CN110698550A

CN110698550A - Molecular detection method for rapidly identifying real plum/apricot plum strain

Info

Publication number: CN110698550A
Application number: CN201911096082.3A
Authority: CN
Inventors: 郑唐春; 张启翔; 李平; 李璐璐; 程堂仁; 王佳; 袁存权
Original assignee: Beijing Forestry University
Current assignee: Beijing Forestry University
Priority date: 2019-11-11
Filing date: 2019-11-11
Publication date: 2020-01-17
Anticipated expiration: 2039-11-11
Also published as: CN110698550B

Abstract

The invention relates to the technical field of plant genetic engineering and molecular biology, in particular to a molecular detection method for rapidly identifying a real plum/apricot plum line. The invention provides a plum blossom PmeIL protein, the sequence of which is shown in SEQ ID NO. 1. The PmEIL protein participates in plum blossom response seasonal climate change, and the regulation and control modes of seasonal climate change such as temperature, sunlight and the like in the real plum and the plum line have obvious difference, so that the PmEIL protein can be used for identifying the real plum/plum line, realizing the rapid and accurate identification of the plum line in the seedling stage of plum blossom, shortening the breeding period and greatly reducing the breeding workload. The invention also provides an SNP molecular marker related to the PmeIL gene and related to the plum blossom stem color, and provides a basis and a molecular tool for identifying the plum blossom stem color character and the real plum/apricot strain.

Description

Molecular detection method for rapidly identifying real plum/apricot plum strain

Technical Field

The invention relates to the technical field of plant genetic engineering and molecular biology, in particular to a plum blossom ethylene insensitive protein PmEIL, an SNP molecular marker related to the plum blossom ethylene insensitive protein PmEIL and the plum blossom stem color, and a molecular detection method for rapidly identifying a real plum/apricot strain by utilizing the ethylene insensitive protein PmEIL and the SNP molecular marker related to the plum blossom stem color.

Background

Plum blossom (Prunus mume Sieb. et Zucc.) is a Prunus plant of Rosaceae (Rosaceae), is one of ten traditional famous flowers in China, has important ornamental and economic values, originates in southwest China, and has been introduced and cultivated for more than 3000 years to date (Chenjun happy. Chinese plum blossom variety, image [ M ]. Chinese forestry publishing house, 2010). Plum blossom is divided into three major systems, namely a real plum system (real plum), a plum system (apricot plum) and a plum system (cherry plum). The real plum system has the characteristics of early flowering phase, various flower colors, rich flower type/plant type and the like, and the plum of the apricot plum system has stronger cold resistance (Chen and happiness, China plum variety, J.M., China forestry publishing company, 2010). At present, more than 400 varieties of the real plum system and the apricot plum system which are internationally registered exist, the property difference of part of varieties is small, and the varieties are difficult to distinguish particularly in the seedling stage. The conventional identification method of the apricot plum is to identify according to the characteristics of plant types of perennial plants, the color of branches after leaves fall in autumn or the shape of flower parts after flowering and the like, and has strong subjectivity and long period. With the wide application of plum blossom in landscaping, the plum blossom strain background needs to be accurately evaluated according to the principle of fitting trees appropriately.

Plants respond to seasonal climatic changes, creating an apparent phenomenon that adapts to climatic changes. The falling leaf time of plum blossom in the apricot plum system in late autumn and early winter is earlier than that of the real plum system, and the color of annual branches is obviously changed along with the falling leaf. In recent years, although plum blossom has completed genome sequencing and genome re-sequencing, there are still a large number of genes and their functions to be discovered, especially genes related to plum blossom response seasonal climate change trait and related to plum blossom strains. The research on the gene function related to the seasonal climate change character of plum blossom response and the SNP molecular marker has important significance on seed selection and breeding, variety identification, landscaping and the like.

