CN104131076B - A kind of method utilizing miRNA166b gene forecast bacterial blight of rice - Google Patents
A kind of method utilizing miRNA166b gene forecast bacterial blight of rice Download PDFInfo
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Abstract
The invention discloses a kind of method utilizing miRNA166b gene forecast bacterial blight of rice, belong to biological technical field。Said method comprising the steps of: choose Oryza sativa L. to be measured and comparison Oryza sativa L., the described comparison Oryza sativa L. chosen is be uninfected by bacterial leaf-blight but the Oryza sativa L. cultivated under the same conditions with Oryza sativa L. to be measured；It is individually separated the total miR-96 gene in described Oryza sativa L. to be measured and described comparison Oryza sativa L.；Detect the expression of miRNA166b gene in the total miR-96 gene in described Oryza sativa L. to be measured and described comparison Oryza sativa L. respectively, in the sequence such as sequence table of described miRNA166b gene shown in SEQ ID NO:1；Expression according to the miRNA166b gene in described Oryza sativa L. to be measured and described comparison Oryza sativa L., it is judged that whether experiment is successful, if success, judges whether described plant to be measured infects bacterial leaf-blight further。Forecasting Methodology provided by the invention obtains successfully, it is possible to before bacterial blight of rice disease occurs, a couple of days realizes accurate forecast, for early controlling the time won, decreases the loss that Oryza sativa L. is caused by bacterial leaf-blight anti-morning。
Description
Technical field
The present invention relates to biological technical field, particularly to a kind of method utilizing miRNA166b gene forecast bacterial blight of rice。
Background technology
Oryza sativa L. is China's staple food crop, and bacterial leaf-blight is one of big Major Diseases of Oryza sativa L. two, belongs to worldwide disease, and outstanding weight occurs in Rice In Asian Cultivated Rice district。Bacterial leaf-blight sickness rate is high, it is fast to infect, and fall ill rice field underproduction 20-30%, and even 50%。The same with major part disease, bacterial leaf-blight morbidity early prevention and treatment effect is preferably。Morbidity middle and late stage, pathogenic bacteria is amount reproduction, Oryza sativa L. is caused injury, and prevention effect is poor。As can be seen here, early prediction is the key point of bacterial blight of rice prevention。
Tradition bacterial blight of rice forecast Main Basis weather, weather, varietal resistance, nitrogen application situation and disease history etc. speculate, it is possible to according to field water bacterial blight of rice Symptoms, later stage PD is forecast。
In the process realizing the present invention, inventor have found that prior art at least there is problems in that
It is very fuzzy according to the forecast result that weather, weather, varietal resistance, Planting status and history etc. carry out, the probability that disease occurs in certain area with time range can be described, but can not forecasting concrete time and place that disease occurs, the applicability of forecast result is poor。Such as, the weather conditions of the hot and humid epidemics forecast frequently as bacterial blight of rice, but not can determine that whether hot and humid lower disease necessarily occurs, occurs in that block field, occurs in that day。Due to above uncertainty, peasant can not decide whether to start to prevent and treat bacterial leaf spot, which block field is prevented and treated and when prevented and treated。Meanwhile, disease occurs showing that bacterial leaf-blight has been enter into middle and late stage, and pathogenic bacteria is amount reproduction, and bacterial leaf-blight is popular to be difficult to avoid that so that utilize the work that disease investigation carries out forecasting also without too big practical significance。
Summary of the invention
In order to solve in prior art bacterial blight of rice and forecast not in time, inaccurate shortcoming, embodiments provide a kind of method utilizing miRNA166b gene forecast bacterial blight of rice。Described technical scheme is as follows:
Choose 9311 rice varieties as Oryza sativa L. to be measured and comparison Oryza sativa L.；
Cultivate described Oryza sativa L. to be measured and described comparison Oryza sativa L.；
It is individually separated the total miR-96 gene in described Oryza sativa L. to be measured and described comparison Oryza sativa L.；
Detecting the expression of miRNA166b gene in the described total miR-96 gene in described Oryza sativa L. to be measured and described comparison Oryza sativa L. respectively, the sequence of described miRNA166b gene is such as shown in SEQ ID NO:1；
