaaccaaaa 1860

ctagttgctt tagacctgca gtgtagcgaa ctcaaaatag tttggaagga tagcaagttg 1920

catcagaatt tgaaaatcct taatctcagt ggttcctata agctaacaaa atcaccagac 1980

ttttcaaaac tcccaaatct ggaggaattg atattggaag actgtgagag tttgtctgag 2040

gttcactcat ccatcgggga tcttggaaga ctttctttgg taaatcttaa aggctgcata 2100

atgctaaagg atctcccact gaatttctat aaatccaagt ctattgaaac tcttatactg 2160

aataagtgta ggagttttgt aaagttggct gagggcttag gggacatggt atcattgaca 2220

actctgaaag cggatgagac agccataaga caaataccat cttccatatt aaaattgaag 2280

aaactgaaag ttttatcatt atgtaatgtg aatgggtcgc catcaacaaa tcttttgcct 2340

ccttcgttgc aaagtttaag ctctttaaga gaattagctc ttgcagactg gagtttaact 2400

gatgatgcat tccccaagga tctcggtagc ctaatttcct tagaaaattt agatcttgca 2460

ggaaatgatt tttgcagcct accaagcttc agtcgtcttt caaagcttca tgatttgtct 2520

ttaagtaagt gcaaaaatct tcgtgcaatc ccagatttac caacaaattt gaaagtctta 2580

aaagcagaat attgctttga attggaaaaa atgccagatt tttcagaaat gtcaaatata 2640

aaagaattgt atctaagtgg ttcggacaaa gtcactgaga ttccaggcct ggataagtca 2700

ttaaactcca tgacaatgat ttctatggat ggctgcacta atctcactgc tgattttagg 2760

aagaacatcc tacagggatg gacttcttgc ggatatggtg gcatttttct cagtggaaat 2820

gatattcctg attggttcga ctgcgtccat gacgacgata ttgtgtattt cactgtgcct 2880

cgaagtgttg gtcgtaattt taaagggtta actttgtcct tcgtttcctc tccaggcttt 2940

ttaagtcgtc ctattagcat tagcattaaa aacatgacca agggggctga gcttgaagcc 3000

aggatcatac ccgattgtcc aattgaccaa gggggctggc tcatttacaa tccagtttct 3060

tatctttggc agggacagtt atcaaatgac gagctcaaat tgcaagacgg tgataaagtc 3120

ttgattgaaa taatagtgga ggattattat agggtgaagg tcaaggtgaa gaaaacaggt 3180

gttagtctgg tatgggacaa atttatgaac gaaaatatga ttgattacca tctttgtgcg 3240

tatgaacgac gcccatctca aaatctggtc aatgatgatg acatcattca tgtcgaagat 3300

gataatcaca taacaaaatc accagacttt tcaaaattcc caaatctcga gaagttgata 3360

ttgaaaggtt gtaagaagtt gattaaggtt cactcataca tcggggatct tggaagactt 3420

tctttggtaa atcttgaaga ctgcgaaatg ctaagggatc tcccactgaa tttctataaa 3480

tccaagtcta ttgaaactct tatactgaat ggttgttcaa gatttgaaga cttggctgat 3540

ggcttagggg acatggtatc attaacagtt ttggaagcaa ataagacagc catcagacga 3600

attccatctt ccatagtaaa attgaagaac ttagaacatc tgcttcttgc aaacaattac 3660

tttcgtagcc taccaagtct cgctggtctt tcaaagctca aggtcttgtc tttaaatgca 3720

tgcagagaac ttcgtgcaat cccagattta ccaaccaatt tgtatgttct gaaagcaaat 3780

ggctgcccaa atttggaaac aattccagat ttttcaaaaa tgtcgaatat gagaaaattg 3840

tatctccgtg attcggtcaa actcactgag gttccaggct tggataagtc gttaaactcc 3900

atgacaagga ttcgtatgga aggctgcacc aatctgactg ccgattttag gaacaacatc 3960