Disclosure of Invention

In order to solve the technical problems in the prior art, the invention aims to provide an ethylene-insensitive protein PmeIL of plum blossom responding to seasonal climatic change, a coding gene and a related SNP molecular marker thereof, and a method for identifying a real plum/apricot strain by using the PmeIL gene or the related SNP molecular marker.

In order to achieve the purpose, the technical scheme of the invention is as follows: with seasonal climatic changes, the plums fallen earlier than the real plums. The color of annual branches of apricot plum branches is obviously changed in the process of leaf falling, a great number of plum blossom stalk color phenotypes are obtained by using British Royal color comparison card comparison and data statistics, and GWAS association analysis is further carried out on the basis of phenotype data to mine SNP sites related to the stalk color phenotypes. SNP loci with significant association of stemmed characters exist on

chromosomes

1, 3 and 5 of apricot plum and genuine plum through GWAS association analysis, and the genome sequence annotation result shows that part of related genes of the SNP loci participate in the plum abiotic stress process. The invention further discloses a plum blossom PmEIL gene on the basis of the 2 SNP sites, and through deep research on the sequence, bioinformatics prediction and expression mode of the gene, the invention discovers that the mode of responding seasonal climate change (temperature, sunlight and the like) of the gene in apricot plum and plum is obviously different, the characteristic and related SNP molecular markers thereof can be used for screening and identifying plum blossom/plum strains, the breeding period is obviously shortened, and the method has practical application value.

Specifically, the technical scheme of the invention is as follows:

the invention provides a plum blossom PmEIL protein, which has any one of the following amino acid sequences (1) to (3):

(1) an amino acid sequence shown as SEQ ID NO. 1;

(2) the amino acid sequence of the protein with the same function is obtained by deletion, substitution and insertion of one or more amino acids of the amino acid sequence shown as SEQ ID NO. 1;

(3) has at least 90 percent of homology with the amino acid sequence shown as SEQ ID NO.1 and obtains the amino acid sequence of the protein with the same function.

According to the invention, through functional domain prediction analysis, the plum blossom PmeIL protein belongs to an ethylene-insensitive protein family (EIN3), and therefore, the plum blossom PmeIL protein is presumed to be possibly involved in the regulation and control of plum blossom ethylene signal transduction pathways.

In the above (2), the deletion, substitution or insertion of an amino acid that does not affect the function of the protein may be performed in the inactive region, or the substitution of a conservative amino acid that does not affect the function of the protein may be performed in the active region.

In the above (3), the homology is preferably at least 95%, more preferably at least 99%.

The invention also provides a gene for coding the plum blossom PmEIL protein.

Specifically, the gene encoding the plum blossom PmEIL protein has any one of the following nucleotide sequences (1) to (3):

(1) a nucleotide sequence shown as SEQ ID NO. 2;

(2) the nucleotide sequence shown as SEQ ID NO.2 is obtained by replacing, deleting or inserting one or more nucleotides in the nucleotide sequence to encode the same functional protein;

(3) a nucleotide sequence which can hybridize with the nucleotide sequence described in (1) or (2) under strict conditions and encodes the same functional protein.

It is understood by those skilled in the art that, based on the principle of codon degeneracy, different gene sequences encoding the plum blossom PmEIL protein can be designed according to the codon preference of different species, and all genes encoding the plum blossom PmEIL protein are within the protection scope of the present invention.

The invention also provides a biological material containing the gene for coding the plum blossom PmEIL protein, and the biological material comprises an expression cassette, a vector, a host cell, an engineering bacterium or a transgenic plant cell line.

The above vectors include but are not limited to cloning vectors, expression vectors, plasmid vectors, and all vectors containing the gene encoding the plum blossom PmeIL protein are within the scope of the present invention.

The host cell or the engineering bacterium can be derived from microorganism, plant or animal cells, and all the host cells or the engineering bacteria containing the gene for coding the plum blossom PmeIL protein are in the protection scope of the invention.

Furthermore, the invention also provides a specific amplification primer of the gene for coding the plum blossom PmeIL protein, which has a sequence shown as SEQ ID NO.3-4 or a sequence shown as SEQ ID NO. 5-6.

