CN104141005B - MiRNA827 gene is utilized to forecast the method for bacterial blight of rice - Google Patents

MiRNA827 gene is utilized to forecast the method for bacterial blight of rice Download PDF

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CN104141005B
CN104141005B CN201410309695.1A CN201410309695A CN104141005B CN 104141005 B CN104141005 B CN 104141005B CN 201410309695 A CN201410309695 A CN 201410309695A CN 104141005 B CN104141005 B CN 104141005B
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王沁
张继
彭海
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Abstract

The invention discloses the method utilizing miRNA827 gene to forecast bacterial blight of rice, belong to biological technical field.Said method comprising the steps of: choose paddy rice to be measured and contrast paddy rice, the described contrast paddy rice chosen is the paddy rice not infecting bacterial leaf-blight but cultivate under the same conditions with paddy rice to be measured; Be separated the total miR-96 gene in described paddy rice to be measured and described contrast paddy rice respectively; Detecting the expression amount of the miRNA827 gene in the total miR-96 gene in described paddy rice to be measured and described contrast paddy rice respectively, is the sequence of described miRNA827 gene as SEQ in sequence table? ID? shown in NO:1; According to the expression amount of the miRNA827 gene in described paddy rice to be measured and described contrast paddy rice, whether judgment 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, can bacterial blight of rice disease occur before a couple of days realize accurate forecast, for morning anti-morning control the time won, decrease the loss that bacterial leaf-blight causes paddy rice.

Description

MiRNA827 gene is utilized to forecast the method for bacterial blight of rice
Technical field
The present invention relates to biological technical field, particularly utilize miRNA827 gene to forecast the method for bacterial blight of rice.
Background technology
Paddy rice is China's staple food crop, and bacterial leaf-blight is one of large Major Diseases of paddy rice two, belongs to worldwide disease, and Rice In Asian Cultivated Rice district occurs outstanding heavy.Bacterial leaf-blight sickness rate is high, it is fast to infect, morbidity rice field underproduction 20-30%, and even 50%.The same with most of disease, bacterial leaf-blight morbidity early prevention and treatment effect is better.The morbidity middle and later periods, germ is amount reproduction, causes injury to paddy rice, 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. are inferred, also according to field water bacterial blight of rice Symptoms, can forecast later stage PD.
Realizing in process of the present invention, contriver finds that prior art at least exists following problem:
Very fuzzy according to the forecast result that weather, weather, varietal resistance, Planting status and history etc. are carried out, the probability that disease occurs in certain area and time range can be described, but can not forecast the concrete time that disease occurs and place, the applicability of forecast result is poor.Such as, the weather condition of the epidemics forecast of hot and humid Chang Zuowei bacterial blight of rice, but can not determine whether hot and humid lower disease necessarily occurs, occurs in that block field, occurs in that day.Due to above uncertainty, peasant can not determine whether start to prevent and treat bacterial leaf spot, prevent and treat which block field and when prevent and treat.Meanwhile, illness occurs showing that bacterial leaf-blight enters the middle and later periods, and germ is amount reproduction, and bacterial leaf-blight popular being difficult to is avoided, and makes the work utilizing illness investigation to carry out forecasting also without too large practical significance.
