CN110982926A - Application and screening method of corn sheath blight disease resistance related gene ZmPR1a - Google Patents
Application and screening method of corn sheath blight disease resistance related gene ZmPR1a Download PDFInfo
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Abstract
The invention relates to a corn sheath blight disease resistance related gene ZmPR1a, and application of the relative expression quantity of the ZmPR1a gene in rapid screening of corn sheath blight resistance. Further provides a rapid screening method of corn sheath blight resistance, which comprises the steps of inoculating rhizoctonia solani in a corn seedling stage, sampling for RNA extraction and cDNA synthesis, and performing fluorescence quantitative PCR detection on the relative expression quantity of the ZmPR1a gene by taking the extracted cDNA as a template; and screening the disease resistance of the corn variety according to the change of the relative expression quantity of the ZmPR1a gene with the inoculation time. The screening method is quick, and seedlings are cultured in a greenhouse without being limited by the growing season; the method can accurately reflect the disease resistance of the corn variety infected by pathogenic bacteria, and is favorable for screening the variety with good disease resistance for later-stage disease-resistant variety breeding.
Description
Technical Field
The invention belongs to the technical field of plant genetic engineering, relates to a corn sheath blight disease resistance related gene ZmPR1a and application thereof, and further relates to a rapid screening method of corn sheath blight disease resistance.
Background
Corn is a main grain and feed crop and an important industrial raw material in China, and plays an important role in economic development and the national civilians. However, due to overlarge planting density and planting area, lack of disease-resistant varieties, outbreak of various epidemic races of diseases and change of pathogenicity of the disease-resistant varieties, the corn production in each corn production area is seriously damaged by the diseases, so that a large amount of corn yield is lost, and the quality is sharply reduced. The corn diseases in China are various, and the yield loss of king rice caused by various diseases accounts for more than the total yield every year. Corn sheath blight is a high-morbidity disease in corn producing areas in the world, corn sheath blight occurs in large scale in the United states earlier than 1934, then is transmitted to countries such as India, Russia, south Africa and the like, and in 1996, the corn sheath blight appears in Jilin of China for the first time and is rapidly transmitted to places such as Hebei, Henan, Shandong, Shaanxi, Zhejiang, Sichuan, Shandong and the like, so that the production of corns in China is seriously influenced.
In recent years, the corn planting area in China is high, and the incidence of corn sheath blight is increased year by year. The investigation shows that the incidence rate of most regions is between 40 and 70 percent, the yield is reduced by more than 10 percent after the incidence of the disease is usually, and the disease is not harvested in serious cases. In view of the harm of the corn sheath blight, a high-sense one-vote rejection system of the sheath blight is added in the approval of corn varieties in Zhejiang province at present, and breeding experts are forced to pay more attention to the resistance traits in the breeding of materials. In future breeding, the identification of disease resistance of materials should be widely carried out, the overall resistance level of the corn is gradually improved, and the harm of sheath blight is effectively controlled.
The control methods currently in common use are planting resistant varieties, rational agricultural measures and chemical control, with planting resistant varieties being the most effective and safe method. In the breeding process of resistant varieties, resistance identification needs to be carried out on the bred varieties. The current method for identifying the sheath blight resistance of corn mainly refers to the part 9 of the corn insect resistance identification technical specification (NY/T1248.1-2006). According to the method, sorghum seeds with rhizoctonia solani are inoculated in the large-horn-mouth stage of the corn, and investigation is carried out in the late stage of the maturity of the corn. The disease level is not higher than 5, and the resistant variety is determined. However, the method is long in time consumption, only two-season resistance identification can be carried out in one year, and the method is not beneficial to the rapid identification of resistant varieties. And the identification method results are greatly influenced by the plant height and the ear height of the corn, so that the defense reaction of the corn to pathogenic bacteria cannot be accurately reflected, and the screening of varieties really resisting the corn leaf blight is not facilitated.
Disclosure of Invention
The invention aims to provide a corn sheath blight disease resistance related gene ZmPR1a and application thereof, and also provides a rapid screening method for corn sheath blight disease resistance.