caacagagat ggacttcttg cggatttggt ggaatttatt tgaatggaat ttatgatatt 4020

cctgagtggt tcaaaatcct caatgatgtg ggcaatatcg tcttctttga agttcctcaa 4080

agaatcatgg gtcgtgattt aaaagggttg actatatgct tcgtttactc ttttgttgtt 4140

tttggccgag aacttgaagg tcctattggc attatcgtta gaaatcttac caaacaaact 4200

gctttgcaca ccaagatagc atttgccaga tgcggaagac cagaaccgga tttgcttatt 4260

tggagactat tgtcaaccgg acttgaagac cgttatcttt ggcagggaca attgtcaaac 4320

gatgtgctct gtttgcaagc cggggaccaa gtctccattc ttgtaaggcc tctagttgat 4380

tttgtgatag tgaagaagac aggggttcat ctagaatggg acaaagtcat gaaggagaat 4440

atggataatc tggatcctca tttgtatgat tggaaaacga atcgggattt ttga 4494

<211>1497

<212>PRT

<213> birthday star peach

<221> PpRm3 protein amino acid sequence

<400>3

Met Asp Ala Arg Lys Ala His Lys Ala Ser Ser Ser Ser Ser Pro

1 5 10 15

Ser Ser Ser Ser Lys Arg Arg Glu Tyr Gln Val Phe Leu Ser Phe

20 25 30

Arg Gly Glu Asp Thr Arg Lys Gly Phe Thr Gly His Leu His Ala

35 40 45

Ala Leu Ser Gly Asp Gly Phe Arg Ala Phe Leu Asp Asp Asn Glu

50 55 60

Leu Lys Arg Ala Glu Phe Ile Lys Thr Gln Leu Glu Gln Ala Ile

65 70 75

Asp Gly Ser Met Ile Ser Ile Ile Val Phe Ser Lys Arg Tyr Ala

80 85 90

Asp Ser Ser Trp Cys Leu Asp Glu Leu Val Lys Ile Met Glu Cys

95 100 105

Arg Glu Arg Gln Gln Val Phe Pro Leu Phe Tyr Asn Val Asp Ala

110 115 120

Ser Asp Val Arg Lys Gln Thr Gly Ser Phe Ala Gln Ala Phe Glu

125 130 135

Lys His Glu Ala Gly Ile Cys Glu Gly Lys His Glu Lys Glu Lys

140 145 150

Val Gln Arg Trp Arg Asn Ala Leu Thr Gln Ala Ala Asp Leu Cys

155 160 165

Gly Glu Asp Leu Lys Asn Ala Asp Gly His Glu Ala Lys Phe Ile

170 175 180

Lys Lys Ile Leu Gly Lys Val Asn Asn Leu Val Asn Ser Lys Tyr

185 190 195

Gln Leu Asp Thr Glu Asp Leu Val Gly Ile Thr Ser Arg Val Asn

200 205 210

Asp Val Val Arg Met Ile Gly Ile Glu Asn Ser Gly Ser Lys Asp

215 220 225

Val Val Arg Met Ile Gly Ile Leu Gly Met Gly Gly Ile Gly Lys

230 235 240

Thr Thr Leu Ala Lys Thr Ile Tyr Asn Lys Phe Gly Pro Ile Phe

245 250 255

Glu Gly Arg Ser Phe Leu Ala Asp Val Arg Glu Val Phe Ala Asn

260 265 270

Gln Arg Ser Asn Gly Leu Val Gly Leu Gln Glu Gln Leu Leu Asn

275 280 285

Asp Ile Leu Lys Asn Glu Gly Ile Lys Val Gly Ser Val Ala Lys

290 295 300

Gly Ile Asp Met Ile Arg Glu Arg Leu Cys Cys Lys Arg Ala Leu

305 310 315

Val Ile Ile Asp Asp Ala Asp Asp Leu Gln Gln Leu Lys Ala Ile

320 325 330

Ala Arg Ala Arg Asp Trp Phe Gly Pro Gly Ser Arg Ile Val Ile

335 340 345

Thr Thr Arg Asn Gln His Leu Leu Asp Gln Val Gly Val Asp Ser

350 355 360

Thr Tyr Met Ala Gln Ala Met Asp Glu Glu Glu Ala Leu Glu Leu

