CN105367640A - Pea powdery mildew resisting er1 allele er1-8 and coding protein thereof - Google Patents

Abstract

The invention relates to a pea powdery mildew resisting er1 allele er1-8 and coding protein thereof. The allele is located on an er1 gene seat of a linkage group VI of a pea genetic map. Powdery mildew isolate EPYN is artificially inoculated to pea sources G0004389, and it is shown that G0004389 generates immunoreactions on the powdery mildew isolate EPYN. The PsMLO1cDNA sequence of er1 candidate genes of the pea powdery mildew resisting resources G0004389 is measured, the obtained sequence and the PsMLO1cDNA sequence of a wild susceptible variety are subjected to comparative analysis, it is found that when the PsMLO1CDNA sequence of G0004389 is compared with the PsMLO1cDNA sequence of a wild susceptible gene, three bases GTG are deleted at the position of 1,340 bp to 1,342 bp, frameshift mutation in the translation process is caused by the deletion, the deficiency of 447th amino acid (valine) of PsMLO1 protein is caused, and therefore the change of the function of PsMLO1 protein is caused. The allele is different from seven identified er1 alleles in mutation position and mutation mode, it is indicated that the allele is a new allele of er1 and named er1-8, and therefore new gene resources are provided for molecular breeding of powdery mildew resisting of the pea resources.

Description

Pea mildew-resistance er1 allelotrope er1-8 and proteins encoded thereof

Technical field

The present invention relates to plant pathology, genetic breeding and biology field, specifically, relate to pea mildew-resistance er1 neomorph er1-8 and proteins encoded thereof.

Background technology

Pea (PisumsativumL.) is Food Legume crop important in the world.China is first garden pea producing country and the third-largest dry pea producing country (FAOSTAT data) in the world.The Powdery Mildew caused by powdery mildew (ErysiphepisiD.C.) is one of Major Diseases of pea, causes serious financial consequences (Nisaretal., 2011 in the world; Fondevillaetal., 2012).

Control powdery mildew of pea is the most economical, effective and the method for environmental safety is plantation disease-resistant variety.At present, identify a large amount of mildew-resistance pea resources abroad, and in Resistance resource, identify mildew-resistance gene (er1, er2 and Er3) (Harlandetal., 1948:Heringaetal., 1969 of 3 independent inheritances; Fondevillaetal., 2007).So far, except identifying pea mildew-resistance gene er2 and identify Er3 in pea resource JI2480 in pea wild species P.fulvum, the research of a large amount of Resistance resource screening and genetic analysis aspect shows, the resistance of the mildew-resistance pea resource of many different geographic origin controls (Tiwarietal., 1997 by er1; Ghafooretal., 2012; Liuetal., 2003; Vaidetal., 1997).The resistance of the mildew-resistance pea resource applied in current production controls by er1.Along with the exploitation of pea molecule marker and the structure of genetic map, disease-resistant gene er1 is positioned in the 6th linkage group (LGVI) (Timmermanetal., 1994) of pea genetic map.Disease-resistant gene er1 is suppress pathogenic bacteria to the invasion of the epidermic cell of host to the disease-resistant or immune mechanism of powdery mildew performance.Disease-resistant gene er1 has lastingly disease-resistant and not by the impact of external environment, has been widely used in breeding and cultivation in states such as European Union.Recent research shows that er1 gene produces because the transgenations such as the insertion of pea PsMLO Homologous gene sequences producer, disappearance, displacement, replacement cause afunction.At present, position based on sudden change is different with mode has found 7 er1 allelotrope, er1-1, er1-2, er1-3, er1-4, er1-5, er1-6 and er1-7 respectively, allelotrope er1-2 is wherein only had to produce owing to inserting large unknown gene fragment, other er1 allelotrope is all lacked by single base mutation or small segment and causes (Humphryetal., 2011; Pavanetal., 2011; Sunetal., 2015a; 2015b).

Pea has the cultivation history of more than 2000 year in China, and powdery mildew of pea has been produced China's pea and caused serious threat.But, the research of China to pea mildew-resistance is less, mainly concentrate on the Screening germplasm of pea mildew-resistance at present, and in Chinese pea resource, find the better resource (Peng Huaxian etc., 1991 that Chinese powdery mildew isolate EPYN and EPBJ are all showed to immunity; Zeng Liang etc., 2012; Wang Zhongyi etc., 2013; Pay Haining etc., 2014).Recently, in these Resistant germplasm, identify disease-resistant gene er1-1, er1-2, er1-6 and er1-7.

Summary of the invention

The object of this invention is to provide pea mildew-resistance er1 neomorph er1-8 and proteins encoded thereof.

In order to realize the object of the invention, er1-8 albumen provided by the invention, its aminoacid sequence is as shown in SeqIDNo.1, or this sequence is through replacing, lacking or add one or several amino acids formed aminoacid sequence with same function.

The present invention also provides the pea mildew-resistance er1 allelotrope er1-8 of encoding said proteins, and its cDNA sequence is as shown in SeqIDNo.2.

