CN104131077B - Utilize the method for miRNA408 gene forecast bacterial blight of rice - Google Patents
Utilize the method for miRNA408 gene forecast bacterial blight of rice Download PDFInfo
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Abstract
The present invention discloses the method utilizing miRNA408 gene forecast bacterial blight of rice, belongs to biological technical field. Described method comprises the following steps: choose paddy rice to be measured and comparison paddy rice, and the described comparison paddy rice chosen is the paddy rice not infecting bacterial leaf-blight but cultivating under the same conditions with paddy rice to be measured; The total miR-96 gene being separated in described paddy rice to be measured and described comparison paddy rice respectively; Detecting the expression amount of the miRNA408 gene in the total miR-96 gene in described paddy rice to be measured and described comparison paddy rice respectively, is the sequence of described miRNA408 gene such as SEQ in sequence table? ID? shown in NO:1; Expression amount according to the miRNA408 gene in described paddy rice to be measured and described comparison 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, it is possible within several days before bacterial blight of rice disease occurs, realizes accurately forecast, for early controlling the time won, decreases the loss that paddy rice is caused by bacterial leaf-blight anti-morning.
Description
Technical field
The present invention relates to biological technical field, in particular to the method utilizing miRNA408 gene forecast bacterial blight of rice.
Background technology
Paddy rice is China's main grain food crop, and bacterial leaf-blight is one of big main disease of paddy rice two, belongs to worldwide disease, and Asia Dao Qu occurs outstanding heavy. Bacterial leaf-blight sickness rate height, infect fast, morbidity rice field underproduction 20-30%, even 50%.The same with major part disease, bacterial leaf-blight morbidity early prevention and treatment effect is better. The morbidity middle and later periods, germ breeds in a large number, paddy rice is caused injury, and prevention effect is poor. It thus is seen that early prediction is the key point of bacterial blight of rice prevention.
Tradition bacterial blight of rice forecast is main to be speculated according to weather, weather, kind resistance, nitrogen application situation and disease history etc., it is possible to according to the performance of field water bacterial blight of rice symptom, later stage state of an illness development is forecast.
In the process realizing the present invention, contriver finds that prior art at least exists following problem:
It is very fuzzy according to the forecast result that weather, weather, kind resistance, Planting status and history etc. carry out, the probability that disease occurs in certain region and time range can be described, but can not forecasting 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, which block field is prevented and treated and when prevented and treated. Meanwhile, illness occurs showing that bacterial leaf-blight has entered the middle and later periods, and germ breeds in a large number, and bacterial leaf-blight is popular to be difficult to avoid so that utilize illness to investigate the work carrying out forecasting also without too big actual meaning.
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 miRNA408 gene forecast bacterial blight of rice. Described technical scheme is as follows:
Choose 9311/Xa23 rice varieties as paddy rice to be measured and comparison paddy rice;
Cultivate described paddy rice to be measured and described comparison paddy rice;
The total miR-96 gene being separated in described paddy rice to be measured and described comparison paddy rice respectively;
The expression amount of paddy rice to be measured described in repeated detection and the miRNA408 gene in the described total miR-96 gene in described comparison paddy rice respectively, the sequence of described miRNA408 gene is as shown in SEQ ID NO:1;
Expression amount according to the miRNA408 gene in described paddy rice to be measured and described comparison 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 miRNA408 gene of described comparison 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 miRNA408 gene of described comparison paddy rice and the ratio of expression amount minimum value 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 miRNA408 gene of described paddy rice to be measured is greater than the expression amount of the miRNA408 genes of 4 times of described comparison paddy rice, then described paddy rice to be measured is not susceptible; If the expression amount of the miRNA408 gene of described paddy rice to be measured is less than or equals the expression amount of the miRNA408 gene of the described comparison paddy rice of 4 times, then described paddy rice to be measured is susceptible.
Specifically; when the described paddy rice to be measured of described cultivation and described comparison paddy rice; described comparison 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 comparison 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.
Specifically, 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 comparison paddy rice.
Specifically, adopt real time quantitative PCR method, detect the expression amount of described miRNA408 gene in described paddy rice to be measured and described comparison paddy rice.