Expression according to the miRNA166b gene in described Oryza sativa L. to be measured and described comparison Oryza sativa L., judge that whether experiment is successful, if success, then judge whether described plant to be measured infects bacterial leaf-blight further, described judge to test whether successful methods as: if the ratio of the expression maximum of the miRNA166b gene of described comparison Oryza sativa L. and expression minima is less than 1.5, then Success in Experiment；If expression maximum and the expression minima of the miRNA166b gene of described comparison Oryza sativa L. be more than or equal to 1.5, then test unsuccessful。
Judge described plant to be measured whether infect the method for bacterial leaf-blight as: if the expression of the miRNA166b gene of the expression of the miRNA166b gene of described Oryza sativa L. to be measured comparison described less than 2 times Oryza sativa L., then described Oryza sativa L. to be measured is not susceptible；If the expression of the miRNA166b gene of the described comparison Oryza sativa L. that the expression of the miRNA166b gene of described Oryza sativa L. to be measured is more than or equal to 2 times, then described Oryza sativa L. to be measured is susceptible。
Specifically; when the described Oryza sativa L. to be measured of described cultivation and described comparison Oryza sativa L.; described comparison Oryza sativa L. adopts protectiveness cultivation step prevent and treat bacterial leaf-blight when cultivation, and except described protectiveness cultivation step with except preventing and treating bacterial leaf-blight, described comparison Oryza sativa L. is consistent with the maintenance of the cultivation condition of described Oryza sativa L. to be measured。
Further, described protectiveness cultivation step includes soil isolation, water source isolation and spatial separation。
Specifically, the morning 8:55～9:05 utilize described Oryza sativa L. to be measured to carry out the separation of described total miR-96 gene with the same area of the identical blade of described comparison Oryza sativa L.。
Specifically, adopt real time quantitative PCR method, detect described miRNA166b gene expression in described Oryza sativa L. to be measured and described comparison Oryza sativa L.。
Further, the pre-treatment step of described real time quantitative PCR method includes:
Utilize spectrophotometric determination and obtain the concentration of described total miR-96 gene of separation；
Outer source reference miR-96 gene is added in described total miR-96 gene, obtain the first mixed liquor, the addition of described outer source reference miR-96 gene is the 0.05% of described total miR-96 gene quality, and the sequence of described outer source reference miR-96 gene is such as shown in SEQ ID NO:2；
5 ' ends of described total miR-96 gene and described outer source reference miR-96 gene are connected with 3 ' ends, obtain described total miR-96 gene of cyclisation and the mixed liquor of the described outer source reference miR-96 gene of cyclisation；
The mixed liquor of total miR-96 gene of described cyclisation and the outer source reference miR-96 gene of described cyclisation is carried out reverse transcription, and the reverse transcription product obtained is for carrying out described real-time quantitative PCR detection。
Further, described employing real time quantitative PCR method, detect described miRNA166b gene expression in described Oryza sativa L. to be measured and described comparison Oryza sativa L., including:
By the ROX fluorescence correction dyestuff mix homogeneously of primer, 10 μ l quantitative PCR mixture and 0.4 μ l50 times that reverse transcription product described in 2 μ l, 3 μ l concentration are 1 μM, obtain the second mixed liquor, being reacted in real-time PCR by described second mixed liquor, described response procedures is: 50 DEG C 2 minutes；95 DEG C 10 minutes；95 DEG C 45 seconds, 56 DEG C 45 seconds, 66 DEG C 30 seconds, 67 DEG C 30 seconds, totally 45 circulations, wherein, 66 DEG C of 30 seconds and 67 DEG C of 30 seconds these two steps increase by 0.1 DEG C and 0.2 DEG C respectively after every circulation primary, collect fluorescence signal in the final step that circulates each time, the power of described fluorescence signal for weigh described expression number；
Described primer includes: sequence such as the miRNA166b gene forward primer shown in SEQ ID NO:3, sequence such as the miRNA166b gene reverse primer shown in SEQ ID NO:4, the sequence such as outer source reference miR-96 gene forward primer shown in SEQ ID NO:5 and the sequence such as outer source reference miR-96 gene reverse primer shown in SEQ ID NO:6。
Further, the step that 5 ' ends of described total miR-96 gene are connected with 3 ' ends is included:
