CN102146439B

CN102146439B - Detection primers and molecular detection method for pathogenic fungi of corn common rust

Info

Publication number: CN102146439B
Application number: CN 201010606639
Authority: CN
Inventors: 郑文明; 邢国珍; 蒋士君; 许君; 袁红霞; 王明凤; 杨文博; 张磊
Original assignee: Henan Agricultural University
Current assignee: Henan Agricultural University
Priority date: 2010-12-27
Filing date: 2010-12-27
Publication date: 2013-04-03
Anticipated expiration: 2030-12-27
Also published as: CN102146439A

Abstract

The invention relates to specific primers and a molecular detection method for pathogenic fungi of corn common rust. The specific primers comprise an upstream primer: 5'-TTTAGTAGTCTCTATCTCAACAACA-3', and a reverse primer: 5'-AAGACTCTTTTGCATGGTTT-3'. PCR (Polymerase Chain Reaction) amplification and agarose gel electrophoresis are performed on the specific primers, wherein the amplification parameters are as follows: pre-denaturating for 5 minutes at 95 DEG C, then denaturating for 45 seconds at 94 DEG C, annealing for 45 seconds at 55 DEG C, extending for 90 seconds at 72 DEG C, circulating for 30 times, and extending for 10 minutes at 72 DEG C, and the germ carrying condition of field corn is detected by the amplified product (with the fragment length of about 480bp). The primer provided by the invention has strong specificity on corn strip rust fungi and can be used for detection of the corn rust with the sensitivity of 0.001ng/mu L, and the detection method provided by the invention has good practicability and is simple and quick to operate.

Description

Detection primer and the molecular detecting method of coventional type corn rust pathogenic fungi

Technical field

The present invention relates to biology field, be specifically related to a kind of special primer and molecular detecting method of coventional type corn rust pathogenic fungi.

Background technology

Corn rust is a kind of corn fungal disease more and more serious in the global range.So far the corn rust of having reported has 3 kinds, the conventional corn rust that is namely caused by corn handle rest fungus (Puccinia sorghi Schw.), the southern corn rust that is caused by Puccinia polysora Underw (Puccinia polysora Underw.) and the tropical corn rust that is caused by corn husk rest fungus [Physopella zeae (Mains) Cummins ﹠ Ramachar].The coventional type corn rust is to occur the most general one type on the corn.

China's corn rust occurrence scope is wider, spreads all over Major Maize producing region, north and south, mainly contains two types of coventional type rust and southern type rust, and the coventional type rust mostly occurs in the northeast of low temperature and high relative humidity, the northwest Maize Region; The southern type rust occurs at first at China Taiwan, Hainan, Yuanjiang County of Yunnan.In recent years, corn rust increases the weight of gradually in the harm of China, as along with the popularization of hybrid maize and the variation of corn planting system, corn rust breaks out in province's big area such as China Hainan, Jiangsu, Henan, Shandong, Shanxi, Hebei, Zhejiang, causes local corn yield to fall sharply.The field of corn rust morbidity moderate can the underproduction 10～20%, susceptiblely heavier can reach more than 50% part plot even total crop failure.

Corn rust fungi infestation can be observed scab at maize leaf after 14 days, but carry out pesticide application this moment, be not enough to control spreading of the state of an illness, if can be early stage at the corn rust pathogenic bacterial infection, in time, detect accurately evaluation, can accomplish to prevent in advance, as select responsive effectively mycocide, with the popular great outburst of the control state of an illness.Therefore, setting up a kind of detection method that can monitor fast and accurately and differentiate the corn rust germ is significant for the safety in production of China's corn.

In recent years, along with the continuous infiltration of Protocols in Molecular Biology to the plant pathology subject, particularly development and the application of relevant molecular marking technique is for the diagnostic detection of phytopathogen provides new approach.Round pcr is with its high specificity, very early studied diagnosis for pathogenic fungi of characteristics that susceptibility is high.Relevant report in recent years emerges in an endless stream especially, has shown very wide application prospect, as disclosing a kind of molecular detecting method of Fusarium circinatum among the Chinese patent literature CN1880476C; A kind of phytophthora infestans molecular detection primer and using method thereof is disclosed among the CN101643788 A.But the Molecular Detection research of relevant corn rust bacterium there is not yet report at present both at home and abroad.

Summary of the invention

The technical problem to be solved in the present invention provides the detection primer of the good coventional type corn rust pathogenic fungi of a species specificity, and provided a kind of can be fast, accurately differentiate the molecular detecting method that detects coventional type corn rest fungus.