Further, the pre-treatment step of described real time quantitative PCR method comprises:
Utilize spectrophotometric determination and obtain the concentration of described total miR-96 gene of separation;
External source is added with reference to miR-96 gene in described total miR-96 gene, obtain the first mixed solution, described external source is the 0.05% of described total miR-96 gene quality with reference to the add-on of miR-96 gene, and the sequence of described external source reference miR-96 gene is as shown in SEQ ID NO:2;
5 ' end of described total miR-96 gene and described external source reference miR-96 gene is connected with 3 ' end, obtains described total miR-96 gene of cyclisation and the mixed solution of the described external source reference miR-96 gene of cyclisation;
The mixed solution of total miR-96 gene of described cyclisation and the external 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, detects the expression amount of described miRNA408 gene in described paddy rice to be measured and described comparison paddy rice, comprising:
By reverse transcription product described in 2 �� l, 3 �� l concentration be the primer of 1 ��M, 10 �� l quantitative PCR mixtures and 0.4 �� l50 ROX fluorescence correction dyestuff doubly mix, obtain the 2nd mixed solution, being reacted in real-time PCR by described 2nd 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: miRNA408 gene reverse primer as shown in SEQ ID NO:4 of the miRNA408 gene forward primer of sequence as shown in SEQ ID NO:3, sequence, the sequence external source as shown in SEQ ID NO:5 with reference to external source as shown in SEQ ID NO:6 of miR-96 gene forward primer and sequence with reference to miR-96 gene reverse primer.
Further, the step that 5 ' end of described total miR-96 gene is connected with 3 ' end is comprised:
Get the MnCl of the first mixed solution described in 5ng, 2 �� l10 �� reaction buffers, 1 �� l50mM2, the trimethyl-glycine of 4 �� l5M and the cyclase of 1 �� l5u/ �� l, mix even after supplying 20 �� 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 total miR-96 gene of described cyclisation and the mixed solution of the external source reference miR-96 gene of cyclisation.
Further, the step mixing liquid of total miR-96 gene of described cyclisation and the external source reference miR-96 gene of cyclisation carrying out reverse transcription comprises:
Get the mixed solution 2 �� l of external source with reference to miR-96 gene of total miR-96 gene of described cyclisation and described cyclisation, the reversed transcriptive enzyme of DTT and 20U that dNTP that reverse transcriptase primer that 5 �� l concentration are 1 ��M, 2 �� l concentration are 10mM, 5 �� l concentration are 100mM, with water supply 50 �� l mix even after, obtain the 4th mixed solution, described 4th mixed solution 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 comprises the reverse transcriptase primer of miRNA408 gene and the reverse transcriptase primer of external source reference miR-96 gene, the reverse transcriptase primer sequence of described miRNA408 gene is as shown in SEQ ID NO:7, and the reverse transcriptase primer sequence of described external source reference miR-96 gene is as shown in SEQ ID NO:8.
The useful effect that the technical scheme that the embodiment of the present invention provides is brought is: the method utilizing miRNA408 gene forecast bacterial blight of rice provided by the invention, the expression change of miRNA408 gene can be can be used as the mark that paddy rice infects hoja blanca bacterium, in order to whether paddy rice clearly to be measured catches an illness, and the time clearly fallen ill and field, avoid the fuzzy property of tradition disease forecast. Meanwhile, the predicted time of the present invention early, can infect bacterial leaf-blight in paddy rice and make forecast in 24 hours.