Take the MnCl of the first mixed liquor described in 5ng, 2 μ l10 × reaction buffer, 1 μ l50mM2, the glycine betaine of 4 μ l5M and 1 μ l5u/ μ l cyclase, mix after supplying 20 μ l with water, obtain the 3rd mixed liquor, after described 3rd mixed liquor is incubated 15 minutes in 60 DEG C, 80 DEG C are incubated 10 minutes, make enzyme deactivation, obtain total miR-96 gene of described cyclisation and the mixed liquor of the outer source reference miR-96 gene of cyclisation。
Further, the step that the mixing liquid of the outer source reference miR-96 gene of total miR-96 gene of described cyclisation and cyclisation carries out reverse transcription includes:
Take total miR-96 gene of described cyclisation and the reverse transcriptase of DTT and the 20U of reverse transcriptase primer that the mixed liquor 2 μ l of the outer source reference miR-96 gene of described cyclisation, 5 μ l concentration are 1 μM, 2 μ l concentration to be the dNTP of 10mM, 5 μ l concentration be 100mM, after supplying 50 μ l mixings with water, obtain the 4th mixed liquor, described 4th mixed liquor is incubated 2 hours in 42 DEG C, 75 DEG C are incubated 15 minutes, make enzyme deactivation, obtain described reverse transcription product；Described reverse transcriptase primer includes the reverse transcriptase primer of miRNA166b gene and the reverse transcriptase primer of outer source reference miR-96 gene, the reverse transcriptase primer sequence of described miRNA166b gene is such as shown in SEQ ID NO:7, and the reverse transcriptase primer sequence of described outer source reference miR-96 gene is such as shown in SEQ ID NO:8。
The technical scheme that the embodiment of the present invention provides has the benefit that a kind of method utilizing miRNA166b gene forecast bacterial blight of rice provided by the invention, expressing of miRNA166b gene can be changed the mark that can infect hoja blanca bacterium as Oryza sativa L., in order to whether Oryza sativa L. clearly to be measured catches an illness, and the time clearly fallen ill and field, it is to avoid the ambiguity of traditional disease forecasting。Meanwhile, the predicted time of the present invention early, can make forecast in Oryza sativa L. infects bacterial leaf-blight 24 hours。
Detailed description of the invention
For making the object, technical solutions and advantages of the present invention clearly, below embodiment of the present invention is described further in detail。The reagent not marking explanation in the present invention is conventional commercial reagent, is all commercially available and effect is almost without difference in most of biotech companies。
Embodiment
The embodiment of the present invention is using 9311 rice varieties as Oryza sativa L. to be measured and comparison Oryza sativa L., and wherein, Oryza sativa L. to be measured refers to be planted under the normal cultivation condition in land for growing field crops, it is necessary to monitor whether to infect the Oryza sativa L. of bacterial leaf-blight；Comparison Oryza sativa L. refers to by protectiveness technical measures and bacterial leaf-blight prophylactico-therapeutic measures; guarantee that it is the healthy Oryza sativa L. not infecting bacterial leaf-blight; except protectiveness cultivation step with except preventing and treating bacterial leaf-blight; comparison Oryza sativa L. keeps consistent with the cultivation condition of Oryza sativa L. to be measured; wherein, protectiveness cultivation step includes soil isolation, water source isolation and spatial separation。
Choosing of seed: the seed of the seed of Oryza sativa L. to be measured and comparison Oryza sativa L. is all harvested from 9311 rice plants of health, and the surface of the seed is without obvious pest and disease damage hazard symptoms。
The pre-treatment of seed, sowing and cultivation condition: compare that seed and seed to be measured are used that concentration is 70% alcohol-pickled 2 minutes, 2 times are washed again with deionized water, soaked overnight in the water of 30 DEG C, carries out accelerating germination to seed at the temperature of 30 DEG C, sows after seed germination。
Fetch earth earth from the field of paddy growth to be measured, utilizes autoclave (Shanghai Medical Equipment Plant of Bo Xun Industrial Co., Ltd. produces, and model is YSQ-LS-50S11) that soil carries out high-temperature high-voltage sterilizing sterilizing, and sterilization condition is: 120 DEG C, 30 minutes。Soil after sterilization is as the planting soil of comparison Oryza sativa L.。Comparison Oryza sativa L. is sowed when isolation, transplants, is grown in the plastic tub being filled with disinfection soil; Oryza sativa L. to be measured is then planted in the environment of land for growing field crops; when cultivating Oryza sativa L. to be measured and comparison Oryza sativa L.; except protectiveness cultivation step and bacterial leaf-blight prophylactico-therapeutic measures, Oryza sativa L. to be measured keeps consistent with the cultivation condition of Oryza sativa L. to be measured。