Summary of the invention
In order to solve, bacterial blight of rice in prior art is forecast not in time, inaccurate shortcoming, embodiments provides a kind of method utilizing miRNA827 gene to forecast bacterial blight of rice.Described technical scheme is as follows:
Choose 9311/Xa23 rice varieties as paddy rice to be measured and contrast paddy rice;
Cultivate described paddy rice to be measured and described contrast paddy rice;
Be separated the total miR-96 gene in described paddy rice to be measured and described contrast paddy rice respectively;
Detect the expression amount of the miRNA827 gene in the described total miR-96 gene in described paddy rice to be measured and described contrast paddy rice respectively, the sequence of described miRNA827 gene is as shown in SEQ ID NO:1;
According to the expression amount of the miRNA827 gene in described paddy rice to be measured and described contrast paddy rice, judge that whether experiment is successful, if success, then judge whether described plant to be measured infects bacterial leaf-blight further, wherein, described judge to test whether successfully method as: if the expression amount maximum value of miRNA827 gene of described contrast paddy rice and the ratio of expression amount minimum value are less than 1.5, then Success in Experiment; If the expression amount maximum value of miRNA827 gene and the ratio of expression amount minimum value of described contrast paddy rice are more than or equal to 1.5, then test unsuccessful;
Judge method that whether described plant to be measured infect bacterial leaf-blight as: if the expression amount of the miRNA827 gene of described paddy rice to be measured is greater than the expression amounts of the miRNA827 gene of 2 times of described contrast paddy rice, then described paddy rice to be measured is not susceptible; If the expression amount of the miRNA827 gene of described paddy rice to be measured is less than or equal to the expression amount of the miRNA827 gene of the described contrast paddy rice of 2 times, then described paddy rice to be measured is susceptible.
Particularly; when the described paddy rice to be measured of described cultivation and described contrast paddy rice; described contrast paddy rice adopts protectiveness cultivation step when cultivating and prevents and treats bacterial leaf-blight; except described protectiveness cultivation step and prevent and treat except bacterial leaf-blight, the cultivation condition of described contrast paddy rice and described paddy rice to be measured is consistent.
Further, described protectiveness cultivation step comprises soil isolation, water source isolation and spatial separation.
Particularly, the morning 8:55 ~ 9:05 utilize described paddy rice to be measured to carry out being separated of described total miR-96 gene with the same area of the identical blade of described contrast paddy rice.
Particularly, adopt real time quantitative PCR method, detect the expression amount of described miRNA827 gene in described paddy rice to be measured and described contrast paddy rice.
Further, the pre-treatment step of described real time quantitative PCR method comprises:
Utilize spectrophotometric determination and the concentration of described total miR-96 gene of acquisition separation;
Outer source reference miR-96 gene is added in described total miR-96 gene, obtain the first mixed solution, the add-on of described outer source reference miR-96 gene is 0.05% of described total miR-96 gene quality, and the sequence of described outer source reference miR-96 gene is as shown in SEQ ID NO:2;
Described total miR-96 gene is held with the 5 ' end and 3 ' of described outer source reference miR-96 gene and is connected, obtain the mixed solution of described total miR-96 gene of cyclisation and the described outer source reference miR-96 gene of cyclisation;
The mixed solution of the outer source reference miR-96 gene of total miR-96 gene of described cyclisation and 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, detects the expression amount of described miRNA827 gene in described paddy rice to be measured and described contrast paddy rice, comprising:
Be that the primer of 1 μM, 10 μ l quantitative PCR mixtures and 0.4 μ l50 ROX fluorescence correction dyestuff doubly mix by reverse transcription product described in 2 μ l, 3 μ l concentration, obtain the second mixed solution, reacted in real-time PCR by described second mixed solution, 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 fluorescent signal in the final step that circulating each time, the power of described fluorescent signal for weigh described expression amount number;
Described primer comprises: outer source reference miR-96 gene forward primer as shown in SEQ ID NO:5 of the miRNA827 gene forward primer of sequence as shown in SEQ ID NO:3, the sequence miRNA827 gene reverse primer as shown in SEQ ID NO:4, sequence and the outer source reference miR-96 gene reverse primer of sequence as shown in SEQ ID NO:6.
Further, the step be connected is held to comprise with 3 ' at 5 ' of described total miR-96 gene end:
Get the MnCl of the first mixed solution described in 5ng, 2 μ l10 × reaction buffers, 1 μ l50mM 2, the trimethyl-glycine of 4 μ l5M and the cyclase of 1 μ l5u/ μ l, mix after supplying 20 μ l with water, obtain the 3rd mixed solution, after described 3rd mixed solution is incubated 15 minutes in 60 DEG C, 80 DEG C are incubated 10 minutes, make enzyme deactivation, obtain the mixed solution of total miR-96 gene of described cyclisation and the outer source reference miR-96 gene of cyclisation.