In order to achieve the purpose, the invention provides the following technical scheme:
1. the corn sheath blight disease resistance related gene is ZmPR1 a.
2. Application of a corn sheath blight disease resistance related gene ZmPR1a in rapid screening of corn sheath blight disease resistance.
3. A rapid screening method for corn sheath blight resistance, which comprises the following steps:
a. inoculating rhizoctonia solani in a seedling stage of the corn;
b. sampling and extracting RNA;
c, synthesizing cDNA;
d. detecting the relative expression quantity of the ZmPR1a gene by fluorescent quantitative PCR;
e. the corn variety is screened for disease resistance according to the change of the relative expression quantity of the ZmPR1a gene with the inoculation time.
Further, in the step e, the disease resistance of the corn variety is screened according to the change of the relative expression quantity of the ZmPR1a gene along with the inoculation time as follows: the relative expression quantity of the ZmPR1a gene is C n hours after the inoculation of the rhizoctonia solani in the seedling stage of the maizenWithin the range of n being more than 0 and less than or equal to 24, the corn variety with the Cn being more than or equal to 4 at least once and the Cn not less than C0 is the corn sheath blight disease resistant variety.
Further, n is equal to 6, 12 and/or 24.
Further, relative expression amount CnCalculated by the △△ Ct method.
Further, phaseFor expression amount Cn＝2- (△ Ct treatment sample- △ Ct 0 hours)△ Ct is Ct target gene-Ct reference gene.
Further, the inoculation of rhizoctonia solani in the corn seedling stage in the step a is to place sorghum grains with rhizoctonia solani strains in the first leaf sheath of the corn seedlings in the trefoil stage and culture the sorghum grains at room temperature.
Further, step b is to take plant tissues of 1cm above and below the inoculated part, quickly freeze the plant tissues by using liquid nitrogen, then grind the plant tissues into powder, take 50-100mg and add 1ml of Trizol, and stand the plant tissues at room temperature; adding 200 μ l of precooled chloroform, shaking vigorously for 15s, and standing at room temperature for 10-15 min; 12000g, centrifuging for 5min at 4 ℃; transferring the upper layer water phase into a 1.5ml centrifuge tube, adding 500 μ l isopropanol, and standing at-20 deg.C for precipitation for 1 hr; 12000g, 4 ℃ centrifugation for 10min, carefully remove the supernatant, prevent sucking away the precipitate; washing twice with 75% ethanol, 7500g, and centrifuging at 4 deg.C for 5 min; centrifuging for 2min in an empty tube; carefully removing the supernatant, inverting and drying for 2 min; the RNA concentration was determined by adding 30. mu.l DEPC-H2O to the solution.
Further, step c is adding 4 XgDNA wiper Mix4 μ l to each 1 μ g of extracted RNA, supplementing to 16 μ l with RNase-freeddH2O, mixing well, reacting at 42 deg.C for 2 min; then directly adding 4 mul of 5 XHiScript III qRT SuperMix, uniformly mixing, reacting for 15min at 37 ℃, and denaturing for 5sec at 85 ℃ to obtain a product for later use.
Further, the 20 μ l reaction system of the fluorescence quantitative PCR is: 2 XChamQ Universal SYBR qPCRMaster Mix 10. mu.l, Primer F0.4. mu.l, Primer R0.4. mu.l, Template cDNA 1. mu.l, ddH2O 8.2.2. mu.l; wherein the content of the first and second substances is controlled,
primer F of Actin: GGTTCTATTCCAGCCATCCTTCATTG the flow of the air in the air conditioner,
primer R of Actin: TCTCCTTGCTCATGCGGTCAC the flow of the air in the air conditioner,
primer F of ZmPR1 a: GGCGAGAGCCCCTACTAGAC
Primer R of ZmPR1 a: AAATCGCCTGCATGGTTTTA are provided.
Further, the fluorescent quantitative PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 30s, pre-denaturation at 95 ℃ for 5s, and pre-denaturation at 58 ℃ for 30s for 45 cycles; the temperature of the dissolution curve was 95 ℃ for 15s, 58 ℃ for 60s, and 95 ℃ for 15 s.