Protectiveness cultivation step includes: (1) soil is isolated: utilize potted plant, makes soil and the field soil isolation of comparison paddy growth, it is to avoid pass disease by soil and infect bacterial leaf-blight；(2) water source isolation: adopt tap water to irrigate comparison Oryza sativa L., it is ensured that water source, land for growing field crops does not enter comparison Oryza sativa L., it is to avoid infect bacterial leaf-blight by water source；(3) spatial separation: do not plant other Oryza sativa L. for 10 meters around comparison Oryza sativa L., it is to avoid rub with the rice leaf with bacterial leaf spot bacterium and infect bacterial leaf-blight。Oryza sativa L. to be measured does not prevent and treat bacterial leaf-blight; the cultivation condition compareing Oryza sativa L. and Oryza sativa L. to be measured except protectiveness cultivation step except bacterial leaf-blight prophylactico-therapeutic measures keeps consistent as far as possible, including sowing, transplanting, fertilising, insect protected, diseases prevention (except bacterial leaf-blight other disease) etc. the while of production management's measure, is synchronously performed。
Specifically, around comparison Oryza sativa L. within the scope of 10 meters, do not sow or plant other Oryza sativa L.；Compare the clean tap water of Rice irrigation hydromining；Except conditions above, compare identical with common land for growing field crops conventional cultivation condition with other cultivation condition of Oryza sativa L. to be measured, but all cultivation steps are in comparison Oryza sativa L. and keep consistent between Oryza sativa L. to be measured, as the measure of the production management such as sowing, transplanting, fertilising, insect protected, diseases prevention (except hoja blanca other disease) while, be synchronously performed。In this test, sowing time is on November 27th, 2010, and place is Qionghai。
Comparison Oryza sativa L. is adopted with Oryza sativa L. to be measured identical cultivation condition plantation, enables comparison Oryza sativa L. and Oryza sativa L. to be measured to grow at identical conditions, it is ensured that the expression of comparison Oryza sativa L. and Oryza sativa L. to be measured is not by the impact of cultivation condition。
Needing forecast bacterial leaf-blight period of whether occurring, such as seedling stage and cut phase to heading flowering period, at the blade of the 8:55～9:05 position at second middle part 2/3 of inverted respectively in the morning。Each comparison Oryza sativa L. respectively takes at least 3 blades with Oryza sativa L. to be measured and mixes, for representing the sample of comparison Oryza sativa L. and Oryza sativa L. to be measured。Taken blade is immediately placed in liquid nitrogen and preserves the extraction to total miR-96 gene。In the present embodiment, sample time, section was that on February 28th, 2011 is to March 3 days then, continuous sampling 5 days, obtain comparison Oryza sativa L. and each 5 samples of Oryza sativa L. to be measured altogether, it is numbered C1～C5 and T1～T5 respectively, wherein C represents Control, T represents Treatment, wherein, comparison Oryza sativa L. is consistent with the time point that Oryza sativa L. to be measured samples, leaf position selected by comparison Oryza sativa L. and Oryza sativa L. to be measured is also identical, this can make the biological clock between comparison Oryza sativa L. and Oryza sativa L. to be measured be identical with developmental condition, only choose the biological clock blade all identical with developmental condition and just can be further ensured that the miRNA expression compareed between Oryza sativa L. and Oryza sativa L. to be measured is than less by the impact of biological clock with growth。
Separate total miR-96 gene: (miR-96 gene separating kit is commercially available to utilize miR-96 gene separating kit, article No.: R6727, production company: Omega, this test kit specifically includes: RNA centrifugal column, genomic DNA remove centrifugal column, centrifuge tube, MCL lysis buffer, XD binding buffer liquid, RNA eluent II and DEPC water) separate the total miR-96 gene in above-mentioned C1～C5 and T1～T5 rice leaf。