Further, the step of the mixing liquid of the outer source reference miR-96 gene of total miR-96 gene of described cyclisation and cyclisation being carried out reverse transcription comprises:
Get the mixed solution 2 μ l of total miR-96 gene of described cyclisation and the outer source reference miR-96 gene of described cyclisation, reversed transcriptive enzyme that dNTP that reverse transcriptase primer that 5 μ l concentration are 1 μM, 2 μ l concentration are 10mM, 5 μ l concentration are DTT and 20U of 100mM, after supplying 50 μ l mixings with water, obtain the 4th mixed solution, by described 4th mixed solution in 42 DEG C of insulations 2 hours, 75 DEG C are incubated 15 minutes, make enzyme deactivation, obtain described reverse transcription product; Described reverse transcriptase primer comprises the reverse transcriptase primer of miRNA827 gene and the reverse transcriptase primer of outer source reference miR-96 gene, the reverse transcriptase primer sequence of described miRNA827 gene is as shown in SEQ ID NO:7, and the reverse transcriptase primer sequence of described outer source reference miR-96 gene is as shown in SEQ ID NO:8.
The beneficial effect that the technical scheme that the embodiment of the present invention provides is brought is: the method utilizing miRNA827 gene to forecast bacterial blight of rice provided by the invention, the expression of miRNA827 gene change can be can be used as the mark that paddy rice infects hoja blanca bacterium, whether catch an illness in order to paddy rice clearly to be measured, and the time of clearly falling ill and field, avoid the ambiguity of traditional disease forecasting.Meanwhile, predicted time of the present invention early, can infect bacterial leaf-blight in 24 hours in paddy rice and make forecast.
Embodiment
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, all can buy and effect almost indifference in most of biotech company.
Embodiment
The embodiment of the present invention is using 9311/Xa23 rice varieties as paddy rice to be measured and contrast paddy rice, and wherein, paddy rice to be measured refers to be planted under the normal cultivation condition in land for growing field crops, needs monitoring whether to infect the paddy rice of bacterial leaf-blight; Contrast paddy rice refers to by protectiveness technical measures and bacterial leaf-blight prophylactico-therapeutic measures; guarantee that it is the healthy paddy rice not infecting bacterial leaf-blight; except protectiveness cultivation step and prevent and treat except bacterial leaf-blight; the cultivation condition of contrast paddy rice and paddy rice to be measured is consistent; wherein, protectiveness cultivation step comprises soil isolation, water source isolation and spatial separation.
Choosing of seed: the seed of paddy rice to be measured and the seed of contrast paddy rice are all harvested from healthy 9311/Xa23 rice plant, and seed-coat is without obvious disease and pest hazard symptoms.9311/Xa23 rice varieties is the rice varieties formed after Xa23 gene being proceeded to 9311 paddy rice by back cross breeding, and concrete preparation process is: utilize the donor parents CBB23 containing Xa23 gene and receptor parent 9311 to hybridize and obtain F1 generation plant; Utilize 9311 for recurrent parent, backcross with the F1 generation plant obtained, obtain BC 1f 1colony; To BC 1f 1colony inoculation bacterial leaf spot bacterium, identifies its disease resistance, from BC 1f 1select resisting bacterial leaf-blight in colony and the individual plant the most similar to 9311, hybridize with 9311, obtain BC 2f 1colony; Again to BC 2f 1colony inoculation bacterial leaf spot bacterium, identifies its disease resistance, from BC 2f 1select resisting bacterial leaf-blight in colony and the individual plant the most similar to 9311, hybridize with 9311, obtain BC 3f 1colony; Circulation like this, until obtain BC 6f 1colony, then to BC 6f 1colony inoculation bacterial leaf spot bacterium, identifies its disease resistance, from BC 6f 1select resisting bacterial leaf-blight in colony and the individual plant selfing the most similar to 9311, gather in the crops seed from the individual plant of each selfing, obtain S1 colony; To S1 colony inoculation bacterial leaf spot bacterium, identify its disease resistance, filter out resistance unseparated S1 colony (i.e. each individual plant resisting bacterial leaf-blight in colony) and the individual plant the most similar to 9311, be and cultivate successful 9311/Xa23 kind.
