阅读说明：本技术 一种与青稞条纹病相关的miRNAs及其挖掘方法 (miRNAs related to highland barley stripe disease and mining method thereof ) 是由 姚晓华 吴昆仑 姚有华 王越 苏乐平 侯璐 白羿雄 于 2019-09-25 设计创作，主要内容包括：本发明提供一种与青稞条纹病相关的miRNAs及其挖掘方法,通过从青稞条纹病叶上分离纯化青稞条纹病菌株,构建实验室条纹病病原菌侵染体系,采集发病和未发病的幼叶,进行高通量测序(miRNA-seq),构建了青稞miRNA的数据库,通过与miRBase 22、PMRD、mRNA/EST和rRNAetc数据库进行比对,挖掘出与青稞条纹病相关的miRNAs。(The invention provides miRNAs related to highland barley stripe disease and an excavating method thereof, wherein a highland barley stripe disease strain is separated and purified from highland barley stripe disease leaves, a laboratory stripe disease pathogenic bacteria infection system is constructed, diseased and non-diseased young leaves are collected, high-throughput sequencing (miRNA-seq) is carried out, a highland barley miRNA database is constructed, and miRNAs related to highland barley stripe disease are excavated by comparing with miRBase22, PMRD, mRNA/EST and rRNAetc databases.)

1. A mining method of miRNAs related to highland barley stripe disease is characterized by comprising the following steps:

Step 1, collecting highland barley leaves infected with stripe disease;

Step 2, construction of a pathogenic bacteria infection system of the stripe disease

step 2.1, culturing and separating the highland barley leaves infected with the streaky disease to obtain highland barley streaky disease pathogenic bacteria;

Step 2.2, infecting seeds of the highland barley varieties with high stripe disease infection and the highland barley varieties with high stripe disease resistance by using the pathogenic bacteria of the highland barley stripe disease, and then culturing;

Step 3, after the highland barley leaves grow out and are infected with diseases in the step 2.2, collecting infected and uninfected leaves of the highland barley variety with high susceptibility to the stripe disease and the highland barley variety with high resistance to the stripe disease, respectively extracting total RNA, constructing a Small RNA library, and performing miRNA high-throughput sequencing;

Step 4, excavating miRNA related to stripe disease

According to the miRNA high-throughput sequencing result, the highland barley genome is used as a reference, the difference of miRNA before and after the stripe disease high-susceptibility highland barley variety and the stripe disease high-resistance highland barley variety are infected is analyzed by comparing with a miRBase22, PMRD, mRNA/EST and rRNAetc database, so as to obtain difference miRNAs, and the miRNAs related to the highland barley stripe disease are screened out through the expression quantity difference.

2. The mining method of miRNAs related to barley stripe disease as claimed in claim 1, wherein in step 1, the collected barley leaves infected with stripe disease are stored in an ultra low temperature freezer of-80 ℃ for later use.

3. The mining method of miRNAs related to highland barley stripe disease as claimed in claim 1, wherein step 2.1 is specifically; inoculating the highland barley leaves infected with the streak disease on a culture medium, sealing, culturing in a biochemical incubator at 22-25 ℃, and separating to obtain highland barley streak disease pathogenic bacteria.

4. The mining method of miRNAs related to highland barley stripe disease as claimed in claim 3, wherein the highland barley leaves infected with stripe disease are treated as follows before being inoculated on the culture medium: washing highland barley leaves infected with stripe disease with water, treating with 75% alcohol and 2% sodium hypochlorite, and washing with water to remove water on the surface of highland barley leaves.

5. the mining method of miRNAs related to highland barley stripe disease as claimed in claim 1, wherein step 2.2 is specifically; inoculating the separated highland barley streak germs into a culture dish, sealing, culturing in a biochemical culture box at 22-25 ℃, taking seeds of a highland barley variety with high susceptibility to streak diseases and a highland barley variety with high resistance to streak diseases after the germs overgrow the whole culture dish, sealing, infecting in the culture box until the seeds germinate, and then planting the germinated seeds in a container filled with nutrient soil for culturing.