Concrete operation method is as follows: takes out C1～C5 and T1～T5 rice leaf sample from liquid nitrogen respectively, is respectively placed in 10 mortars, and pours liquid nitrogen in mortar immediately, be fully ground, and grinds respectively and can avoid cross-contamination。In each mortar, take powder ground for about 100mg be placed in the centrifuge tube of 1.5ml, add the lysate of 700 μ L, vortex 30 seconds is to mix sample, 55 DEG C are incubated 30 minutes, it is centrifuged 5 minutes under the room temperature condition that centrifugal force is 12000 × g, obtain supernatant, and it is centrifugal to be transferred to by supernatant in centrifugal column, for removing the DNA in genome, it is centrifuged 2 minutes through 12000 × g room temperature, and the liquid of outflow is transferred in the centrifuge tube of a new 1.5mL, dehydrated alcohol the vortex mixing in 20 seconds of this liquid volume 1.1 times is added in the liquid of this outflow, liquid after this vortex is transferred in RNA centrifugal column and is centrifuged 1 minute through 12000 × g room temperature, abandon centrifugal liquid, add ethanol that 500 μ L concentration are 96-100% in RNA centrifugal column, it is centrifuged 1 minute then through 12000 × g room temperature, abandon centrifugal liquid, add 500 μ LXD binding buffer liquid in RNA centrifugal column, centrifugal 1 minute of 12000 × g room temperature, abandon centrifugal liquid, add in 750 μ LRNA eluent II to RNA centrifugal columns, centrifugal 1 minute of 12000 × g room temperature, abandon centrifugal liquid, add in 750 μ LRNA eluent II to RNA centrifugal columns, centrifugal 1 minute of 12000 × g room temperature, abandon centrifugal liquid, by RNA centrifugal column under the maximal rate more than 12000 × g, centrifugal 2 minutes of room temperature, 30-50 μ LDEPC water is added in RNA centrifugal column, after ambient temperatare puts 5 minutes, under the maximal rate more than 12000 × g, centrifugal 1 minute of room temperature, the centrifugal liquid obtained is the solution containing total miR-96 gene, this solution is stored in-70 DEG C standby。
The pre-treatment step of real-time quantitative PCR (polymerasechainreaction, polymerase chain reaction) method includes:
Total miR-96 gene quantitative: (spectrophotometer is produced by Quawell company of the U.S. to utilize spectrophotometer, model is Q5000) in RNA quant program, measure and obtain the concentration of total miR-96 gene of separation, the quality according to concentration Yu the total miR-96 gene of volume computing。
Outer source reference miR-96 gene is added in the total miR-96 gene separated, obtain the first mixed liquor: the addition of outer source reference miR-96 gene is the 0.05% of the total miR-96 gene quality separated, the sequence of outer source reference miR-96 gene is such as shown in SEQ ID NO:2, and it is synthesized by Shanghai JiMa pharmacy Technology Co., Ltd。
MiR-96 gene cyclisation: take containing the first mixed liquor of the total miR-96 gene of about 5ng, 2 μ l10 × reaction buffer, 1 μ l50mM MnCl2, the glycine betaine of 4 μ l5M and 1 μ l5u/ μ l cyclase, supply mixing after 20 μ l with water, obtain the 3rd mixed liquor；After the 3rd mixed liquor is incubated 15 minutes in 60 DEG C, 80 DEG C are incubated 10 minutes, make enzyme deactivation。5 ' ends of total miR-96 gene and outer source reference miR-96 gene are connected with 3 ' ends, it is thus achieved that the mixed liquor of total miR-96 gene of cyclisation and the outer source reference miR-96 gene of cyclisation, this mixed liquor is used for rolling ring reverse transcription。Wherein, cyclase is produced by Epicentre company of the U.S., and article No. is CL9021K。With this enzyme provide also have: 10 × reaction buffer, 50mM MnCl2, 5M glycine betaine and without enzyme water。
The reverse transcription of the mixed liquor of total miR-96 gene of cyclisation and the outer source reference miR-96 gene of cyclisation: take total miR-96 gene of cyclisation and the mixed liquor 2 μ l of the outer source reference miR-96 gene of cyclisation, 5 μ l concentration are the reverse transcriptase primer of 1 μM, 2 μ l concentration are the dNTP of 10mM, 5 μ l concentration are the DTT of 100mM, (American I nvitrigen company produces the reverse transcriptase of 20U, article No. is 18064-014) and 5 μ l10 × RT Buffer (providing with reverse transcriptase), after supplying 50 μ l mixings with water, 42 DEG C are incubated 2 hours, 75 DEG C are incubated 15 minutes, make enzyme deactivation。This reverse transcriptase primer includes the reverse transcriptase primer of miRNA166b gene and the reverse transcriptase primer of outer source reference miR-96 gene, and the sequence of miRNA166b gene is such as shown in SEQ ID NO:1；The reverse transcriptase primer of the miRNA166b gene designed accordingly is such as shown in SEQ ID NO:7；The reverse transcriptase primer sequence of outer source reference miR-96 gene is such as shown in SEQ ID NO:8, and above-mentioned reverse transcriptase primer synthesizes by American I nvitrigen company。Wherein, reverse transcription includes two classes, one class is target gene, the i.e. reverse transcription of miRNA166b gene, the another kind of reverse transcription for outer source reference miR-96 gene (outer source reference miR-96 gene called after ECK gene), two process of reverse-transcription are parallel to carry out, and responded composition is all identical with condition, and only reverse transcriptase primer is inconsistent。