365 370 375

Phe Ser Trp His Ala Phe Glu Ile Gly Tyr Pro Asp Gln Glu Tyr

380 385 390

Leu Asn Leu Ser Lys Arg Val Ile Arg Tyr Cys Gln Gly Leu Pro

395 400 405

Leu Ala Leu Arg Val Val Gly Ser Phe Leu Ile Lys Arg Ser Ile

410 415 420

Val Glu Trp Glu Ser His Leu Glu Lys Leu Glu Arg Ser Pro Pro

425 430 435

Asp Gly Glu Ile Gln Lys Val Leu Arg Ile Ser Phe Asp Leu Leu

440 445 450

Pro Asp Gln Glu Lys Arg Glu Ile Phe Leu Asp Ile Ser Cys Phe

455 460 465

Phe Ile Gly Met Asp Lys Asp Tyr Val Thr Gln Ile Leu Lys Gly

470 475 480

Cys Asp Phe Ser Ala Thr Ile Gly Ile Ser Val Leu Ile Glu Arg

485 490 495

Cys Leu Val Thr Val Ser Glu Glu Lys Leu Arg Met His Asp Leu

500 505 510

Leu Arg Asp Met Gly Arg Glu Ile Val Arg Glu Lys Ser Thr Gly

515 520 525

Arg Ala Glu Lys Phe Ser Arg Leu Trp Lys Gly Glu Asp Val Ile

530 535 540

Asp Val Leu Ser Asp Glu Ser Gly Thr Lys Lys Ile Gly Gly Val

545 550 555

Ala Leu Asp Ser Asp Leu Asp Phe Ile Ser Phe Arg Ala Gln Ala

560 565 570

Phe Thr Asn Met Lys Lys Leu Arg Leu Leu His Leu Ser Gly Val

575 580 585

Glu Leu Thr Gly Glu Tyr Lys Asp Phe Pro Gln Asn Leu Ile Trp

590 595 600

Leu Ser Trp Leu Arg Phe Pro Leu Glu Ser Ile Pro Asp Asp Phe

605 610 615

Pro Val Gln Pro Lys Leu Val Ala Leu Asp Leu Gln Cys Ser Glu

620 625 630

Leu Lys Ile Val Trp Lys Asp Ser Lys Leu His Gln Asn Leu Lys

635 640 645

Ile Leu Asn Leu Ser Gly Ser Tyr Lys Leu Thr Lys Ser Pro Asp

650 655 660

Phe Ser Lys Leu Pro Asn Leu Glu Glu Leu Ile Leu Glu Asp Cys

665 670 675

Glu Ser Leu Ser Glu Val His Ser Ser Ile Gly Asp Leu Gly Arg

680 685 690

Leu Ser Leu Val Asn Leu Lys Gly Cys Ile Met Leu Lys Asp Leu

695 700 705

Pro Leu Asn Phe Tyr Lys Ser Lys Ser Ile Glu Thr Leu Ile Leu

710 715 720

Asn Lys Cys Arg Ser Phe Val Lys Leu Ala Glu Gly Leu Gly Asp

725 730 735

Met Val Ser Leu Thr Thr Leu Lys Ala Asp Glu Thr Ala Ile Arg

740 745 750

Gln Ile Pro Ser Ser Ile Leu Lys Leu Lys Lys Leu Lys Val Leu

755760 765

Ser Leu Cys Asn Val Asn Gly Ser Pro Ser Thr Asn Leu Leu Pro

770 775 780

Pro Ser Leu Gln Ser Leu Ser Ser Leu Arg Glu Leu Ala Leu Ala

785 790 795

Asp Trp Ser Leu Thr Asp Asp Ala Phe Pro Lys Asp Leu Gly Ser

800 805 810

Leu Ile Ser Leu Glu Asn Leu Asp Leu Ala Gly Asn Asp Phe Cys

815 820 825

Ser Leu Pro Ser Phe Ser Arg Leu Ser Lys Leu His Asp Leu Ser

830 835 840

Leu Ser Lys Cys Lys Asn Leu Arg Ala Ile Pro Asp Leu Pro Thr

845 850 855

Asn Leu Lys Val Leu Lys Ala Glu Tyr Cys Phe Glu Leu Glu Lys

860 865 870

Met Pro Asp Phe Ser Glu Met Ser Asn Ile Lys Glu Leu Tyr Leu