For solving the problems of the technologies described above, the technical solution used in the present invention is:

For a pair of detection primer of designing of coventional type corn rust pathogenic fungi, its nucleotide sequence following (SEQ ID NO:2,3):

Upstream primer: 5 '-TTTAGTAGTCTCTATCTCAACAACA-3',

Downstream primer: 5 '-AAGACTCTTTTGCATGGTTT-3'.

The nucleotide sequence of the dna characteristics fragment of the coventional type corn rust pathogenic fungi corresponding with above-mentioned detection primer is shown in SEQ ID NO:1.

Described upstream primer and downstream primer go out the product of about 483bp to coventional type corn rust pathogenic fungi specific amplification.

Described detection primer can be to the discriminating of coventional type corn rust pathogenic fungi with the application in detecting.

A kind of molecular detecting method of coventional type corn rust pathogenic fungi may further comprise the steps:

Extract the maize leaf genomic dna;

Pcr amplification, in the PCR thin-walled tube, put into successively respectively:

10 ' Buffer, 2.5 μ L, 25mmol/LMgCL ₂2.0 μ L, 2.5mmol/LdNTP 2.0 μ L, aforementioned upstream primer and downstream primer 2mmol/L each 2.5 μ L, maize leaf genomic dna 10～50ng, Taq archaeal dna polymerase 1.0U are at last with aseptic deionized water polishing to 25 μ L; The laggard performing PCR amplification of centrifugal 10sec; Amplification condition is: 95 ℃ of denaturation 5min of the first circulation; Then 94 ℃ of sex change 45sec, 55 ℃ of annealing 45sec, 72 ℃ are extended 90sec, totally 30 circulations; Last circulation, 72 ℃ are extended 10min polishing end, and amplification is finished;

Upper step gained PCR product 1.5 ℅ sepharoses with 1 * TAE damping fluid in electrophoretic analysis, voltage is 4-5V/cm, analyzes with gel imaging system after electrophoresis finishes;

4. specific band occurs at the 483bp place, maize leaf band coventional type corn rust pathogenic fungi is described, otherwise, this pathogenic fungi then do not carried.

The present invention has actively useful effect:

1. the used probe of the present invention (primer) has very strong specificity to the corn strip rest fungus, can be used in difference corn Puccinia sorghi germ and corn southern rust germ and Exserohilum turcicum, southern corn leaf blight, the common germ on the corns such as Curvularia mould; Can be used for the detection of corn rust, and its sensitivity can reach 0.001ng/ μ L.

2. detection method practicality of the present invention is good, and is easy and simple to handle quick, and testing cost is lower.

Description of drawings

Fig. 1 is the electrophoretogram of primer of the present invention (probe) specific amplified under 55 ℃ of annealing temperatures; Wherein, 1～10 swimming lane is respectively southern corn rust fungal spore sample 1, corn southern rust fungal spore sample 2, corn southern rust blade, corn Puccinia sorghi fungal spore, corn Puccinia sorghi blade, Exserohilum Turcicum, stigma germ, curved spore mould, the corn healthy leaves, aseptic deionized water; Corn Puccinia sorghi special primer amplification;

Fig. 2 is the sensitivity electrophoretogram of corn Puccinia sorghi probe (primer) specific amplified under 55 ℃ of annealing temperatures among the present invention; Wherein, 1～8 swimming lane is respectively 10ng/ μ L, 1ng/ μ L, 0.1ng/ μ L, 0.01ng/ μ L, 0.001ng/ μ L, 0.0001ng/ μ L, 0.00001ng/ μ L, 0.000001ng/ μ L, CK: aseptic deionized water.

Embodiment

Further set forth the present invention below in conjunction with specific embodiment.Test method among the following embodiment if no special instructions, is ordinary method; Used test materials and reagent among the following embodiment, if no special instructions, all available from routine biochemistry reagent company.

Embodiment 1 field corn rust monitoring simultaneous test

(1) sample collecting

On February 17th, 2009, punching slope, Sanya, Hainan Province town, August 26, Changchun City, September 24, Chongqing qian Jiang Xinhua takes respectively 10,7,4 strain maize leaf samples at random, and-80 ℃ save backup.

(2) detect primer (together lower) by the synthetic coventional type rust of following nucleotide sequence:

Upstream primer: 5 '-TTTAGTAGTCTCTATCTCAACAACA-3',

Downstream primer: 5 '-AAGACTCTTTTGCATGGTTT-3'.