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 comparison paddy rice, and wherein, paddy rice to be measured refers to be planted under the normal cultivation condition in land for growing field crops, it is necessary to whether monitoring infects the paddy rice of bacterial leaf-blight; Comparison 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 comparison 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 the seed of paddy rice to be measured and comparison paddy rice is all harvested from healthy 9311/Xa23 rice plant, and seed-coat is without obvious disease and pest hazard symptoms. Wherein, 9311/Xa23 rice varieties is the kind formed after Xa23 gene having been proceeded to 9311 paddy rice by the method for back cross breeding, and concrete preparation process is: utilize the donor parents CBB23 containing Xa23 gene and receptor parent 9311 hybridization to obtain F1 generation plant; Utilize 9311 for recurrent parent, backcross with the F1 generation plant obtained, obtain BC1F1Colony; To BC1F1Colony inoculation bacterial leaf spot bacterium, identifies its disease resistance, from BC1F1Colony selects resisting bacterial leaf-blight and the single strain the most similar to 9311, with 9311 hybridization, obtains BC2F1Colony; Again to BC2F1Colony inoculation bacterial leaf spot bacterium, identifies its disease resistance, from BC2F1Colony selects resisting bacterial leaf-blight and the single strain the most similar to 9311, with 9311 hybridization, obtains BC3F1Colony; So circulation, until obtaining BC6F1Colony, then to BC6F1Colony inoculation bacterial leaf spot bacterium, identifies its disease resistance, from BC6F1Colony selects resisting bacterial leaf-blight and the individual plant selfing the most similar to 9311, from single strain of each selfing, gathers in the crops seed, obtain S1 colony; S1 colony is inoculated bacterial leaf spot bacterium, identifies its disease resistance, filter out S1 colony (i.e. each single strain resisting bacterial leaf-blight in colony) and the single strain the most similar to 9311 that resistance is not separated, be and cultivate successful 9311/Xa23 kind.
The pre-treatment of seed, sowing and cultivation condition: compare seed and seed to be measured is all alcohol-pickled 2 minutes of 70% by concentration, 2 times are washed again with deionized water, soaked overnight in the water of 30 DEG C, carries out vernalization 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, (Shanghai Medical Equipment Plant of Bo Xun Industrial Co., Ltd. produces to utilize Autoclave, model is YSQ-LS-50S11) soil is carried out high-temperature high-voltage sterilizing sterilizing, sterilization condition is: 120 DEG C, 30 minutes. The planting soil of the paddy rice in contrast of the soil after sterilization. Comparison paddy rice is sowed when isolation, transplants, is grown in the plastic tub being filled with disinfection soil; 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 comparison 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, makes soil and the field soil isolation of comparison paddy growth, avoids passing sick infection bacterial leaf-blight by soil; (2) water source isolation: adopt tap water to irrigate comparison paddy rice, it is ensured that water source, land for growing field crops does not enter comparison paddy rice, avoids infecting bacterial leaf-blight by water source; (3) spatial separation: do not plant other paddy rice for 10 meters around comparison paddy rice, avoids the friction of the rice leaf with band bacterial leaf spot bacterium to infect bacterial leaf-blight. Paddy rice to be measured does not prevent and treat bacterial leaf-blight; the cultivation condition compareing 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, the while of comprising the production management measures such as sowing, transplanting, fertilising, insect protected, diseases prevention (except bacterial leaf-blight other disease), synchronously carries out.
Specifically, around comparison paddy rice within the scope of 10 meters, do not sow or plant other paddy rice; Comparison Rice irrigation water adopts clean tap water; Except above condition, comparison paddy rice other cultivation condition with paddy rice to be measured is identical with common land for growing field crops conventional cultivation condition, but all cultivation steps are consistent between comparison 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.
Comparison paddy rice and paddy rice to be measured are adopted identical cultivation condition plantation, comparison paddy rice and paddy rice to be measured are grown at identical conditions, ensure that the expression amount of comparison 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 comparison paddy rice is respectively taken to few 3 blades and mixes with paddy rice to be measured, for representing the sample of comparison paddy rice and paddy rice to be measured. got blade and it is placed in the extraction that liquid nitrogen is saved to total miR-96 gene immediately. in the present embodiment, sample time, section was that on February 28th, 2011 is to March 3 days then, serial sampling 5 days, obtain comparison paddy rice and each 5 samples of paddy rice to be measured altogether, it is numbered C1��C5 and T1��T5 respectively, wherein C represents Control, T represents Treatment, wherein, comparison paddy rice is consistent with the time point that paddy rice to be measured samples, it is also identical for compareing the leaf position selected by paddy rice and paddy rice to be measured, this can make the circadian clock body clock between comparison paddy rice and paddy rice to be measured and developmental condition be identical, only choose miRNA expression amount that the circadian clock body clock blade all identical with developmental condition could ensure to compare between paddy rice and paddy rice to be measured further more by the impact of circadian clock body clock and growth.