875 880 885

Ser Gly Ser Asp Lys Val Thr Glu Ile Pro Gly Leu Asp Lys Ser

890 895 900

Leu Asn Ser Met Thr Met Ile Ser Met Asp Gly Cys Thr Asn Leu

905910 915

Thr Ala Asp Phe Arg Lys Asn Ile Leu Gln Gly Trp Thr Ser Cys

920 925 930

Gly Tyr Gly Gly Ile Phe Leu Ser Gly Asn Asp Ile Pro Asp Trp

935 940 945

Phe Asp Cys Val His Asp Asp Asp Ile Val Tyr Phe Thr Val Pro

950 955 960

Arg Ser Val Gly Arg Asn Phe Lys Gly Leu Thr Leu Ser Phe Val

965 970 975

Ser Ser Pro Gly Phe Leu Ser Arg Pro Ile Ser Ile Ser Ile Lys

980 985 990

Asn Met Thr Lys Gly Ala Glu Leu Glu Ala Arg Ile Ile Pro Asp

995 1000 1005

Cys Pro Ile Asp Gln Gly Gly Trp Leu Ile Tyr Asn Pro Val Ser

1010 1015 1020

Tyr Leu Trp Gln Gly Gln Leu Ser Asn Asp Glu Leu Lys Leu Gln

1025 1030 1035

Asp Gly Asp Lys Val Leu Ile Glu Ile Ile Val Glu Asp Tyr Tyr

1040 1045 1050

Arg Val Lys Val Lys Val Lys Lys Thr Gly Val Ser Leu Val Trp

10551060 1065

Asp Lys Phe Met Asn Glu Asn Met Ile Asp Tyr His Leu Cys Ala

1070 1075 1080

Tyr Glu Arg Arg Pro Ser Gln Asn Leu Val Asn Asp Asp Asp Ile

1085 1090 1095

Ile His Val Glu Asp Asp Asn His Ile Thr Lys Ser Pro Asp Phe

1100 1105 1110

Ser Lys Phe Pro Asn Leu Glu Lys Leu Ile Leu Lys Gly Cys Lys

1115 1120 1125

Lys Leu Ile Lys Val His Ser Tyr Ile Gly Asp Leu Gly Arg Leu

1130 1135 1140

Ser Leu Val Asn Leu Glu Asp Cys Glu Met Leu Arg Asp Leu Pro

1145 1150 1155

Leu Asn Phe Tyr Lys Ser Lys Ser Ile Glu Thr Leu Ile Leu Asn

1160 1165 1170

Gly Cys Ser Arg Phe Glu Asp Leu Ala Asp Gly Leu Gly Asp Met

1175 1180 1185

Val Ser Leu Thr Val Leu Glu Ala Asn Lys Thr Ala Ile Arg Arg

1190 1195 1200

Ile Pro Ser Ser Ile Val Lys Leu Lys Asn Leu Glu His Leu Leu

12051210 1215

Leu Ala Asn Asn Tyr Phe Arg Ser Leu Pro Ser Leu Ala Gly Leu

1220 1225 1230

Ser Lys Leu Lys Val Leu Ser Leu Asn Ala Cys Arg Glu Leu Arg

1235 1240 1245

Ala Ile Pro Asp Leu Pro Thr Asn Leu Tyr Val Leu Lys Ala Asn

1250 1255 1260

Gly Cys Pro Asn Leu Glu Thr Ile Pro Asp Phe Ser Lys Met Ser

1265 1270 1275

Asn Met Arg Lys Leu Tyr Leu Arg Asp Ser Val Lys Leu Thr Glu

1280 1285 1290

Val Pro Gly Leu Asp Lys Ser Leu Asn Ser Met Thr Arg Ile Arg

1295 1300 1305

Met Glu Gly Cys Thr Asn Leu Thr Ala Asp Phe Arg Asn Asn Ile

1310 1315 1320

Gln Gln Arg Trp Thr Ser Cys Gly Phe Gly Gly Ile Tyr Leu Asn

1325 1330 1335

Gly Ile Tyr Asp Ile Pro Glu Trp Phe Lys Ile Leu Asn Asp Val

1340 1345 1350

Gly Asn Ile Val Phe Phe Glu Val Pro Gln Arg Ile Met Gly Arg

13551360 1365

Asp Leu Lys Gly Leu Thr Ile Cys Phe Val Tyr Ser Phe Val Val

1370 1375 1380