On the other hand, the invention also provides an SNP molecular marker related to plum blossom stalk color, which is located in the 16248370 bp-16251843 bp interval of plum blossom chromosome 1 and is any one or the combination of two of the 2 SNP molecular markers in the following (1) and (2):

(1) the polymorphism is T/C and is located at 16248370bp of the plum blossom chromosome 1;

(2) located at 16251843bp of plum blossom chromosome 1, and the polymorphism is A/G.

The position of the SNP molecular marker is the position on the plum blossom chromosome 1 with the Genome version of Prunus mumme Genome v1.0(http:// prunuusmumgenome. bjfu. edu. cn /).

The SNP site in the (1) is positioned at the upstream 214bp position of the plum blossom PmeIL gene, namely the 215 th position of the sequence shown as SEQ ID NO. 7; the SNP site in the above (2) is located at 1449bp downstream of the plum blossom PmeIL gene, i.e. 266 th position of the sequence shown in SEQ ID NO. 8.

Furthermore, the invention also provides a detection primer of the SNP molecular marker, which has a sequence shown as SEQ ID NO. 9-12.

Wherein, the primers of SEQ ID NO.9 and SEQ ID NO.10 are used for detecting the SNP molecular marker with the polymorphism of T/C at 16248370bp of the plum blossom chromosome 1. The primers of SEQ ID NO.11 and SEQ ID NO.12 are used for detecting the SNP molecular marker with polymorphism A/G at 16251843bp of the plum blossom chromosome 1.

Experiments prove that the expression quantity of the PmeIL gene in the real plum and the apricot plum is changed by changing the temperature and the sunshine conditions, the PmeIL gene is induced or inhibited by different environmental factors in the real plum and the apricot plum to form an expression mode with obvious difference, and the differential expression mode of the PmeIL gene in the real plum and the apricot plum can be used for quickly identifying the real plum and the apricot plum strain and the apricot plum hybrid offspring, or the PmeIL gene is used for regulating and controlling the seasonal climate change response character of the plum blossom.

In another aspect, the invention provides the use of the gene encoding the PmEIL protein or the biological material containing the gene as any one of the following:

(1) the application in regulating and controlling the stem color and the leaf fall character of the plum blossom or responding to the seasonal climate change character;

(2) the application in plum blossom genetic breeding.

Experiments prove that the SNP molecular marker related to the plum blossom stem color can be used for identifying the real plum/apricot plum strain and has higher accuracy.

In another aspect, the invention provides the use of the gene encoding the PmEIL protein or its specific amplification primer or the SNP molecular marker or its detection primer as any one of the following:

(1) the application in identifying the apricot plum/real plum strain;

(2) the application in the seedling stage identification of the hybrid progeny of apricot and plum;

(3) the application in identifying plum stem color, defoliation character or seasonal climate change response character.

Specifically, the applications include: detecting the regulation and control condition of the PmEIL gene in the plum blossom to be detected by seasonal climate change, and identifying the plum blossom/real plum blossom strain according to the differential expression condition of the PmEIL gene under different climate conditions.

Alternatively, the above applications include: and detecting the genotype of the SNP molecular marker related to the plum blossom stem color by taking plum blossom genome DNA as a template, and identifying the apricot plum/real plum blossom strain.

The invention provides a method for identifying a real plum/apricot strain, which is one of parallel technical schemes: the identification method comprises the following steps: culturing the plum blossom to be detected under different seasonal climatic conditions, extracting RNA of the plum blossom to be detected, analyzing the differential expression condition of the gene coding the PmeIL protein in the plum blossom to be detected cultured under different seasonal climatic conditions, and judging the strain of the plum blossom to be detected;

the coding protein of the PmeIL gene has any one of the following amino acid sequences (1) to (3):

(1) an amino acid sequence shown as SEQ ID NO. 1;

As a second parallel technical solution, the identification method comprises: and (3) taking the genome of the plum blossom to be detected as a template, carrying out PCR amplification by adopting the detection primer of the SNP molecular marker related to the plum blossom stem color, analyzing the sequence of a PCR amplification product, and judging the genotype of the plum blossom to be detected.