It is 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: the centrifugal post of RNA, genomic dna remove centrifugal post, centrifuge tube, MCL lysis buffer, XD binding buffer liquid, RNA elutriant II and DEPC water) total miR-96 gene of being separated 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 placed in 10 mortars respectively, and pours liquid nitrogen in mortar 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, the lysate of the �� L that adds 700, vortex 30 seconds is with mixed even sample, 55 DEG C are incubated 30 minutes, it is under the room temperature condition of 12000 �� g centrifugal 5 minutes at centrifugal force, obtain supernatant liquor, and supernatant liquor is transferred in centrifugal post centrifugal, for the DNA removed in genome, through 12000 �� g room temperature centrifugal 2 minutes, and by the centrifuge tube of the new 1.5mL of divert away to flowed out, the dehydrated alcohol of this liquid volume 1.1 times is added and vortex 20 seconds is mixed even in the liquid of this outflow, by in the divert away after this vortex to the centrifugal post of RNA through 12000 �� g room temperature centrifugal 1 minute, abandon centrifugate, add ethanol that 500 �� L concentration are 96-100% in the centrifugal post of RNA, through 12000 �� g room temperature centrifugal 1 minute again, abandon centrifugate, add 500 �� LXD binding buffer liquid in the centrifugal post of RNA, centrifugal 1 minute of 12000 �� g room temperature, abandon centrifugate, add in the 750 �� LRNA centrifugal posts of elutriant II to RNA, centrifugal 1 minute of 12000 �� g room temperature, abandon centrifugate, add in the 750 �� LRNA centrifugal posts of elutriant II to RNA again, centrifugal 1 minute of 12000 �� g room temperature, abandon centrifugate, by centrifugal for RNA post under the top speed being greater than 12000 �� g, centrifugal 2 minutes of room temperature, 30-50 �� LDEPC water is added in the centrifugal post of RNA, 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) in the quantitative program of RNA, measure and obtain the concentration of total miR-96 gene of separation, according to the quality of the total miR-96 gene of concentration and volume computing.
External source is added with reference to miR-96 gene in total miR-96 gene of separation, obtain the first mixed solution: external source is the 0.05% of the total miR-96 gene quality being separated with reference to the add-on of miR-96 gene, the sequence of external 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: the MnCl getting the first mixed solution containing the total miR-96 gene of the 5ng that has an appointment, 2 �� l10 �� reaction buffers, 1 �� l50mM2, the trimethyl-glycine of 4 �� l5M and the cyclase of 1 �� l5u/ �� l, mixed even after supplying 20 �� l with water, obtain the 3rd mixed solution; After the 3rd mixed solution is incubated 15 minutes in 60 DEG C, 80 DEG C are incubated 10 minutes, make enzyme deactivation. Being connected with 3 ' end by 5 ' end of total miR-96 gene and external source reference miR-96 gene, obtain total miR-96 gene of cyclisation and the mixed solution of the external 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. With also having that this enzyme provides: the MnCl of 10 �� reaction buffer, 50mM2, 5M trimethyl-glycine and without enzyme water.
The reverse transcription of the mixed solution of total miR-96 gene of cyclisation and the external source reference miR-96 gene of cyclisation: get total miR-96 gene of cyclisation and the mixed solution 2 �� l of the external source reference miR-96 gene of cyclisation, 5 �� l concentration are the reverse transcriptase primer of 1 ��M, 2 �� l concentration are the dNTP of 10mM, 5 �� 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 �� l10 �� RT Buffer (providing with reversed transcriptive enzyme), with water supply 50 �� l mix even after, 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 miRNA408 gene and the reverse transcriptase primer of external source reference miR-96 gene, and the sequence of miRNA408 gene is as shown in SEQ ID NO:1; The reverse transcriptase primer of the miRNA408 gene designed accordingly is as shown in SEQ ID NO:7; The reverse transcriptase primer sequence of external 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 miRNA408 gene, another class is the reverse transcription of external source with reference to miR-96 gene (external source is with reference to 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 miRNA408 gene, connection contact when designed reverse transcriptase primer has striden across miRNA408 gene cyclisation, therefore, when reverse transcription, only the successful miRNA408 gene of cyclisation is reversed record, thus reduces the interference of other genes.