The pre-treatment of seed, sowing and cultivation condition: contrast alcohol-pickled 2 minutes that seed and seed to be measured by concentration are all 70%, 2 times are washed again with deionized water, soaked overnight in the water of 30 DEG C, carries out vernalization to seed, sows after seed germination at the temperature of 30 DEG C.
Fetch earth earth from the field of paddy growth to be measured, (Shanghai Medical Equipment Plant of Bo Xun Industrial Co., Ltd. produces to utilize Autoclave, model is YSQ-LS-50S11) high-temperature high-voltage sterilizing sterilizing is carried out to soil, sterilization condition is: 120 DEG C, 30 minutes.The planting soil of the paddy rice in contrast of the soil after sterilization.Contrast paddy rice is sowed, transplants, is grown on and is filled with in the plastic tub of disinfection soil under isolation condition; paddy rice to be measured is then planted in the environment of land for growing field crops; when cultivating paddy rice to be measured and contrast paddy rice; except protectiveness cultivation step and bacterial leaf-blight prophylactico-therapeutic measures, the cultivation condition of paddy rice to be measured and paddy rice to be measured is consistent.
Protectiveness cultivation step comprises: (1) soil is isolated: utilize potted plant, the soil of contrast paddy growth and field soil are isolated, and avoids passing sick infection bacterial leaf-blight by soil; (2) water source isolation: adopt tap water to irrigate contrast paddy rice, guarantees that water source, land for growing field crops does not enter contrast paddy rice, avoids infecting bacterial leaf-blight by water source; (3) spatial separation: do not plant other paddy rice for 10 meters around contrast paddy rice, avoiding rubs with the rice leaf with bacterial leaf spot bacterium infects bacterial leaf-blight.Paddy rice to be measured does not prevent and treat bacterial leaf-blight; the cultivation condition contrasting paddy rice and paddy rice to be measured except protectiveness cultivation step except bacterial leaf-blight prophylactico-therapeutic measures is consistent as far as possible, comprises the production management measure such as sowing, transplanting, fertilising, insect protected, diseases prevention (except bacterial leaf-blight other disease) simultaneously, synchronously carry out.
Particularly, around contrast paddy rice within the scope of 10 meters, do not sow or plant other paddy rice; The clean tap water of contrast Rice irrigation hydromining; Except above condition, contrast paddy rice is identical with common land for growing field crops conventional cultivation condition with other cultivation condition of paddy rice to be measured, but all cultivation steps are consistent between contrast paddy rice and paddy rice to be measured, as the production management measure such as sowing, transplanting, fertilising, insect protected, diseases prevention (except hoja blanca other disease) while, synchronously carry out.In this test, sowing time is on November 27th, 2010, and place is Qionghai.
Contrast paddy rice and paddy rice to be measured are adopted identical cultivation condition plantation, contrast paddy rice and paddy rice to be measured are grown at identical conditions, ensure that the expression amount of contrast paddy rice and paddy rice to be measured is not by the impact of cultivation condition.