The invention has the beneficial effects that:

(1) the PmEIL gene provided by the invention has obvious difference in the regulation and control modes of seasonal climate change such as temperature, sunlight and the like in the real plum and the apricot plum strains, so that the PmEIL gene and the corresponding amplification primer thereof, the corresponding SNP molecular marker of the gene and the detection primer thereof can be used for identifying the plum strains of natural hybrid groups, the real plum/apricot plum strain can be quickly and accurately identified in the seedling stage of plum blossom, the breeding period is shortened, the breeding workload is greatly reduced, the foundation is laid for accelerating the screening and breeding of the plum strains, and the PmEIL gene has guiding significance for the selection of the plum strains for landscaping;

(2) the PmEIL gene provided by the invention can respond to seasonal climate changes such as temperature, sunlight and the like in true plum and apricot strains, and can quickly distinguish the plum strains by utilizing the relative expression quantity of the PmEIL gene, thereby providing a foundation for quick breeding of excellent plum strains.

(3) The SNP molecular marker related to the plum blossom stem color provided by the invention is used for identifying characters such as the plum blossom stem color and the like and identifying the apricot plum/true plum strain, has higher accuracy, and can realize the quick and early identification of the plum strain.

Drawings

Figure 1 is a Manhattan plot of the GWAS analysis in example 1 of the present invention.

FIG. 2 shows the leaf drop phenomenon of plum blossom in response to seasonal climate change in example 1 of the present invention; in the figure, the apricot plum is on the left side, and the real plum is on the right side.

FIG. 3 is a phylogenetic tree of the plum blossom PmeIL protein and ethylene insensitive protein of other species in example 2 of the present invention.

FIG. 4 shows the expression level changes of plum blossom PmeIL gene under low temperature and short day treatment in example 3 of the present invention.

FIG. 5 is the sequencing peak diagram of 2 SNP molecular markers in example 4 of the present invention, wherein A, C are true plum ('jiangmei'), B, D are apricot plum ('swallow apricot').

FIG. 6 shows the statistical results of the breeding accuracy of the plum blossom line assisted selection of 2 SNP molecular markers in example 5.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 mining of SNP molecular markers related to plum blossom stalk color trait based on GWAS analysis

1. Plum blossom Stem color phenotype Change assay

When the plum blossom responds to seasonal climate change, the color of annual branches of the apricot plum obviously changes along with the occurrence of fallen leaves, and the annual branches of the real plum keep the original green character. The color of annual branches before and after the leaves fall of different lines of plum blossoms is determined by using the British Royal horticulture Association color comparison card (RHS). The results show that the color difference of annual branches before the fallen leaves of different lines of plum blossoms is not obvious, the character difference after the fallen leaves is obvious, the annual stem color of apricot plum is dark purple (the color comparison card of the Imperial horticulture Association is 183-B), the annual stem color of real plum is green (the color comparison card of the Imperial horticulture Association is 137-A), and the phenotypic characters are statistically analyzed on the basis.

2. GWAS mining trait-marker association site

And (4) taking the measured phenotypic characters as quality characters, and performing association by using a Mixed Linear Model (MLM). The structure matrix (Q) and the affinity relation matrix (K) of the plum blossom population are calculated by using ADMIXTURE1.3 and SPMGedi v.1.4b software, GWAS association is carried out through an MLM model combining phenotype, mark, genetic effect and the like, and the GWAS association result is shown in figure 1.

Through GWAS association analysis, SNP loci with significant association of traits exist on

chromosomes

1, 3 and 5 respectively for the differential traits of apricot plum and authentic plum, wherein annotation genes of most of the SNP loci are related to plum abiotic stress. With seasonal climatic change, the fallen leaves of the apricot strain are earlier than those of the true plum strain, 2 SNP loci exist in the 16248370 bp-16251843 bp region of the No.1 chromosome, and the gene comprises an ethylene insensitive protein (EIN3) gene related to the ripening of plant fruits and the aging of leaves, and accords with the fallen leaves characteristics of the apricot (figure 2).