6. The mining method of miRNAs related to highland barley stripe disease as claimed in claim 1, wherein the highland barley variety with high stripe disease susceptibility is ZDM6006, and the highland barley variety with high stripe disease resistance is Kunlun No. 14.

7. the mining method of miRNAs related to highland barley stripe disease as claimed in claim 1, wherein in step 3, extracting total RNA, constructing Small RNA library, and performing miRNA high throughput sequencing specifically are:

(1) small RNA library construction

Respectively extracting total RNA of susceptible and unaffected leaves of a stripe disease high-susceptibility highland barley variety and a stripe disease high-resistance highland barley variety, and constructing a Small RNA library by using a Small RNA Sample Pre Kit;

(2) MiRNA high throughput sequencing

And after the Small RNA library is qualified, carrying out miRNA high-throughput sequencing.

8. The method for mining miRNAs related to barley stripe disease As claimed in claim 1, wherein in step 4, the miRNAs related to barley stripe disease screened from differential miRNAs are specifically: and evaluating the difference multiple and the corrected significant level, and comparing the results of the susceptible group and the non-susceptible group of the stripe disease high-susceptibility highland barley variety and the stripe disease high-resistance highland barley variety to obtain the difference miRNAs common to the stripe disease high-susceptibility highland barley variety and the stripe disease high-resistance highland barley variety, namely the miRNAs related to the highland barley stripe disease.

9. miRNAs related to highland barley stripe disease are characterized in that the RNA sequence of hvu-novel-11 or hvu-novel-91, hvu-novel-11 is shown as SEQ No. 1, and the RNA sequence of hvu-novel-91 is shown as SEQ No. 2.

The application of miRNAs in breeding new highland barley varieties resisting stripe diseases is characterized in that the miRNAs are hvu-miR168-5p or hvu-miR 6198.

Technical Field

the invention relates to a highland barley stripe disease, in particular to miRNAs related to the highland barley stripe disease and an excavating method thereof.

Background

Highland barley (Hordeum vulgare L. var. nudum) belongs to the barley genus of the family gramineae, is a variation of barley (Hordeum vulgare L.), has a genetic background which is highly similar to that of barley, and is also called naked barley because glumes and caryopses are separated and grains are naked. The highland barley is the most distinctive crop in the Qinghai-Tibet plateau, and the production position is very important.

The highland barley stripe disease is a fungal disease which affects the highland barley production most seriously (figure 1). At present, the main method for preventing the stripe disease of the highland barley is the seed soaking treatment of medicines. However, the existing seed soaking medicines such as rickxiu, triadimefon, seed soaking agent and the like (the effective components are tebuconazole, triadimefon and dithiocyano methane respectively) can obviously influence the germination rate of the highland barley, and the seed soaking warm soup needs to be prepared and used under the technical guidance, so that the seed soaking warm soup is difficult to be generally realized in the vast highland barley planting area mainly comprising Tibetan. In addition, the ecological environment of the plateau area is fragile, pesticide and fertilizer are strictly controlled, and the drug control is difficult to implement (Lidanhui et al, 2016). Therefore, the breeding of new disease-resistant varieties is a general consensus of the academia on the prevention and control of the barley stripe disease.

The pathogen of the highland barley stripe disease is amethystoides graminiformis [ Drechslera graminea (Rabenh. ex Schl.) Shoemaker, synonym Helminthosporium Rabenh ], belonging to fungi of deuteromycotina; a characteristic of the fungus is Pyrenophora graminea (Rabenh.) Ito et Kurib, which is a fungus of the subdivision Ascomycotina (Gatti et al, 1992; Wu-Banana et al, 2013).

Although the pathogeny of the highland barley stripe disease has been confirmed for many years, the molecular mechanism of the interaction between the pathogeny and the host in the stripe disease process is still unclear at present. The researchers located three disease-resistance-associated genes (QTL) Rdg1a, Rdg2a and Rdg3 on the 2HL and 7HS chromosomes, respectively, by crossing varieties of barley resistant and non-resistant to stripe disease and performing QTL (quantitative trait locus) analysis on the hybrid progeny (Arru et al, 2002; Arru et al, 2003; Zhang, 2016). Functional studies were performed on Rdg1a (Biselli et al,2010) and Rdg2a (Haegi et al, 2008; Bulgarelli et al,2010), respectively, indicating that overexpression of these two genes can significantly improve the resistance of Mediterranean variety barley to stripe disease strains. However, to date, no similar studies have been reported on the mechanism of action of barley stripe (Wu Widan et al, 2013). Therefore, a bottleneck possibly exists when the highland barley stripe disease is researched by a traditional gene localization analysis method, and the research needs to be carried out from a new angle.