Needing to forecast bacterial leaf-blight period of whether occurring, if seedling stage and cut phase are to heading flowering period, the morning 8:55 ~ 9:05 get the blade at the position at second from the bottom middle part 2/3 respectively.Each contrast paddy rice and paddy rice to be measured are respectively taken to few 3 blades and mix, for representing the sample of contrast paddy rice and paddy rice to be measured.Get blade and be placed in the extraction that liquid nitrogen is saved to total miR-96 gene immediately.In the present embodiment, sample time, section was March 3 extremely then on February 28th, 2011, serial sampling 5 days, obtain contrast paddy rice and each 5 samples of paddy rice to be measured altogether, be numbered C1 ~ C5 and T1 ~ T5 respectively, wherein C represents Control, T represents Treatment, wherein, contrast paddy rice is consistent with the time point that paddy rice to be measured samples, contrast paddy rice and the leaf position selected by paddy rice to be measured are also identical, this can make the circadian clock body clock between contrast paddy rice and paddy rice to be measured and developmental condition be identical, only choose the circadian clock body clock blade all identical with developmental condition and could ensure that the miRNA expression amount contrasted between paddy rice and paddy rice to be measured is not more subject to the impact of circadian clock body clock and growth further.
Be separated 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 comprises: RNA centrifugal column, genomic dna remove centrifugal column, centrifuge tube, MCL lysis buffer, XD binding buffer liquid, RNA elutriant II and DEPC water) be separated total miR-96 gene in above-mentioned C1 ~ C5 and T1 ~ T5 rice leaf.
Concrete operation method is as follows: from liquid nitrogen, take out C1 ~ C5 and T1 ~ T5 rice leaf sample respectively, be placed in 10 mortars respectively, and in mortar, pour liquid nitrogen into immediately, fully grind, and grinding can avoid crossed contamination respectively.The centrifuge tube that the ground powder of about 100mg is placed in 1.5ml is got in each mortar, add the lysate of 700 μ L, vortex 30 seconds is to mix sample, 55 DEG C are incubated 30 minutes, be under the room temperature condition of 12000 × g centrifugal 5 minutes at centrifugal force, obtain supernatant liquor, and supernatant liquor is transferred in centrifugal column centrifugal, for removing the DNA in genome, through 12000 × g room temperature centrifugal 2 minutes, and the liquid rotating of outflow is moved in the centrifuge tube of a new 1.5mL, the dehydrated alcohol of this liquid volume 1.1 times is added and vortex mixing in 20 seconds in the liquid of this outflow, liquid rotating after this vortex to be moved in RNA centrifugal column through 12000 × g room temperature centrifugal 1 minute, abandon centrifugate, adding 500 μ L concentration is that the ethanol of 96-100% is in RNA centrifugal column, through 12000 × g room temperature centrifugal 1 minute again, abandon centrifugate, add 500 μ LXD binding buffer liquid in RNA centrifugal column, centrifugal 1 minute of 12000 × g room temperature, abandon centrifugate, add in 750 μ LRNA elutriant II to RNA centrifugal columns, centrifugal 1 minute of 12000 × g room temperature, abandon centrifugate, add in 750 μ LRNA elutriant II to RNA centrifugal columns again, centrifugal 1 minute of 12000 × g room temperature, abandon centrifugate, by RNA centrifugal column under the top speed being greater 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 top speed being greater than 12000 × g, centrifugal 1 minute of room temperature, the liquid of centrifugal acquisition is the solution containing total miR-96 gene, this solution is stored in-70 DEG C for subsequent use.
The pre-treatment step of real-time quantitative PCR (polymerasechainreaction, polymerase chain reaction) method comprises:
Total miR-96 gene quantitative: (spectrophotometer is produced by Quawell company of the U.S. to utilize spectrophotometer, model is Q5000) middle RNA quant program, measure and obtain the concentration of total miR-96 gene be separated, according to the quality of concentration and the total miR-96 gene of volume computing.
Outer source reference miR-96 gene is added in the total miR-96 gene be separated, obtain the first mixed solution: the add-on of outer source reference miR-96 gene is 0.05% of the total miR-96 gene quality be separated, the sequence of outer source reference miR-96 gene is as shown in SEQ ID NO:2, and it is synthesized by Shanghai JiMa pharmacy Technology Co., Ltd.