Example 2 GWAS-associated region PmeIL Gene cloning and analysis

The extraction of total RNA from the plum blossom variety ('Jiangmei') was performed according to the instructions of the plant total RNA extraction kit from Tiangen Biochemical technology (Beijing) Co., Ltd.

The total RNA is reversely transcribed into cDNA by a kit, and the ORF of the EIN3 gene is cloned by PCR by using the cDNA as a template. The reaction system comprises 100-150 ng of template (cDNA), 25 muL of PrimeSTAR HS (Premix), 0.3 muM of upstream primer (EIL _ F, SEQ ID NO.3), 0.3 muM of downstream primer (EIL _ R, SEQ ID NO.4) and ddH₂O was added to 50. mu.L. Reaction conditions are as follows: 2min at 98 ℃; 98 ℃ 10s, 56 ℃ 15s, 72 ℃ 60s (35 cycles); 5min at 72 ℃; storing at 4 ℃.

And recovering the target band through gel electrophoresis, connecting, transforming and sequencing to obtain the target sequence. Sequence comparison is carried out on EIN3 gene ORF by combining DNAMAN software, and the matching degree is 98.87%. Therefore, the EIN3 gene was confirmed to be located in a SNP region gene associated with GWAS, and the EIN3 gene was named PmeIL.

Taking the amino acid sequence of the plum blossom PmeL protein as a target sequence, obtaining 22 species of homologous amino acids of arabidopsis, peach, plum and the like by utilizing Blastp search in an NCBI database, comparing by MEGA7.0 software, and establishing a phylogenetic tree, and finding that the plum blossom PmeL protein has closer relationship with ethylene-insensitive proteins of peach (XP _020421398.1) and cherry (PQQ06442.1) (figure 3).

Example 3 analysis of variation in expression level of PmeIL Gene under conditions simulating seasonal climate Change

1. Low temperature short sun treatment plum blossom

The true plum ('Jiangmei') and apricot ('Yangming') with consistent growth vigor and 4 weeks age are selected as test materials. Firstly, adaptively culturing for 2 days under the uniform condition of 24 ℃ long sunshine (16/8h light/dark), then respectively carrying out 24 ℃ long sunshine (16/8h light/dark), 24 ℃ short sunshine (8/16h light/dark), 6 ℃ long sunshine (16/8h light/dark) and 6 ℃ long sunshine (8/16h light/dark) condition treatment, continuously culturing for 4 weeks, observing the growth vigor of the apricot plum and the real plum, and counting and comparing the plant height and the leaf number.

2. Analysis of PmeIL Gene expression level

The extraction of total RNA from plum blossom is carried out by using an RNAscope total RNA extraction kit of Tiangen Biotechnology (Beijing) Co., Ltd, and the extraction method is carried out by referring to the kit specification. cDNA Synthesis and real-time fluorescent quantitative PCR Using PrimeScript from Bao bioengineering (Dalian) Ltd^TMII 1st Strand cDNA Synthesis Kit and TB

Premix Ex Taq^TM(Tli RNaseH Plus) kit, the assay being performed with reference to kit instructions.

The PmeIL gene is subjected to relative quantitative test by utilizing a qPCR method, and a detection primer of the qPCR is shown as SEQ ID NO. 5-6. As a result, the expression level of the PmeIL gene of the apricot strain is found to be remarkably different under different conditions (figure 4). Under the condition of short-day treatment, the expression level of the PmeEIL gene of the prunus armeniaca is obviously up-regulated, and the expression quantity in the true prunus armeniaca is almost kept unchanged; in the low-temperature treatment test, the genuine plum PmeL gene is obviously inhibited. Example 4GWAS associated region SNP site cloning and analysis

According to the nucleic acid sequence of the plum blossom stalk color associated region, a marker specific Primer is designed by using Primer Premier 5.0 software, and the specific Primer is shown in table 1:

TABLE 12 primer information for Sanger sequencing

Randomly selecting different varieties of seedlings of true plum ('Jiangmei') and apricot plum ('Yangapricot'), and extracting DNA of young leaves according to the instruction of a plant genome DNA extraction kit of Tiangen Biochemical technology (Beijing) Co.