The expression amount of real time quantitative PCR method detection reverse transcription product: real-time quantitative PCR comprises two classes equally, one class is target gene, the i.e. real-time quantitative of miRNA408 gene, another class is the real-time quantitative of external source with reference to miR-96 gene, the two is parallel carries out, all reaction compositions and condition are all identical, and only primer is not identical. Primer comprises: miRNA408 gene forward primer sequence is as shown in SEQ ID NO:3; MiRNA408 gene reverse primer sequence is as shown in SEQ ID NO:4. Primer also comprises: external source reference miR-96 gene forward primer sequence is as shown in SEQ ID NO:5; External source reference miR-96 gene reverse primer sequence 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 2 above-mentioned reverse transcription product of �� l, 3 �� l concentration are that (primer here refers to forward primer and reverse primer equimolar ratio mixture for the primer of 1 ��M, namely the mixture of the equimolar ratio of miRNA408 gene reverse primer as shown in SEQ ID NO:4 of the miRNA408 gene forward primer of sequence as shown in SEQ ID NO:3 and sequence is referred to, or refer to that the external source of sequence as shown in SEQ ID NO:5 is with reference to the mixture of external source as shown in SEQ ID NO:6 of miR-96 gene forward primer and sequence with reference to the equimolar ratio of miR-96 gene reverse primer), the ROX fluorescence correction dyestuff of the quantitative PCR mixture (article No. is QPS-201) that 10 �� l Toyobo, Japan produce and 0.4 �� l50 times (is produced by Toyobo, Japan, and provide with QPS-201) mix, obtain the 2nd mixed solution, 2nd 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 by MicrosoftExcel2010 process, during data processing taking external source with reference to miR-96 gene as the reference gene of miRNA408 expression analysis.The results are shown in Table 1:
The relative expression quantity of table 1miRNA408 gene between comparison paddy rice and paddy rice to be measured
Note: expression amount *=2(CT(miRNA408)-CT(ECK)), wherein, CT (miRNA408) and CT (ECK) is respectively during real-time quantitative PCR reacts the miRNA408 gene obtained and the CT value of external source reference miR-96 gene.
By table 1 data it will be seen that the expression amount maximum value of 5 time points of comparison paddy rice in detection time section is 79.35, expression amount minimum value is 74.45, and ratio therebetween is 1.07, and this ratio is less than 1.5. Showing in this experiment, without the expression of bacterial leaf-blight or other factors impact comparison paddy rice miRNA408 gene, better, experiment is successful, and experimental result can be used for the forecast of bacterial blight of rice in experiment condition control. In paddy rice to be measured, from February 28 to these three days March 1, the expression amount relative constancy of miRNA408 gene, the condition control that this experiment is described further is better. To March 2, the expression amount of miRNA408 gene in paddy rice to be measured is lower more than 4 times than comparison paddy rice, this kind is expressed change and was maintained March 4, shows that detected paddy rice miRNA408 changes in gene expression to be measured is unlikely that the factors such as detection error cause. Can judging, paddy rice to be measured probably infects bacterial leaf spot bacterium, it is necessary to medical treatment. Predicting further accordingly, other paddy rice in this area's plantation also has the danger of recent infection bacterial leaf-blight, it is necessary to control. Starting to March 6, paddy rice to be measured starts bacterial leaf-blight symptom occur, and to March 12, symptom was quite obvious. Meanwhile, what serious bacterial leaf-blight and bacterial stripe had also occurred in other rice varieties local mixes harm, the situation that the appearance of part field is had no harvest. As can be seen here, the expression amount of miRNA408 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 whether paddy rice clearly to be measured catches an illness, and clear and definite bacterial leaf spot disease time and place, avoid the fuzzy property of tradition disease forecast.