Reverse transcription reaction for miRNA166b gene, last base (determining the critical base of primer specificity) of designed reverse transcriptase primer can not with two member's pairings of miRNA166c and miRNA166g in miRNA166 family, thus they can not be carried out reverse transcription, reduce they interference to result。Early-stage Study finds: except miRNA166b, miRNA166c and miRNA166g, other member or infecting in bacterial leaf-blight 24 hours in miRNA166 family, express and also rise more than 2 times, about the 1/20 of expression only miRNA166b, and miRNA166b is infecting after bacterial leaf spot in 24 hours, expression rises more than 4 times, and therefore, rising 2 times is a loose criterion。Therefore, when reverse transcriptase primer designs, deliberately do not distinguish other member of miRNA166 family except miRNA166c and miRNA166g, because they do not disturb the judgement to result。
The expression of real time quantitative PCR method detection reverse transcription product: real-time quantitative PCR includes two classes equally, one class is target gene, the i.e. real-time quantitative of miRNA166b gene, the another kind of real-time quantitative for outer source reference miR-96 gene, the two is parallel carries out, responded composition is all identical with condition, and only primer differs。Primer includes: miRNA166b gene forward primer sequence is such as shown in SEQ ID NO:3；MiRNA166b gene reverse primer sequences is such as shown in SEQ ID NO:4, the reverse primer of the miRNA166b wherein designed and last base (determining the critical base of primer specificity) can not with two member's pairings of miRNA166c and miRNA166g in miRNA166 family, thus they can not be carried out pcr amplification, reduce they interference to result。Early-stage Study finds: except miRNA166b, miRNA166c and miRNA166g, in miRNA166 family other member otherwise infect bacterial leaf-blight 24 little constantly in, express and also rise more than 2 times, about the 1/20 of expression only miRNA166b, and miRNA166b is infecting after bacterial leaf spot in 24 hours, expression rises more than 4 times, and therefore, rising 2 times is a loose criterion。Therefore, when quantification PCR primer designs, deliberately do not distinguish miRNA166 family except miRNA166c and miRNA166g other member they because they do not disturb the judgement to result。Primer also includes: outer source reference miR-96 gene forward primer sequence is such as shown in SEQ ID NO:5；Outer source reference miR-96 gene reverse primer sequences is for such as shown in SEQ ID NO:6。Real-time quantitative PCR primer synthesizes by American I nvitrigen company。
Specifically comprising the following steps that of real time quantitative PCR method takes the 2 above-mentioned reverse transcription product of μ l, 3 μ l concentration are that (primer here refers to forward primer and reverse primer equimolar ratio mixture for the primer of 1 μM, namely the mixture of sequence such as the miRNA166b gene forward primer shown in SEQ ID NO:3 and the sequence such as equimolar ratio of the miRNA166b gene reverse primer shown in SEQ ID NO:4 is referred to, or refer to the mixture of the sequence such as outer source reference miR-96 gene forward primer shown in SEQ ID NO:5 and the sequence such as equimolar ratio of the outer source reference miR-96 gene reverse primer shown in SEQ ID NO:6), quantitative PCR mixture (article No. is QPS-201) and the ROX fluorescence correction dyestuff of 0.4 μ l50 times that 10 μ l Toyobo, Japan produce (are produced by Toyobo, Japan, and provide with QPS-201) mix homogeneously, obtain the second mixed liquor, this second mixed liquor is carried out real-time quantitative PCR detection by following program in ABIStepOne real-time PCR: 50 DEG C 2 minutes；95 DEG C 10 minutes；95 DEG C 45 seconds, 56 DEG C 45 seconds, 66 DEG C 30 seconds, 67 DEG C 30 seconds, totally 45 circulations, wherein, 66 DEG C increase by 0.1 DEG C and 0.2 DEG C respectively in 30 seconds in 30 seconds and 67 DEG C after every circulation primary, collect fluorescence signal in the final step that circulates each time, the power of fluorescence signal for weigh expression number。The result of real-time quantitative is by MicrosoftExcel2010 process, and beyond when data process, source reference miR-96 gene is the reference gene of miRNA166b expression analysis。Result is in Table 1:
Table 1miRNA166b gene relative expression quantity between comparison Oryza sativa L. and Oryza sativa L. to be measured
Note: expression *=2(CT(miRNA166b)-CT(ECK)), wherein, the CT value of CT (miRNA166b) and the miRNA166b gene obtained in the reaction of CT (ECK) respectively real-time quantitative PCR and outer source reference miR-96 gene。