MiR-96 gene cyclisation: get the MnCl containing the first mixed solution of the total miR-96 gene of the 5ng that has an appointment, 2 μ l10 × reaction buffers, 1 μ l50mM 2, the trimethyl-glycine of 4 μ l5M and the cyclase of 1 μ l5u/ μ l, mix after supplying 20 μ l with water, obtain the 3rd mixed solution; After 3rd mixed solution is incubated 15 minutes in 60 DEG C, 80 DEG C are incubated 10 minutes, make enzyme deactivation.Total miR-96 gene held with the 5 ' end and 3 ' of outer source reference miR-96 gene and be connected, obtain the mixed solution of total miR-96 gene of cyclisation and the outer source reference miR-96 gene of cyclisation, this mixed solution is used for rolling ring reverse transcription.Wherein, cyclase is produced by Epicentre company of the U.S., and article No. is CL9021K.What provide with this enzyme also has: the MnCl of 10 × reaction buffer, 50mM 2, 5M trimethyl-glycine and without enzyme water.
The reverse transcription of the mixed solution of total miR-96 gene of cyclisation and the outer source reference miR-96 gene of cyclisation: the mixed solution 2 μ l getting total miR-96 gene of cyclisation and 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 reversed transcriptive enzyme of 20U, article No. is 18064-014) and 5 μ l10 × RT Buffer (providing with reversed transcriptive enzyme), 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 comprises the reverse transcriptase primer of miRNA827 gene and the reverse transcriptase primer of outer source reference miR-96 gene, and the sequence of miRNA827 gene is as shown in SEQ ID NO:1; The reverse transcriptase primer of the miRNA827 gene designed accordingly is as shown in SEQ ID NO:7; The reverse transcriptase primer sequence of outer source reference miR-96 gene is as shown in SEQ ID NO:8, and above-mentioned reverse transcriptase primer synthesizes by American I nvitrigen company.Wherein, reverse transcription comprises two classes, one class is target gene, the i.e. reverse transcription of miRNA827 gene, another kind of is the reverse transcription of 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 all reaction compositions are all identical with condition, and only reverse transcriptase primer is inconsistent.For the reverse transcription reaction of miRNA827 gene, designed reverse transcriptase primer has striden across the connection contact during cyclisation of miRNA827 gene, therefore, when reverse transcription, only the successful miRNA827 gene of cyclisation is reversed record, thus reduces the interference of other genes.
Real time quantitative PCR method detects the expression amount of reverse transcription product: real-time quantitative PCR comprises two classes equally, one class is target gene, the i.e. real-time quantitative of miRNA827 gene, another kind of is the real-time quantitative of outer source reference miR-96 gene, the two is parallel carries out, all reaction compositions are all identical with condition, and only primer is not identical.Primer comprises: miRNA827 gene forward primer sequence is as shown in SEQ ID NO:3; MiRNA827 gene reverse primer sequences is as shown in SEQ ID NO:4.Primer also comprises: outer source reference miR-96 gene forward primer sequence is 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.
The concrete steps of real time quantitative PCR method are as follows: get the above-mentioned reverse transcription product of 2 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 the equimolar ratio of the miRNA827 gene forward primer of sequence as shown in SEQ ID NO:3 and the miRNA827 gene reverse primer of sequence as shown in SEQ ID NO:4 is referred to, or refer to the mixture of the equimolar ratio of the outer source reference miR-96 gene forward primer of sequence as shown in SEQ ID NO:5 and the outer source reference miR-96 gene reverse primer of sequence as shown in SEQ ID NO:6), the quantitative PCR mixture (article No. is QPS-201) that 10 μ l Toyobo, Japan produce and 0.4 μ l50 ROX fluorescence correction dyestuff doubly (are produced by Toyobo, Japan, and provide with QPS-201) mix, obtain the second mixed solution, this second mixed solution 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 fluorescent signal in the final step that circulating each time, the power of fluorescent signal for weigh expression amount number.The result of real-time quantitative is by MicrosoftExcel2010 process, and beyond during data processing, source reference miR-96 gene is the reference gene of miRNA827 expression analysis.The results are shown in Table 1:
The relative expression quantity of table 1miRNA827 gene between contrast paddy rice and paddy rice to be measured
Note: expression amount *=2 (CT (miRNA827)-CT (ECK)), wherein, CT (miRNA827) and CT (ECK) is respectively the CT value of miRNA827 gene and the outer source reference miR-96 gene obtained in real-time quantitative PCR reaction.