MicroRNA (miRNA) is a kind of 21-24 nt non-coding small-molecule RNA, and plays an important role in plant pest and disease resistance (Sun, 2012; Chinen et al, 2012; Rawat et al, 2015). However, current reports of plant disease-associated mirnas are mainly from arabidopsis thaliana, and relatively few are studied in monocotyledonous and cereal crops. There is no report of mirnas in highland barley, and only 71 mirnas have been included in barley. Therefore, compared with other crops, the reference information for researching the functions of the highland barley miRNA is less.

Disclosure of Invention

aiming at the problems in the prior art, the invention provides miRNAs related to the highland barley stripe disease and an excavation method thereof, so that miRNAs related to the highland barley stripe disease are excavated, and a theoretical basis is laid for excavation of highland barley stripe disease-resistant genes and breeding of new disease-resistant varieties in future.

The invention is realized by the following technical scheme:

A mining method of miRNAs related to highland barley stripe disease comprises the following steps:

Step 1, collecting highland barley leaves infected with stripe disease;

step 2, construction of a pathogenic bacteria infection system of the stripe disease

Step 2.1, culturing and separating the highland barley leaves infected with the streaky disease to obtain highland barley streaky disease pathogenic bacteria;

Step 2.2, infecting seeds of the highland barley varieties with high stripe disease infection and the highland barley varieties with high stripe disease resistance by using the pathogenic bacteria of the highland barley stripe disease, and then culturing;

Step 3, after the highland barley leaves grow out and are infected with diseases in the step 2.2, collecting infected and uninfected leaves of the highland barley variety with high susceptibility to the stripe disease and the highland barley variety with high resistance to the stripe disease, respectively extracting total RNA, constructing a Small RNA library, and performing miRNA high-throughput sequencing;

Step 4, excavating miRNA related to stripe disease

According to the miRNA high-throughput sequencing result, the highland barley genome is used as a reference, the difference of miRNA before and after the stripe disease high-susceptibility highland barley variety and the stripe disease high-resistance highland barley variety are infected is analyzed by comparing with a miRBase22, PMRD, mRNA/EST and rRNAetc database, so as to obtain difference miRNAs, and the miRNAs related to the highland barley stripe disease are screened out through the expression quantity difference.

Preferably, in the step 1, the collected highland barley leaves infected with the streak disease are stored in an ultralow temperature refrigerator at-80 ℃ for later use.

Preferably, step 2.1 is specifically; inoculating the highland barley leaves infected with the streak disease on a culture medium, sealing, culturing in a biochemical incubator at 22-25 ℃, and separating to obtain highland barley streak disease pathogenic bacteria.

Further, before inoculating the highland barley leaves infected with the streak disease on the culture medium, the following treatment is carried out: washing highland barley leaves infected with stripe disease with water, treating with 75% alcohol and 2% sodium hypochlorite, and washing with water to remove water on the surface of highland barley leaves.

Preferably, step 2.2 is specifically; inoculating the separated highland barley streak germs into a culture dish, sealing, culturing in a biochemical culture box at 22-25 ℃, taking seeds of a highland barley variety with high susceptibility to streak diseases and a highland barley variety with high resistance to streak diseases after the germs overgrow the whole culture dish, sealing, infecting in the culture box until the seeds germinate, and then planting the germinated seeds in a container filled with nutrient soil for culturing.

Preferably, the highland barley variety with high stripe disease susceptibility is ZDM6006, and the highland barley variety with high stripe disease resistance is Kunlun No. 14.