The present invention further provides the application of described allelotrope er1-8 in the molecular breeding of pea resource mildew-resistance.

In order to the disease-resistant er1 allelotrope of clear and definite pea mildew-resistance resource G0004389, the er1 candidate gene PsMLO1cDNA of the present invention to G0004389 checks order.The PsMLO1cDNA sequence of the sequencing results display G0004389 is compared with wild-type susceptible gene PsMLO1cDNA sequence, in 1340-1342bp place existence 3 bases G TG disappearances, this disappearance causes phase shift mutation in translation process and causes PsMLO1 albumen the 447th amino acid (α-amino-isovaleric acid) disappearance, thus causes the change of PsMLO1 protein function.This is all not identical with mutational formats with 7 the allelic mutated site of er1 identified, shows that this is the neomorph of an er1, by its called after er1-8.This equipotential gene position is on the er1 locus of pea genetic map VI linkage group.

The present invention, by the er1 candidate gene PsMLO1cDNA sequential analysis to pea mildew-resistance resource G0004389, has found new resistance allele er1-8.Determine the cDNA sequence of neomorph er1-8.The first identified of the present invention neomorph er1-8 of er1, significant to pea mildew-resistance breeding work, the generation of powdery mildew of pea effectively can be controlled by breeding technique, alleviate the financial loss that this disease causes, and provide new genetic resources for the molecular breeding of pea resource mildew-resistance.

Accompanying drawing explanation

Fig. 1 is Resistant germplasm G0004389 (left side) and the phenotype of No. 6, susceptible variety dam pea (right side) inoculation pea powdery mildew isolate after EPYN10 days in the present invention.

Fig. 2 is the structure of the functional gene PsMLO1 controlling powdery mildew of pea resistance in the present invention and Resistant germplasm G0004389 and the susceptible resource of the wild-type pea nucleotide sequence comparison result at the 15th exon of gene PsMLO1.

Embodiment

Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, embodiment is experiment condition all conveniently, as Sambrook equimolecular Cloning: A Laboratory Manual (SambrookJ & RussellDW, Molecularcloning:alaboratorymanual, 2001) condition of, or according to manufacturer's specification sheets advising.

The phenotypic evaluation of embodiment 1 pea mildew-resistance resource G0004389

Disease-resistant pea resource G0004389 and No. 6, susceptible check variety dam pea and disease-resistant check variety YI are seeded in respectively with in the dixie cup of the thick vermiculite 250mL that is matrix, broadcast 5 for every glass, in the hot-house culture of 18 ~ 26 DEG C, when pea seedlings the 3rd or the 4th stipes mounted blade, adopt the conidium (NCBI of method inoculation powdery mildew isolate EPYN, Accessionnumber:KR957355), inoculation is placed on 18 ~ 22 DEG C of hot-house cultures.Adopt the disease severity grade scale of 0 ~ 4 grade, 0 grade: anosis; 1 grade: scab has thin subiculum, visible green blade face, does not produce spore; 2 grades: subiculum is thicker, do not reveal the green, produce a certain amount of spore; 3 grades: mycelia thickness, sporulation quantity is more; 4 grades: sporulation quantity is many, the subiculum on scab is covered (Vaidetal., 1997 by spore entirely; Ranaetal., 2013).Inoculate susceptible check variety disease severity after 10 days and reach 4 grades of " Invest, Then Investigate "s respectively for the incidence of examination resource.Evaluation standard of resistance is: 0 grade is immunity (I), and 1 ~ 2 grade is disease-resistant (R), and 3 ~ 4 grades is susceptible (S).To show as immunity and disease-resistant pea resource carry out repetitive identified.

Inoculate after 10 days, the blade of susceptible check variety dam pea No. 6 all plant, stem stalk and tendril all cover thick subiculum, and produces a large amount of conidium, stem stalk and tendril are also covered with mycelium and conidium, and disease severity is 4 grades, show susceptible; All without Visual symptoms on disease-resistant check variety YI (PI391630) blade, stem stalk and tendril, sick level is 0, performance immunity.Check variety is identical with the qualification result of (2013) such as Wang Zhongyi to the phenotypic response of isolate EPYN.The phenotype of Resistance resource G0004389, with disease-resistant to contrast YI identical, shows immune response to isolate EPYN.

Resistant germplasm G0004389 and the phenotype of No. 6, susceptible variety dam pea inoculation pea powdery mildew isolate after EPYN10 days are as shown in Figure 1.