And (3) carrying out PCR cloning on the SNP site section by taking the genome DNA as a template. The reaction system is that the template (genome DNA) is 100-150 ng, PrimeSTAR HS (Premix) is 25 mu L, the upstream primer (SNP _ F, SEQ ID NO.9/SEQ ID NO.11) is 0.3 mu M, the downstream primer (SNP _ R, SEQ ID NO.10/SEQ ID NO.12) is 0.3 mu M, ddH₂O was added to 50. mu.L. Reaction conditions are as follows: 2min at 98 ℃; 98 ℃ 10s, 56 ℃ 15s, 72 ℃ 60s (35 cycles); 5min at 72 ℃; storing at 4 ℃. The PCR products were then subjected to Sanger sequencing (Beijing department of sequencing, Biotechnology engineering, Inc.).

The sequencing result is subjected to sequence analysis, the nucleic acid position of the marker position really contains real SNP, the 1st SNP site is positioned at 16248370bp of No.1 chromosome of plum blossom (as the 215 th site of the sequence shown in SEQ ID NO. 7), and is 'T' in 'Jiangmei' and 'C' in 'Yangxing apricot'; the 2 nd SNP site is located at 16251843bp of chromosome 1 of plum blossom (266 th position of the sequence shown in SEQ ID NO. 8), is 'A' in 'Jiangmei' and 'G' in 'Yangxing' (FIG. 5).

Example 5 application of SNP marker in identification of plum blossom Strain

Randomly selecting 20 different young leaves of true plum (12) and apricot plum (8) strains, and extracting the young leaf DNA according to the instruction of a plant genome DNA extraction kit of Tiangen Biochemical technology (Beijing) Co., Ltd.

Through sequence analysis of a sequencing result, the 1st SNP locus is found to be 'T' in all real plums detected and 'C' in most apricot plum varieties (the probability is 87.5 percent); the probability of the 2 nd SNP site being "A" in all true plum varieties and "G" in apricot plum varieties is 75%. When the phenotype identification is carried out by using the combination of the two markers, the detection accuracy rate in the apricot plum variety reaches 81.25 percent (figure 6).

In conclusion, the molecular biological method for rapidly identifying the real plum/apricot plum line is established, the seedling stage identification and selection of the plum line can be realized by utilizing the detection of the SNP molecular marker locus and the differential expression pattern analysis of the PmeIL gene under different conditions, the application efficiency of the plum in landscaping is improved, and the screening cost is greatly reduced; and a theoretical basis is laid for plum blossom seed selection, breeding and strain identification.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Beijing university of forestry

<120> molecular detection method for rapidly identifying variety of real plum/apricot plum