Preferably, in step 3, extracting total RNA, constructing a Small RNA library, and performing miRNA high-throughput sequencing specifically:

(1) Small RNA library construction

Respectively extracting total RNA of susceptible and unaffected leaves of a stripe disease high-susceptibility highland barley variety and a stripe disease high-resistance highland barley variety, and constructing a Small RNA library by using a Small RNA Sample Pre Kit;

(2) MiRNA high throughput sequencing

And after the Small RNA library is qualified, carrying out miRNA high-throughput sequencing.

Preferably, in the step 4, the step of screening out the miR NAs related to the highland barley stripe disease from the differential miRNAs specifically comprises the following steps: and evaluating the difference multiple and the corrected significant level, and comparing the results of the susceptible group and the non-susceptible group of the stripe disease high-susceptibility highland barley variety and the stripe disease high-resistance highland barley variety to obtain the difference miRNAs common to the stripe disease high-susceptibility highland barley variety and the stripe disease high-resistance highland barley variety, namely the miRNAs related to the highland barley stripe disease.

miRNAs related to the highland barley stripe disease are hvu-novel-11 or hvu-novel-91, the RNA sequence of hvu-novel-11 is shown as SEQ No. 1, and the RNA sequence of hvu-novel-91 is shown as SEQ No. 2.

The application of miRNAs in breeding new highland barley varieties resisting stripe diseases is hvu-miR168-5p or hvu-miR 6198.

Compared with the prior art, the invention has the following beneficial technical effects:

(1) The research idea is novel: many disease-resistant genes are regulated by miRNA, and are linked with other genes by using miRNA as a medium. The invention discloses a method for excavating miRNAs related to highland barley stripe diseases, which comprises the steps of separating and purifying highland barley stripe disease strains from highland barley stripe disease leaves, constructing a laboratory stripe disease pathogenic bacteria infection system, collecting young leaves with diseases and diseases, carrying out high-throughput sequencing (miRNA-seq), constructing a database of highland barley miRNA, and excavating the miRNAs related to highland barley stripe diseases by comparing the database with miRBase22, PMRD, mRNA/EST and rRNAetc.

(2) The development of research direction: the invention discovers a series of miRNAs related to the barley stripe disease. The research of the plant pathogenesis and disease resistance mechanism starting from the miRNA disease control development is a new angle, so the mining of miRNAs related to the highland barley stripe disease also lays a foundation for the discovery of a new disease resistance gene of the highland barley and also lays a certain foundation for the subsequent breeding of a new highland barley stripe disease resistant variety.

By the method, four miRNAs related to the highland barley stripe disease are excavated, wherein the miRNAs are hvu-miR168-5p, hvu-miR6198, hvu-novel-11 and hvu-novel-91 respectively.

Drawings

FIG. 1 shows barley stripe disease leaves in the middle of field and indoor disease onset, and (a) shows barley single plant in the middle of field disease onset; (b) the middle part is the single plant part in the middle disease period of the field; (c) normal and diseased leaves in the middle stage of disease onset in the laboratory.

FIG. 2 shows pathogenic bacteria of highland barley stripe disease separated indoors.

FIG. 3 shows the purified pathogenic bacteria of barley stripe disease.

FIG. 4 shows seeds infected by pathogenic bacteria of barley stripe disease for 15 days.

FIG. 5 is a diagram of indoor infected 15d highland barley leaves.

FIG. 6 is a volcano map of vs ZDM6006 normal leaves vs ZDM6006 diseased leaves and vs Kunlun 14 normal leaves vs Kunlun 14 diseased leaves; ZN represents ZDM6006 normal leaf; ZS represents ZDM6006 diseased leaves; KL14N represents the normal leaves of kunlun 14; KL14S represents kunlun 14 diseased leaves.

Figure 7 is a graph of differential miRNA venn; ZN represents ZDM6006 normal leaf; ZS represents ZDM6006 diseased leaves; KL14N represents the normal leaves of kunlun 14; KL14S represents kunlun 14 diseased leaves.

FIG. 8 is a graph showing the content of the same differential miRNA in different varieties.

Detailed Description

The present invention will now be described in further detail with reference to specific examples, which are intended to be illustrative, but not limiting, of the invention.