From table 1 data, the expression amount maximum value of 5 time points of contrast paddy rice in detection time section is 8.01, and expression amount minimum value is 6.89, and ratio is therebetween 1.16, and this ratio is less than 1.5.Show in this experiment, contrast the expression of paddy rice miRNA827 gene without bacterial leaf-blight or other factors impact, experiment condition controls better, and experiment is successful, and experimental result can be used for the forecast of bacterial blight of rice.In paddy rice to be measured, from February 28 to these three days March 1, the expression amount relative constancy of miRNA827 gene, the condition further illustrating this experiment controls better.To March 2, the expression amount of miRNA827 gene in paddy rice to be measured is lower more than 2 times than contrast paddy rice, this expression change was maintained March 4, showed that detected paddy rice miRNA827 changes in gene expression to be measured is unlikely detect the factors such as error to cause.Can judge, paddy rice to be measured probably infects bacterial leaf spot bacterium, needs medical treatment.Predict also there is the danger of recent infection bacterial leaf-blight in other paddy rice of this area's plantation, needs control accordingly further.Started to March 6, paddy rice to be measured starts to occur bacterial leaf-blight symptom, and to March 12, symptom was quite obvious.Meanwhile, other rice varieties local has also occurred that serious bacterial leaf-blight endangered with mixing of bacterial stripe, and the situation of total crop failure appears in part field.As can be seen here, the expression amount of miRNA827 gene provided by the invention can be used as early stage (in 24 hours) mark that paddy rice infects white leaf bacterium, can be used for paddy rice clearly to be measured whether to catch an illness, and clear and definite bacterial leaf spot disease time and place, avoid the ambiguity of traditional disease forecasting.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.

Claims (8)

1. utilize miRNA827 gene to forecast a method for bacterial blight of rice, it is characterized in that, said method comprising the steps of:
Choose 9311/Xa23 rice varieties as paddy rice to be measured and contrast paddy rice;
Cultivate described paddy rice to be measured and described contrast paddy rice;
Be separated the total miR-96 gene in described paddy rice to be measured and described contrast paddy rice respectively;
Detect the expression amount of the miRNA827 gene in the described total miR-96 gene in described paddy rice to be measured and described contrast paddy rice respectively, the sequence of described miRNA827 gene is as shown in SEQ ID NO:1;
According to the expression amount of the miRNA827 gene in described paddy rice to be measured and described contrast paddy rice, judge that whether experiment is successful, if success, then judge whether described plant to be measured infects bacterial leaf-blight further, wherein, described judge to test whether successfully method as: if the expression amount maximum value of miRNA827 gene of described contrast paddy rice and the ratio of expression amount minimum value are less than 1.5, then Success in Experiment; If the expression amount maximum value of miRNA827 gene and the ratio of expression amount minimum value of described contrast paddy rice are more than or equal to 1.5, then test unsuccessful;
Judge method that whether described plant to be measured infect bacterial leaf-blight as: if the expression amount of the miRNA827 gene of described paddy rice to be measured is greater than the expression amounts of the miRNA827 gene of 2 times of described contrast paddy rice, then described paddy rice to be measured is not susceptible; If the expression amount of the miRNA827 gene of described paddy rice to be measured is less than or equal to the expression amount of the miRNA827 gene of the described contrast paddy rice of 2 times, then described paddy rice to be measured is susceptible.