<130>KHP191115040.2

<160>12

<170>SIPOSequenceListing 1.0

<210>1

<211>266

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>1

Met Glu Thr Leu Ala Lys His Glu Ala Ser Arg Arg Lys Lys Met Ala

1 5 10 15

Arg Ala Gln Asp Ser Ile Leu Lys Tyr Met Val Glu Asn Met Glu Val

20 25 30

Cys Lys Gly Lys Ala Phe Val Tyr Gly Ile Val Pro Glu Lys Gly Lys

35 40 45

Pro Val Thr Gly Ser Ser Asp Ser Leu Arg Glu Trp Trp Lys Asp Lys

50 55 60

Val Ile Phe Asn Arg Asn Ala Ala Thr Ala Ile Ala Glu Tyr Phe Ala

65 70 75 80

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

85 90 95

Leu Pro Ser Ser Asp Lys Arg Lys Ser Ser Ile Phe Asp Glu Glu Ala

100 105 110

Trp Gln Asn Gly Gln Gly Leu Glu Ser Ser Glu Leu Arg Leu Gly Phe

115 120 125

Val Asp Lys Lys Ser Lys Val Asp His Glu Ser Ala Leu Cys Ala Tyr

130 135 140

Gly Asp Gly Gln Asp Gln Ser Asn Val Ser Gly Leu Leu Ala Asp Gly

145 150 155 160

Lys Phe Pro Leu Ile Asp Glu Phe Pro Ile Asp Thr Arg Val Met Ser

165 170 175

Met Val Asp Trp Asn Asn Met Glu Leu Gln Lys Ala Asn Gln Ile Asp

180 185 190

Gln Asn Ser Gly Ala Phe Val Ile Asn Gly Gln Val Gly Val Glu Val

195 200 205

Ser Asp Thr Thr Val Gly Asp Tyr Gly Ala Ser Tyr Trp Gly Gly Gly

210 215 220

Ile Glu Asp Leu Ala Ile Asp Gly Ala Phe Asp Ile Gln Arg Gly Asn

225 230 235 240

Met Asp Leu Asn Leu Ser Ser Glu Glu Glu Ile Ser His Asn Gln Glu

245 250 255

Ser Thr Ser Ile Cys Asp Leu Gly Tyr Asp

260 265

<210>2

<211>798

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

atggagacct tagcaaagca cgaggcctct cgtcgcaaga aaatggcaag agcccaagat 60

tcaatactca agtacatggt ggaaaacatg gaggtttgca aaggaaaggc ctttgtgtat 120

gggattgtgc ctgagaaggg gaagccagtg acagggtcct ctgatagctt gagagagtgg 180

tggaaggaca aagtgatatt taaccgtaat gccgcaactg ccatagctga gtacttcgca 240

gcattgattt ttgatcaagg caagaataat gggaagcgca tgtgcagctt gccgagcagt 300

gataagagga agagcagcat ctttgatgaa gaggcctggc aaaatggtca gggcttggag 360

agttctgagt tgcgtttagg gtttgtggat aagaaatcaa aggtagacca tgaatctgca 420

ttatgtgctt atggtgatgg ccaagaccaa agtaatgttt ctggtttatt ggctgacggg 480

aaatttcctt tgattgatga attccctatt gatactcgtg ttatgagtat ggtggattgg 540

aataatatgg agctgcagaa ggccaatcag attgatcaga acagcggtgc atttgtaatt 600

aatggtcaag ttggggttga ggtttcagac actacagtgg gagactatgg agcaagctac 660

tggggaggtg gtattgaaga tctggctata gatggagcat ttgatatcca aaggggaaat 720

atggatttaa atctctcttc agaagaagaa atctcacata atcaagaatc aacttcaatt 780

tgcgatttgg gatatgat 798

<210>3

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

atggagacct tagcaaagca c 21

<210>4

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

atcatatccc aaatcgcaaa ttg 23

<210>5

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

atgggattgt gcctgagaag 20

<210>6

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

ctcttcctct tatcactgct cg 22

<210>7

<211>449

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

atttcagtgt gtgaaatgga gagtacgtcg catgaactag gaggaaggac caagtctata 60

tatcattcat catcccctct ccaatgtcca aagtccaaac caaaacgcat cataagtttc 120

ttagtacagc aatttcttac gcatgcaata tgtggtttca gactttcggt ctttgagaat 180

gaagatgggg acacaacttc cttacgtacc acgatgcgaa gcaccctaaa ctcccttaca 240

acggctctaa aaccagaaga tttattgtcg gtaattaatt gtttaatcca gtaaagttgg 300

agtgacaatt tttatatatc ttgcaatgga taaggagctt agcttggcaa tcaaattgac 360

accatgtgtc cctcatgcat gcaacattta aaaagaaaaa aagaaagaaa gaaaaagaaa 420

aaaaacaatg acattgcggc cctggaagc 449

<210>8

<211>637

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

tcaacttcaa tttgggattt gggatatgat tagtgaagca atatatatca aaactattat 60

gtaccaaaga gggagtagct atctaggttg cactaatttt tcagattttt tgttgttgtt 120