The invention has the beneficial effects that: the existing identification method can only carry out identification for two seasons in one year, and is time-consuming and labor-consuming, the method only needs about 15 days when the resistance of the corn variety sheath blight is rapidly screened and identified based on the disease-resistant related gene of the corn sheath blight, and seedlings are cultured in a greenhouse, so that a large amount of time can be saved, and the method is not limited by the growing season; the identification result of the existing identification method is often influenced by the height of the corn plant and the height of the ear, the identification result of the variety with higher plant height and ear height is often shown as disease resistance, and the identification result of the variety with lower plant height and ear height is often shown as disease infection.
Drawings
In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 shows the relative expression level of ZmPR1a gene of corn variety Chang 7-2 within 24 hours after inoculation;
FIG. 2 shows the relative expression level of ZmPR1a gene of maize variety Taxiantan No. 1 within 24 hours after inoculation;
FIG. 3 shows the relative expression level of ZmPR1a gene of maize variety fluid 478 within 24 hours after inoculation;
FIG. 4 shows the relative expression levels of ZmPR1a gene in maize variety Hongyu No. 2 within 24 hours after inoculation;
FIG. 5 is a graph showing the relative expression levels of ZmPR1a gene of Zhengdan 958 of maize variety within 24 hours after inoculation;
FIG. 6 shows the relative expression levels of ZmPR1a gene of maize variety Tansyu No. 8 within 24 hours after inoculation.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The experimental procedures, in which specific conditions are not specified in the examples, are generally carried out under conventional conditions or under conditions recommended by the manufacturers.
Example 1
(1) Strain activation: picking a small block of corn sheath blight strain stored on the inclined plane of PDA by using a sterile inoculating needle, placing the small block of corn sheath blight strain in the center of a PDA flat plate, and culturing in an incubator at 28 ℃. When hyphae cover the area of the flat plate 2/3, taking the edge for switching again, and growing the secondary adapter plate on the flat plate for later use.
(2) Inoculation and sampling: taking 200g of sorghum grains in a 500ml triangular flask, and sterilizing at 121 ℃ for 20 min; and (3) after cooling, adding a 2X 2cm strain-carrying culture medium obtained in the step (1) into a triangular flask, placing the triangular flask in an incubator at 28 ℃ for culture, and shaking every day to enable hyphae to grow uniformly. When each sorghum grain in the triangular flask is uniformly covered by hypha, lightly placing the sorghum grains in the first leaf sheath of the maize seedling in the three-leaf stage, and culturing in a greenhouse. Respectively inoculating 12 seedlings of Chang 7-2, Tai fresh sweet No. 1, Yan 478, Hongyu No. 2, Zheng 958 and Kunshu No. 8, respectively, taking plant tissues of 1cm above and below the inoculated part after 0, 6, 12 and 24 hours of inoculation, quickly freezing with liquid nitrogen, and sampling each variety for 3 times.
(3) Total RNA extraction: grinding the plant tissue liquid nitrogen substance in the step (2) into powder, putting 50-100mg into a 1.5ml centrifuge tube, adding 1ml Trizol, and standing for 10min at room temperature; adding 200 μ l of precooled chloroform, shaking vigorously for 15s, and standing at room temperature for 10-15 min; 12000g, centrifuging for 5min at 4 ℃; transferring the upper layer water phase into a 1.5ml centrifuge tube, adding 500 μ l isopropanol, and standing at-20 deg.C for sufficient precipitation for 1 h; 12000g, 4 degrees C centrifugal 10min, carefully remove the supernatant, prevent from sucking away the precipitation; 75% ethanol (DEPC-H)2O preparation) washing twice, 7500g, centrifuging for 5min at 4 ℃; centrifuging for 2min in an empty tube; carefully removing the supernatant, inverting and drying for 2 min; adding 30. mu.l DEPC-H2Dissolving O, and measuring the concentration of RNA by using an ultraviolet spectrophotometer.