(3) the corn rest fungus detects

1. extract the maize leaf genomic dna;

2. pcr amplification, 25 μ L reaction systems are put in the PCR thin-walled tube respectively successively:

10 ' Buffer, 2.5 μ L, 25mmol/LMgCL ₂2.0 μ L, 2.5mmol/LdNTP 2.0 μ L, above-mentioned coventional type corn rust bacterium upstream primer and downstream primer 2mmol/L each 2.5 μ L, maize leaf genomic dna 10～50ng, Taq archaeal dna polymerase 1.0U use aseptic deionized water polishing to 25 μ L at last; Centrifugal 10sec puts into the PCR instrument with the PCR thin-walled tube and increases; Amplification condition is: 95 ℃ of denaturation 5min of the first circulation; Then 94 ℃ of sex change 45sec, 55 ℃ of annealing 45sec, 72 ℃ are extended 90sec, totally 30 circulations; Last circulation, 72 ℃ are extended 10min polishing end, and amplification is finished;

3. the PCR product 1.5% sepharose with 1 * TAE damping fluid in electrophoretic analysis, voltage is 4-5V/cm, analyzes with gel imaging system after electrophoresis finishes.

4. use the result of the maize leaf DNA cloning of coventional type corn rust bacterium upstream primer and downstream primer to show: 7 samples in Jilin have 5 samples and a special band occurred at 483bp, its recall rate is 71.4%, and 14 sample standard deviations in Hainan and Chongqing do not have band.This result confirms the specificity of primer, can accurately differentiate corn Puccinia sorghi germ, and this result is also basically identical with the result of the generaI investigation of the corn rust bacterium field state of an illness then, can Accurate Prediction forecasts the situation of the then popular outburst of maize diseases.

Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.

Sequence table

SEQUENCE LISTING

＜110〉Agricultural University Of He'nan

＜120〉detection primer and the molecular detecting method of coventional type corn rust pathogenic fungi

＜130〉detection primer and the molecular detecting method of southern type corn rust pathogenic fungi

<160> 3

<170> PatentIn version 3.2

<210> 1

<211> 666

<212> DNA

<213> Puccinia sorghi Schw．

<400> 1

gcacttaatt gtggctcgac cccttttaaa ctcaccccaa actttcaaag actcttttgc 60

atggtttgta acaaatcatt gcacctgagt aaaagtaaca ttcttgattg aatgttacat 120

tacccacccc cttttatttt tccaaaactt tttttttaca catacacaca agtttaaaag 180

aatgtaaaca accaccttta attataaaat aacttttaac aatggatctc taggctctca 240

catcgatgaa gaacacagtg aaatgtgata agtaatgtga attgcagaat tcagtgaatc 300

atcgaatctt tgaacgcatc ttgcgccttt tggtattcca aaaggcacac ctgtttgagt 360

gtcatgaaac cctctcacaa aataaataat ttttattatg atttttgtgg atgttgagtg 420

ctgctgtgtt acacatagct cactttaaat gtataagtca tcttctttat atagcaaaaa 480

agaagagatg gattgacttg atgtgtaata attttttttt catcacattg aggaaagtag 540

caatacttgc catctttata ttattttgtt gttgagatag agactactaa acaaacaatt 600

taaaatttaa gacctcaaat caggtgggac tacccgctga acttaagcat atcaataagc 660

ggagga 666

<210> 2

<211> 25

<212> DNA

＜213〉artificial sequence

<400> 2

tttagtagtc tctatctcaa caaca 25

<210> 3

<211> 20

<212> DNA

＜213〉artificial sequence

<400> 3

aagactcttt tgcatggttt 20

Claims

1. the detection primer of a coventional type corn rust pathogenic fungi, its nucleotide sequence is as follows:

Upstream primer: 5 '-TTTAGTAGTCTCTATCTCAACAACA-3',

Downstream primer: 5 '-AAGACTCTTTTGCATGGTTT-3'.

2. the special primer of described coventional type corn rust pathogenic fungi according to claim 1 is characterized in that the nucleotide sequence of the dna characteristics fragment of the coventional type corn rust pathogenic fungi corresponding with described detection primer is shown in SEQ ID NO:1.

3. the described detection primer of claim 1 is to the discriminating of coventional type corn rust pathogenic fungi with the application in detecting.