Phe Gly Arg Glu Leu Glu Gly Pro Ile Gly Ile Ile Val Arg Asn

1385 1390 1395

Leu Thr Lys Gln Thr Ala Leu His Thr Lys Ile Ala Phe Ala Arg

1400 1405 1410

Cys Gly Arg Pro Glu Pro Asp Leu Leu Ile Trp Arg Leu Leu Ser

1415 1420 1425

Thr Gly Leu Glu Asp Arg Tyr Leu Trp Gln Gly Gln Leu Ser Asn

1430 1435 1440

Asp Val Leu Cys Leu Gln Ala Gly Asp Gln Val Ser Ile Leu Val

1445 1450 1455

Arg Pro Leu Val Asp Phe Val Ile Val Lys Lys Thr Gly Val His

1460 1465 1470

Leu Glu Trp Asp Lys Val Met Lys Glu Asn Met Asp Asn Leu Asp

1475 1480 1485

Pro His Leu Tyr Asp Trp Lys Thr Asn Arg Asp Phe

1490 1495 1497

<211>25

<212>DNA

<213> birthday star peach

<221> upstream primer comprising 20bp insertion sequence

<400>4

gctatacact tgacttggta gcaag 25

<211>24

<212>DNA

<213> Artificial sequence

<221>PpRm3Gene amplification downstream primer

<400>5

catttcaaga aattgttgtc actg 24

<211>26

<212>DNA

<213> Artificial sequence

<221>PpRm3Upstream primer for detecting gene expression level

<400>6

aaagtactca gaataagctt tgactt 26

<211>27

<212>DNA

<213> Artificial sequence

<221>PpRm3Downstream primer for detecting gene expression quantity

<400>7

actcaataca tctattacgt cttcacc 27

Claims (7)

1. The disease-resistant gene for controlling peach aversion resistance peach green aphid resistance character is characterized in that: the nucleotide sequence is shown in SEQ ID NO. 2.

2. The protein for controlling peach green aphid resistance character of peach aversion is characterized in that: the amino acid sequence is shown in SEQ ID NO. 3.

3. The use of the disease-resistant gene for controlling peach aversion resistance green peach aphid trait according to claim 1 in screening single peach green peach aphid resistance plants of peach trees.

4. The use of the disease-resistant gene for controlling peach aversion resistance green peach aphid trait of claim 1 in enhancing green peach aphid resistance trait of peach trees.

5. The use of the disease-resistant gene for controlling peach aversion resistance green peach aphid trait according to claim 1 in peach green peach aphid variety breeding.

6. The use of the protein for controlling peach aversion green aphid resistance traits as claimed in claim 2 in screening single peach green aphid resistance plants of peach trees.

7. The use of the protein for controlling peach aversion resistance green peach aphid trait according to claim 2 for enhancing green peach aphid resistance trait of peach trees.

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