(4) The reverse transcription method comprises the following steps: first, genomic DNA was removed, and 4 XgDNA wiper Mix 4. mu.l was added to a quantitative amount of 1. mu.g of RNA, followed by RNase-free ddH2O, filling the system to 16 mu l, gently sucking and uniformly mixing the system by using a pipette, and reacting for 2min at 42 ℃; and carrying out reverse transcription by using the product of the previous step, directly adding 4 mu l of 5 XHiScript III qRTSuperMix into the reaction tube of the previous step, gently sucking by using a pipette, uniformly mixing, reacting at 37 ℃ for 15min, and carrying out denaturation at 85 ℃ for 5sec, wherein the product is standby or stored in a refrigerator at-20 ℃.
(5) qRT-PCR: the qRT-PCR reaction system (20. mu.l) was as follows: 2 × ChamQ Universal SYBR qPCRMaster Mix 10 μ l, Primer F (10 μ M)0.4 μ l，Primer R(10μM)0.4μl，Template cDNA 1μl，ddH2O8.2. mu.l. The primer sequences used for qRT-PCR are shown in Table 1, and the primers were synthesized by Shanghai Optimus department Biotechnology Co., Ltd.
The reaction conditions were as follows: pre-denaturation at 95 ℃ for 30s, pre-denaturation at 95 ℃ for 5s, and pre-denaturation at 58 ℃ for 30s for 45 cycles; dissolution curve temperature: 95 ℃ for 15s, 58 ℃ for 60s, 95 ℃ for 15 s.
TABLE 1 primer sequences for qRT-PCR
(7) Data analysis, in the method, Actin is used as an internal reference gene, and the change fold condition of the relative expression quantity of the ZmPR1a gene (GeneID:100272820) along with time is calculated through △△ Ct, wherein the calculation formula is that the relative expression quantity is 2- (△ Ct treatment sample- △ Ct 0 hours)△ Ct (Ct target gene) -Ct internal reference gene, processing the sample, namely the sample sampled within 24 hours after inoculation in the step (2), wherein the relative expression quantity of the ZmPR1a gene is up-regulated by 4 times or more within 24 hours after inoculation, and the corn variety with the relative expression quantity of the ZmPR1a gene when the expression quantity of the gene is not less than 0 hour within 24 hours is determined to be a corn sheath blight resistant variety.
The method is used for identifying the sheath blight resistance of six corn varieties, and meanwhile, the field corn sheath blight resistance identification is carried out according to the 9 th part of corn insect resistance identification technical specification (NY/T1248.1-2006). The field identification result shows that Chang 7-2 and Tai Xian sweet No. 1 are identified as corn sheath blight resistant varieties, and Ye 478, Hongyu No. 2, Zheng 958 and Kunshu No. 8 are identified as corn sheath blight susceptible varieties.
The results of identifying corn varieties Chang 7-2, Tan Xian Tian 1, Yan 478, Hongyu 2, Zheng 958 and Kunshu 8 by the method are sequentially shown in the figure 1-figure 6, and the relative expression amounts of ZmPR1a genes of various corn varieties at different time points are shown in the table 1. As can be seen from FIGS. 1-6, the relative expression amounts of ZmPR1a genes of Chang 7-2 and Taxiatan No. 1 are not lower than the expression amount at 0 hour within 24 hours, and at least one up-regulation expression which is more than 4 times is generated; while the relative expression level of ZmPR1a genes of other varieties of HgO 478, HgO No. 2, Zhengdan 958 and Kunshu No. 8 is down-regulated within less than 0 hour within 24 hours. According to the screening method of the invention, the obtained Chang 7-2 and Tai Xian sweet No. 1 are corn sheath blight resistant varieties, the obtained Chang 478, Hongyu No. 2, Zheng 958 and Kunshu No. 8 are corn sheath blight susceptible varieties, and the identification result is completely consistent with the field identification result.
TABLE 2 relative expression of ZmPR1a gene of each maize variety at different time points
According to the screening method, ZmPR1a and the other six genes are selected at the early stage for screening by different methods, and the results show that the ZmPR1a gene can be used as a stable marker gene and is applied to the screening method provided by the invention to quickly and accurately screen the corn sheath blight resistant variety.
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the foregoing preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.