2. method according to claim 1; it is characterized in that; when the described paddy rice to be measured of described cultivation and described contrast paddy rice; described contrast paddy rice adopts protectiveness cultivation step when cultivating and prevents and treats bacterial leaf-blight; except described protectiveness cultivation step and prevent and treat except bacterial leaf-blight, the cultivation condition of described contrast paddy rice and described paddy rice to be measured is consistent.
3. method according to claim 2, is characterized in that, described protectiveness cultivation step comprises soil isolation, water source isolation and spatial separation.
4. method according to claim 1, is characterized in that, the morning 8:55 ~ 9:05 utilize described paddy rice to be measured to carry out being separated of described total miR-96 gene with the same area of the identical blade of described contrast paddy rice.
5. method according to claim 1, is characterized in that, adopts real time quantitative PCR method, detects the expression amount of described miRNA827 gene in described paddy rice to be measured and described contrast paddy rice.
6. method according to claim 5, is characterized in that, the pre-treatment step of described real time quantitative PCR method comprises:
Utilize spectrophotometric determination and the concentration of described total miR-96 gene of acquisition separation;
Outer source reference miR-96 gene is added in described total miR-96 gene, obtain the first mixed solution, the add-on of described outer source reference miR-96 gene is 0.05% of described total miR-96 gene quality, and the sequence of described outer source reference miR-96 gene is as shown in SEQ ID NO:2;
Respectively described total miR-96 gene is held with the 5 ' end and 3 ' of described outer source reference miR-96 gene and be connected, obtain the mixed solution of described total miR-96 gene of cyclisation and the described outer source reference miR-96 gene of cyclisation;
The mixed solution of the outer source reference miR-96 gene of total miR-96 gene of described cyclisation and 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, is characterized in that, described employing real time quantitative PCR method, detects the expression amount of described miRNA827 gene in described paddy rice to be measured and described contrast paddy rice, comprising:
Be that the primer of 1 μM, 10 μ l quantitative PCR mixtures and 0.4 μ l50 ROX fluorescence correction dyestuff doubly mix by reverse transcription product described in 2 μ l, 3 μ l concentration, obtain the second mixed solution, reacted in real-time PCR by described second mixed solution, 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 fluorescent signal in the final step that circulating each time, the power of described fluorescent signal for weigh described expression amount number;
Described primer comprises: outer source reference miR-96 gene forward primer as shown in SEQ ID NO:5 of the miRNA827 gene forward primer of sequence as shown in SEQ ID NO:3, the sequence miRNA827 gene reverse primer as shown in SEQ ID NO:4, sequence and the outer source reference miR-96 gene reverse primer of sequence as shown in SEQ ID NO:6.
8. method according to claim 6, is characterized in that, the step that the mixed solution of the outer source reference miR-96 gene of total miR-96 gene of described cyclisation and described cyclisation carries out reverse transcription is comprised:
Get the mixed solution 2 μ l of total miR-96 gene of described cyclisation and the outer source reference miR-96 gene of cyclisation, reversed transcriptive enzyme that dNTP that reverse transcriptase primer that 5 μ l concentration are 1 μM, 2 μ l concentration are 10mM, 5 μ l concentration are DTT and 20U of 100mM, after supplying 50 μ l mixings with water, obtain the 4th mixed solution, by described 4th mixed solution in 42 DEG C of insulations 2 hours, 75 DEG C are incubated 15 minutes, make enzyme deactivation, obtain described reverse transcription product; Described reverse transcriptase primer comprises the reverse transcriptase primer of miRNA827 gene and the reverse transcriptase primer of outer source reference miR-96 gene, the reverse transcriptase primer sequence of described miRNA827 gene is as shown in SEQ ID NO:7, and the reverse transcriptase primer sequence of described outer source reference miR-96 gene is as shown in SEQ ID NO:8.
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