The er1 candidate gene PsMLO1cDNA Sequence Identification of embodiment 2 Resistance resource

The plant total serum IgE of pea resource G0004389, No. 6, susceptible check variety dam pea and disease-resistant check variety YI (containing er1-4 allelotrope) is extracted with RNAprep plant total RNA extraction reagent box (centrifugal column type, biochemical purchased from sky root).Concrete operation method is as follows:

1) get about 100mg pea young leaflet tablet rapid grind into powder in liquid nitrogen, add 450 μ LRL (adding beta-mercaptoethanol to final concentration before use is 1%), vortex concuss mixes;

2) all solution is transferred to (Filter column CS is placed in collection tube) on Filter column CS, the centrifugal 5min of 12000rpm, the supernatant in careful absorption collection tube is in the centrifuge tube of RNase-free;

3) in centrifuge tube, slowly add the dehydrated alcohol of 0.5 times of volume, mixing, the solution obtained is proceeded in adsorption column CR3 together with precipitation, leave standstill the centrifugal 1min of 2min, 12000rpm, outwell the waste liquid in collection tube;

4) in adsorption column CR3, add 500 μ L protein liquid removal RW1, leave standstill the centrifugal 1min of 2min, 12000rpm, outwell the waste liquid in collection tube;

5) preparation of DNase I working fluid: get 10 μ LDNase I storage liquid and put into new RNase-free centrifuge tube, add 70 μ LRDD solution, softly mix;

6) add DNase I working fluid of 80 μ L to adsorption column CR3 central authorities, room temperature places 20min;

7) in adsorption column CR3, add 350 μ L protein liquid removal RW1, leave standstill the centrifugal 1min of 2min, 12000rpm, outwell the waste liquid in collection tube;

8) in adsorption column CR3, add 500 μ L rinsing liquids RW (adding dehydrated alcohol before use), room temperature leaves standstill 2min, and the centrifugal 1min of 12000rpm, outwells the waste liquid in collection tube;

9) repeating step 8);

10) the centrifugal 2min of 12000rpm, outwells waste liquid, adsorption column CR3 is placed in room temperature and places several minutes, thoroughly dries residual rinsing liquid;

11) adsorption column CR3 is put into a new RNase-free centrifuge tube, the unsettled dropping 50 in the mid-way to adsorption film μ LRNase-freeddH ₂o, room temperature places the centrifugal 2min of 10min, 12000rpm, obtains RNA solution;

12) RNA integrity detection: the gel electrophoresis of plain agar sugar, deposition condition: gum concentration 1.2%; 1 × TBE electrophoretic buffer; 150V; 15min.

MRNA the 1st article of cDNA chain is synthesized with BioRT reverse transcription amplification (RT-PCR) test kit (two-step approach), with PsMLO1 Auele Specific Primer, pcr amplification (Pavenetal., 2013) is carried out to PsMLO1F/PsMLO1R (5 '-AAAATGGCTGAAGAGGGAGTT-3 '/5 '-TCCACAAATCAAGCTGCTACC-3 ').Pcr amplification product detects through 1.5%TBE agarose gel electrophoresis, adopts plain agar sugar gel DNA to reclaim test kit (centrifugal column type, biochemical purchased from sky root) and reclaims and purified pcr product.PCR primer after purifying is cloned in pZeroBack carrier or pMG-T carrier (biochemical purchased from sky root), Beijing Liuhe Huada Genomics Technology Co., Ltd is sent to check order, utilize that ClustalX2 software analysis is disease-resistant, PsMLO1cDNA sequence difference between susceptible variety, and compare (Humphryetal.2011) with known wild-type Foreign Banks' Entries PsMLO1cDNA sequence (GenBank:FJ463618).Control the structure of the functional gene PsMLO1 of powdery mildew of pea resistance as shown in Figure 2, this gene comprises 14 introns and 15 exons.

Result shows that the PsMLO1cDNA sequence of No. 6, susceptible contrast dam pea is identical with wild-type PsMLO1 sequence (FJ463618).The PsMLO1cDNA sequence of disease-resistant contrast YI is different from wild-type PsMLO1 sequence (GenBank:FJ463618), find that the PsMLO1cDNA of Resistant germplasm G0004389 is compared with wild-type PsMLO1cDNA sequence, 1340-1342bp place generation 3 bases G TG disappearance (SEQIDNO.2).This disappearance causes phase shift mutation in translation process and causes the 447th amino acid of PsMLO1 albumen--α-amino-isovaleric acid (V) disappearance (SEQIDNO.1), thus cause the change of PsMLO1 protein function.This is all not identical with mutational formats with 7 the allelic mutated site of er1 identified, shows that this is the neomorph of an er1, by its called after er1-8.This equipotential gene position on the er1 locus of pea genetic map VI linkage group, thus provides new genetic resources for the molecular breeding of pea resource mildew-resistance.

Resistant germplasm G0004389 and wild-type pea susceptible variety the 15th exon of gene PsMLO1 nucleotide sequence comparison result as shown in Figure 2.

Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.

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Claims (3)

1.er1-8 albumen, is characterized in that, its aminoacid sequence is as shown in SeqIDNo.1, or this sequence is through replacing, lacking or add one or several amino acids formed aminoacid sequence with same function.

2. the pea mildew-resistance er1 allelotrope er1-8 of albumen described in coding claim 1.

3. allelotrope er1-8 as claimed in claim 2, it is characterized in that, its cDNA sequence is as shown in SeqIDNo.2.