From the data in table 1, it can be seen that the expression maximum of 5 time points that comparison Oryza sativa L. is in the detection time period is 1.12, expression minima is 1.07, and ratio therebetween is 1.13, and this ratio is less than 1.5。Showing, in this experiment, to affect the expression of comparison Oryza sativa L. miRNA166b gene without bacterial leaf-blight or other factors, experiment condition controls better, and experiment is successful, and experimental result can be used for the forecast of bacterial blight of rice。In Oryza sativa L. to be measured, from February 28 to these three days March 1, the expression relative constancy of miRNA166b gene, the condition further illustrating this experiment controls better。To March 2, miRNA166b gene expression in Oryza sativa L. to be measured exceeds 3.32 times than comparison Oryza sativa L., this expression change was maintained March 4, it was shown that detected Oryza sativa L. miRNA166b changes in gene expression to be measured is unlikely to be the factors such as detection error and causes。May determine that, Oryza sativa L. to be measured is likely to infect bacterial leaf spot bacterium, it is necessary to medical treatment。Predicting further accordingly, other Oryza sativa L. in this area's plantation also has the danger of recent infection bacterial leaf-blight, it is necessary to preventing and treating。Starting to March 6, Oryza sativa L. to be measured starts bacterial leaf-blight symptom occur, and to March 12, symptom was quite obvious。Meanwhile, other rice varieties local also occurs in that the mixing of serious bacterial leaf-blight and bacterial stripe endanger, the situation that the appearance of part field is had no harvest。As can be seen here, the expression of miRNA166b gene provided by the invention can infect early stage (in the 24 hours) mark of white leaf bacterium as Oryza sativa L., can be used for whether Oryza sativa L. clearly to be measured catches an illness, and clear and definite bacterial leaf spot disease time and place, it is to avoid the ambiguity of traditional disease forecasting。
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all within the spirit and principles in the present invention, any amendment of making, equivalent replacement, improvement etc., should be included within protection scope of the present invention。
Claims (8)
1. the method utilizing miRNA166b gene forecast bacterial blight of rice, it is characterised in that said method comprising the steps of:
Choose 9311 rice varieties as Oryza sativa L. to be measured and comparison Oryza sativa L.；
Cultivate described Oryza sativa L. to be measured and described comparison Oryza sativa L.；
It is individually separated the total miR-96 gene in described Oryza sativa L. to be measured and described comparison Oryza sativa L.；
Detecting the expression of miRNA166b gene in the described total miR-96 gene in described Oryza sativa L. to be measured and described comparison Oryza sativa L. respectively, the sequence of described miRNA166b gene is such as shown in SEQ ID NO:1；
Expression according to the miRNA166b gene in described Oryza sativa L. to be measured and described comparison Oryza sativa L., judge that whether experiment is successful, if success, then determine whether whether described plant to be measured infects bacterial leaf-blight, described judge experiment whether successfully method as: if the ratio of the expression maximum of the miRNA166b gene of described comparison Oryza sativa L. and expression minima is less than 1.5, then Success in Experiment；If the ratio of the expression maximum of the miRNA166b gene of described comparison Oryza sativa L. and expression minima is be more than or equal to 1.5, then test unsuccessful；
Judge described plant to be measured whether infect the method for bacterial leaf-blight as: if the expression of the miRNA166b gene of the expression of the miRNA166b gene of described Oryza sativa L. to be measured comparison described less than 2 times Oryza sativa L., then described Oryza sativa L. to be measured is not susceptible；If the expression of the miRNA166b gene of the described comparison Oryza sativa L. that the expression of the miRNA166b gene of described Oryza sativa L. to be measured is more than or equal to 2 times, then described Oryza sativa L. to be measured is susceptible。
2. method according to claim 1; it is characterized in that; when the described Oryza sativa L. to be measured of described cultivation and described comparison Oryza sativa L.; described comparison Oryza sativa L. adopts protectiveness cultivation step when cultivation and prevents and treats bacterial leaf-blight; except described protectiveness cultivation step with except preventing and treating bacterial leaf-blight, described comparison Oryza sativa L. is consistent with the maintenance of the cultivation condition of described Oryza sativa L. to be measured。
3. method according to claim 2, it is characterised in that described protectiveness cultivation step includes soil isolation, water source isolation and spatial separation。