ccaatgcatc aataacatta tttctacgaa aaagatgtag tcaataagat tactaattta 180

tatttattga ccacattgtt taccgcatca ctcacggtta tgatcattat aacatgccga 240

tcacattgtt tagcgctgga cggcgacgta aacggccaca agcttaacgt aacacttttt 300

tcattttaaa aattgaactc tttatattta ggctttgata ctagttttaa agtcaaaaca 360

atttttacaa agacatgaac aaaggtaaat gtaaaattga aagttagtga cctaaactaa 420

agtaattagt ggaacccaag taaagaaagt gaaaaagaaa agtcaattac taaattttct 480

gaaaaaaaaa ttcccaagtt agtatttaat ctaaattttc taatcaatta attagtttga 540

ttgacaggga aggatataag ctaggatacc atgtagagct caagaaaatg gcccaggcct 600

ggcactagtt ttagggatat gcgtggcctg agactaa 637

<210>9

<211>26

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

atttcagtgt gtgaaatgga gagtac 26

<210>10

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

gcttccaggg ccgcaatgtc attg 24

<210>11

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>11

tcaacttcaa tttgggattt ggg 23

<210>12

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>12

ttagtctcag gccacgcata tccc 24

Claims

1. The plum blossom PmeIL protein is characterized by having any one of the following amino acid sequences (1) to (3):

(1) an amino acid sequence shown as SEQ ID NO. 1;

2. A gene encoding the plum blossom PmEIL protein of claim 1.

3. The gene according to claim 2, which has a nucleotide sequence of any one of the following (1) to (3):

(1) a nucleotide sequence shown as SEQ ID NO. 2;

(2) the nucleotide sequence of the nucleotide sequence shown in SEQ ID NO.2 is obtained by replacing, deleting or inserting one or more nucleotides to code the same functional protein;

4. Biomaterial comprising a gene according to claim 2 or 3, characterized in that it comprises an expression cassette, a vector, a host cell, an engineered bacterium or a transgenic plant cell line.

5. Primer for the specific amplification of the gene according to claim 2 or 3, characterized in that it has the sequence shown in SEQ ID No.3-4 or the sequence shown in SEQ ID No. 5-6.

6. The SNP molecular marker related to plum blossom stalk color is characterized in that the SNP molecular marker is any one or the combination of two of the SNP molecular markers in the following (1) and (2) positioned in an 16248370 bp-16251843 bp interval of plum blossom chromosome 1:

7. The primer for detecting a SNP molecular marker according to claim 6, which has a sequence shown in SEQ ID nos. 9 to 12.

8. Use of any one of the genes of claim 2 or 3 or the biological material of claim 4:

(2) the application in plum blossom genetic breeding.

9. Any one of the following applications of the gene of claim 2 or 3, or the specific amplification primer of claim 5, or the SNP molecular marker of claim 6, or the detection primer of claim 7:

(1) the application in identifying the apricot plum/real plum strain;

10. A method for identifying a real plum/apricot strain is characterized by comprising the following steps: culturing the plum blossom to be detected under different seasonal climatic conditions, extracting RNA of the plum blossom to be detected, analyzing the differential expression condition of the PmeIL gene in the plum blossom to be detected cultured under different seasonal climatic conditions, and judging the strain of the plum blossom to be detected;

alternatively, the identification method comprises: using the genome of the plum blossom to be detected as a template, carrying out PCR amplification by using the detection primer of the SNP molecular marker of claim 7, analyzing the sequence of the PCR amplification product, and judging the genotype of the plum blossom to be detected;

(1) an amino acid sequence shown as SEQ ID NO. 1;