The invention discloses a method for excavating miRNAs related to highland barley stripe diseases, which comprises the following steps:

(ii) sample Collection

the experimental materials are a stripe disease high-resistance highland barley hybrid variety Kunlun No. 14 and a stripe disease high-susceptibility highland barley variety ZDM6006, and highland barley stripe disease leaves at the field disease middle stage are adopted and stored in an ultra-low temperature refrigerator at minus 80 ℃ for constructing a laboratory stripe disease pathogenic bacteria infection system.

② separation of pathogenic bacteria of highland barley stripe disease

washing the leaf of disease-resistant variety Kunlun No. 14 and susceptible variety ZDM6006 of semen Avenae Nudae infected with stripe disease with distilled water, shearing the stripe disease leaf (5 × 5mm square) with sterilized scissors, treating with 75% alcohol for 30s and 2% sodium hypochlorite for 30s, washing with distilled water for 3 times, and repeating for 3 times. Absorbing water with filter paper, inoculating on culture medium, sealing with sealing film, culturing in biochemical incubator at 22-25 deg.C for 7 days, and taking out the culture dish.

③ infection of pathogenic bacteria of highland barley stripe disease

And (3) beating the separated highland barley streak germs into fungus cakes by using a puncher, inoculating one fungus cake into a new culture dish by using an inoculating ring, sealing by using a sealing film, and culturing in a biochemical incubator at the temperature of 22-25 ℃ for 6-8 days until the fungus grows over the whole culture dish. And then taking seeds of the required highland barley disease-resistant variety Kunlun No. 14 and susceptible variety ZDM6006 (when the seeds are selected, the seeds with full seeds are selected as much as possible), treating for 1min by using 75% alcohol, washing by using distilled water for 3 times, sucking water by using filter paper, and airing on a table for a while until no water exists on the surfaces of the seeds. And (3) placing the treated highland barley seeds in a culture medium full of highland barley streak germs, sealing by using a sealing film, and just placing the PDA culture medium in an ultralow temperature incubator for dip-dyeing for 15 days. Then, planting the infected seeds in a flowerpot filled with nutrient soil, and putting the flowerpot in a climatic incubator for 14h in daytime at 20 ℃; taking two varieties of infected leaves before and after infection at 10 ℃ for 10 days at night, extracting total RNA of the leaves, detecting the total RNA, and using the leaves for high-throughput sequencing after the total RNA is qualified.

construction of highland barley small RNA library

After the RNA of highland barley disease-resistant variety Kunlun No. 14 and susceptible variety ZDM6006 is detected to be qualified, a Small RNA kit is used for constructing a library, the characteristics of hydroxyl at the 3 'end and complete phosphate group at the 5' end of Small RNA are utilized, RNA is used as an initial sample, joints are directly added at the two ends of the Small RNA, and then reverse transcription is carried out to synthesize cDNA. And performing PCR amplification, polyacrylamide gel electrophoresis separation on a target DNA fragment, cutting and recovering the gel to obtain the highland barley small RNA library. And then, carrying out primary quantification by using a fluorescence quantification instrument, diluting the concentration of the library to 1 ng/mu l, detecting the size of the inserted fragment in the library by using an Agilent 2100 bioanalyzer, and accurately quantifying the effective concentration of the library more than 2nM by using a Q-PCR method after the library conforms to the preset condition so as to ensure the quality of the library.

(v) miRNA high-throughput sequencing and candidate miRNA mining

And after the Small RNA library of the disease-resistant variety Kunlun No. 14 and the susceptible variety ZDM6006 of the highland barley are detected to be qualified, miRNA high-throughput sequencing is carried out, and according to the miRNA high-throughput sequencing result, by taking the highland barley genome as reference, the miRNA difference between the miRNA before infection and the miRNA after infection of the disease-resistant variety Kunlun No. 14 and the susceptible variety ZDM6006 is analyzed by comparing through miRBase22, PMRD, mRNA/EST and rRNAetc databases. The different differences of different varieties are removed, the same difference is taken as the key point of research, the differences which are increased or decreased before and after the different varieties are infected are regarded as the same difference, and the differences which are increased by one variety and decreased by the other variety are regarded as different differences; and (3) mining miRNAs related to the highland barley stripe disease by taking the gene as a screening standard.