4. method according to claim 1, it is characterised in that the morning 8:55～9:05 utilize described Oryza sativa L. to be measured to carry out the separation of described total miR-96 gene with the same area of the identical blade of described comparison Oryza sativa L.。
5. method according to claim 1, it is characterised in that adopt real time quantitative PCR method, detect described miRNA166b gene expression in described Oryza sativa L. to be measured and described comparison Oryza sativa L.。
6. method according to claim 5, it is characterised in that the pre-treatment step of described real time quantitative PCR method includes:
Utilize spectrophotometric determination and obtain the concentration of described total miR-96 gene of separation；
Outer source reference miR-96 gene is added in described total miR-96 gene, obtain the first mixed liquor, the addition of described outer source reference miR-96 gene is the 0.05% of described total miR-96 gene quality, and the sequence of described outer source reference miR-96 gene is such as shown in SEQ ID NO:2；
Respectively 5 ' ends of described total miR-96 gene and described outer source reference miR-96 gene are connected with 3 ' ends, obtain described total miR-96 gene of cyclisation and the mixed liquor of the described outer source reference miR-96 gene of cyclisation；
The mixed liquor of total miR-96 gene of described cyclisation and the outer source reference miR-96 gene of described cyclisation is carried out reverse transcription, and the reverse transcription product obtained is for carrying out described real-time quantitative PCR detection。
7. method according to claim 6, it is characterised in that described employing real time quantitative PCR method, detects described miRNA166b gene expression in described Oryza sativa L. to be measured and described comparison Oryza sativa L., including:
By the ROX fluorescence correction dyestuff mix homogeneously of primer, 10 μ l quantitative PCR mixture and 0.4 μ l50 times that reverse transcription product described in 2 μ l, 3 μ l concentration are 1 μM, obtain the second mixed liquor, being reacted in real-time PCR by described second mixed liquor, described response procedures is: 50 DEG C 2 minutes；95 DEG C 10 minutes；95 DEG C 45 seconds, 56 DEG C 45 seconds, 66 DEG C 30 seconds, 67 DEG C 30 seconds, totally 45 circulations, wherein, 66 DEG C increase by 0.1 DEG C and 0.2 DEG C respectively in 30 seconds in 30 seconds and 67 DEG C after every circulation primary, collect fluorescence signal in the final step that circulates each time, the power of described fluorescence signal for weigh described expression number；
Described primer includes: sequence such as the miRNA166b gene forward primer shown in SEQ ID NO:3, sequence such as the miRNA166b gene reverse primer shown in SEQ ID NO:4, the sequence such as outer source reference miR-96 gene forward primer shown in SEQ ID NO:5 and the sequence such as outer source reference miR-96 gene reverse primer shown in SEQ ID NO:6。
8. method according to claim 6, it is characterised in that the step that the mixed liquor of total miR-96 gene of described cyclisation and the outer source reference miR-96 gene of described cyclisation carries out reverse transcription is included:
Take total miR-96 gene of described cyclisation and the reverse transcriptase of DTT and the 20U of reverse transcriptase primer that the mixed liquor 2 μ l of the outer source reference miR-96 gene of cyclisation, 5 μ l concentration are 1 μM, 2 μ l concentration to be the dNTP of 10mM, 5 μ l concentration be 100mM, after supplying 50 μ l mixings with water, obtain the 4th mixed liquor, described 4th mixed liquor is incubated 2 hours in 42 DEG C, 75 DEG C are incubated 15 minutes, make enzyme deactivation, obtain described reverse transcription product；Described reverse transcriptase primer includes the reverse transcriptase primer of miRNA166b gene and the reverse transcriptase primer of outer source reference miR-96 gene, the reverse transcriptase primer sequence of described miRNA166b gene is such as shown in SEQ ID NO:7, and the reverse transcriptase primer sequence of described outer source reference miR-96 gene is such as shown in SEQ ID NO:8。
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| CN103184286A (en) * | 2013-03-21 | 2013-07-03 | 江汉大学 | Identification method of rice bacterial leaf blight resistance and application of miRNA397a genetic locus |
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| CN103184286A (en) * | 2013-03-21 | 2013-07-03 | 江汉大学 | Identification method of rice bacterial leaf blight resistance and application of miRNA397a genetic locus |
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| Identification of a novel microRNA from rice that targets an alternaticely soliced transcript of the Nramp6 gene involved in pathogen resistance;campo s 等;《New Phytol》;20130429;第199卷(第1期);第212-217页 * |
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