Claims (10)
1. The maize sheath blight disease resistance related gene is ZmPR1 a.
2. The use of the gene according to claim 1, wherein ZmPR1a is used for rapidly screening the resistance of corn sheath blight.
3. A method for rapidly screening resistance to corn sheath blight, which is characterized by comprising the following steps:
a. inoculating rhizoctonia solani in a seedling stage of the corn;
b. sampling and extracting RNA;
c, synthesizing cDNA;
d. detecting the relative expression quantity of the ZmPR1a gene by fluorescent quantitative PCR;
e. the corn variety is screened for disease resistance according to the change of the relative expression quantity of the ZmPR1a gene with the inoculation time.
4. The method for rapidly screening maize sheath blight disease resistance according to claim 3, wherein the disease resistance of maize varieties is screened according to the change of the relative expression level of ZmPR1a gene with the inoculation time in the step e as follows: the relative expression quantity of the ZmPR1a gene is C n hours after the inoculation of the rhizoctonia solani in the seedling stage of the maizenWithin n being more than 0 and less than or equal to 24, there is at least one CnNot less than 4 and CnAre not less than C0The corn variety is a corn sheath blight resistant variety.
5. The method for rapid screening of resistance to sheath blight of corn according to claim 3, wherein n is equal to 6, 12 and/or 24.
6. The method for rapidly screening maize sheath blight disease resistance according to claim 3, wherein the relative expression amount Cn is calculated by △△ CT method.
7. The method for rapidly screening maize sheath blight resistance according to claim 3, wherein the inoculation of rhizoctonia solani in the maize seedling stage in the step a is that sorghum grains with the rhizoctonia solani are placed in the first leaf sheaths of maize seedlings in the trefoil stage and cultured at room temperature.
8. The method for rapidly screening the resistance to sheath blight of corn according to claim 3, wherein the step b comprises the steps of taking 1cm of plant tissues above and below the inoculated part, quickly freezing the plant tissues by using liquid nitrogen, grinding the plant tissues into powder, adding 1ml of Trizol into 50-100mg of the powder, and standing the powder at room temperature; adding 200 μ l of precooled chloroform, shaking vigorously for 15s, and standing at room temperature for 10-15 min; 12000g, centrifuging for 5min at 4 ℃; transferring the upper layer water phase into a 1.5ml centrifuge tube, adding 500 μ l isopropanol, and standing at-20 deg.C for sufficient precipitation for 1 h; 12000g, 4 ℃ centrifugation for 10min, carefully remove the supernatant, prevent sucking away the precipitate; washing twice with 75% ethanol, 7500g, and centrifuging at 4 deg.C for 5 min;centrifuging for 2min in an empty tube; carefully removing the supernatant, inverting and drying for 2 min; adding 30. mu.l DEPC-H2O dissolved, and the RNA concentration was measured.
9. The method for rapidly screening maize sheath blight disease resistance according to claim 3, wherein the step c is to add 4 xgDNA wiper Mix4 μ l to each 1 μ g of the extracted RNA, and to use RNase-free ddH2Supplementing O to 16 μ l, mixing, and reacting at 42 deg.C for 2 min; then directly adding 4 mul of 5 XHiScript III qRT SuperMix, uniformly mixing, reacting for 15min at 37 ℃, and denaturing for 5sec at 85 ℃ to obtain a product for later use.
10. The method for rapidly screening the resistance to the corn sheath blight disease according to claim 3, wherein the 20 μ l reaction system of the fluorescent quantitative PCR is as follows: 2 XChamQ Universal SYBR qPCR Master Mix 10. mu.l, Primer F0.4. mu.l, Primer R0.4. mu.l, Template cDNA 1. mu.l, ddH2O8.2. mu.l; wherein the content of the first and second substances,
primer F of Actin: GGTTCTATTCCAGCCATCCTTCATTG the flow of the air in the air conditioner,
primer R of Actin: TCTCCTTGCTCATGCGGTCAC the flow of the air in the air conditioner,
primer F of ZmPR1 a: GGCGAGAGCCCCTACTAGAC
Primer R of ZmPR1 a: AAATCGCCTGCATGGTTTTA are provided.
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