The foregoing is only the better 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 of doing, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (8)
1. one kind utilizes the method for miRNA408 gene forecast bacterial blight of rice, it is characterised in that, described method comprises the following steps:
Choose 9311/Xa23 rice varieties as paddy rice to be measured and comparison paddy rice;
Cultivate described paddy rice to be measured and described comparison paddy rice, within many days same time periods, gather described paddy rice to be measured and described comparison paddy rice respectively;
The total miR-96 gene being separated in described paddy rice to be measured and described comparison paddy rice respectively;
Detecting the expression amount of the miRNA408 gene in the described total miR-96 gene in described paddy rice to be measured and described comparison paddy rice respectively, the sequence of described miRNA408 gene is as shown in SEQ ID NO:1;
Expression amount according to the miRNA408 gene in described paddy rice to be measured and described comparison 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 miRNA408 gene of described comparison 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 miRNA408 gene of described comparison paddy rice and the ratio of expression amount minimum value 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 miRNA408 gene of described paddy rice to be measured is greater than the expression amount of the miRNA408 genes of 4 times of described comparison paddy rice, then described paddy rice to be measured is not susceptible; If the expression amount of the miRNA408 gene of described paddy rice to be measured is less than or equals the expression amount of the miRNA408 gene of the described comparison paddy rice of 4 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 comparison paddy rice; described comparison 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 comparison paddy rice and described paddy rice to be measured is consistent.
3. method according to claim 2, it is characterised in that, described protectiveness cultivation step comprises 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 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 comparison paddy rice.
5. method according to claim 1, it is characterised in that, adopt real time quantitative PCR method, detect the expression amount of described miRNA408 gene in described paddy rice to be measured and described comparison paddy rice.
6. method according to claim 5, it is characterised in that, the pre-treatment step of described real time quantitative PCR method comprises:
Utilize spectrophotometric determination and obtain the concentration of described total miR-96 gene of separation;
External source is added with reference to miR-96 gene in described total miR-96 gene, obtain the first mixed solution, described external source is the 0.05% of described total miR-96 gene quality with reference to the add-on of miR-96 gene, and the sequence of described external source reference miR-96 gene is as shown in SEQ ID NO:2;
Respectively 5 ' end of described total miR-96 gene and described external source reference miR-96 gene is connected with 3 ' end, obtains described total miR-96 gene of cyclisation and the mixed solution of the described external source reference miR-96 gene of cyclisation;
The mixed solution of total miR-96 gene of described cyclisation and the external 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 the expression amount of described miRNA408 gene in described paddy rice to be measured and described comparison paddy rice, comprising:
By reverse transcription product described in 2 �� l, 3 �� l concentration be the primer of 1 ��M, 10 �� l quantitative PCR mixtures and 0.4 �� l50 ROX fluorescence correction dyestuff doubly mix, obtain the 2nd mixed solution, being reacted in real-time PCR by described 2nd 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: miRNA408 gene reverse primer as shown in SEQ ID NO:4 of the miRNA408 gene forward primer of sequence as shown in SEQ ID NO:3, sequence, the sequence external source as shown in SEQ ID NO:5 with reference to external source as shown in SEQ ID NO:6 of miR-96 gene forward primer and sequence with reference to miR-96 gene reverse primer.
8. method according to claim 6, it is characterised in that, the step that the mixed solution of total miR-96 gene of described cyclisation and the external source reference miR-96 gene of described cyclisation carries out reverse transcription comprises:
Get the mixed solution 2 �� l of external source with reference to miR-96 gene of total miR-96 gene of described cyclisation and cyclisation, the reversed transcriptive enzyme of DTT and 20U that dNTP that reverse transcriptase primer that 5 �� l concentration are 1 ��M, 2 �� l concentration are 10mM, 5 �� l concentration are 100mM, with water supply 50 �� l mix even after, obtain the 4th mixed solution, described 4th mixed solution 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 comprises the reverse transcriptase primer of miRNA408 gene and the reverse transcriptase primer of external source reference miR-96 gene, the reverse transcriptase primer sequence of described miRNA408 gene is as shown in SEQ ID NO:7, and the reverse transcriptase primer sequence of described external source reference miR-96 gene is as shown in SEQ ID NO:8.
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| Characterization and expression profiles of miRNAs in rice seeds;Xue LJ et al.;《Nucleic Acids research》;20081222;第37卷(第3期);916-930 * |
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