CN102206651A - Soybean cyst nematode resistance gene and application thereof - Google Patents
Soybean cyst nematode resistance gene and application thereof Download PDFInfo
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Abstract
The invention relates to a soybean cyst nematode resistance gene and application thereof. The cDNA (complementary deoxyribonucleic acid) fragment (SRC-J-6) provided by the invention is derived from soybean (Glycine max (L.) Merrill.) variety L-10, and has a nucleotide sequence shown in 1) a nucleotide sequence shown in SEQ ID NO. 1; 2) a DNA (deoxyribonucleic acid) molecule capable of hybridizing with the nucleotide sequence shown in SEQ ID NO. 1 and being expressed under strict conditions; 3) an mRNA (messenger ribonucleic acid) molecule, which has homology of more than 90% with the nucleotide sequence shown in 1) and can be expressed; or 4) a DNA (deoxyribonucleic acid) molecule, which has homology of more than 90% with the nucleotide sequence shown in 1) and can be expressed. After the plant tissues are transformed by the gene disclosed by the invention, the resistance to cyst nematode of the transgenic soybean is significantly improved. The soybean cyst nematode resistance gene provided by the invention has important significance, not only can effectively guide conventional breeding, can also provide excellent genetic resources for the transgenic breeding of soybean and has broad application prospects in the soybean production.
Description
Technical field
The present invention relates to a kind of soybean Cyst nematode resistant gene and application thereof, belong to the RNA that contains proteins encoded or the compound library field of DNA.
Background technology
In China, especially in China the Northeast because estrepement, herd excessively, the increasingly sharpening of phenomenon such as the denudation, make the area in farmland desertification, arid and saltings increase year by year.It is reported that national saltings area exceedes 500,000,000 mu, heavy salinized ground, Heilongjiang Province area is 1,600 ten thousand mu (" alkaline land improvement and organic industry development forum ", 2010).Because (humidity 60%, pH:8.0), so the expansion of desertification of land, arid and saltings area has caused the serious day by day of soybean Cyst nematode harm to arid, the alkaline edatope of soybean Cyst nematode happiness.
The soybean Cyst nematode is one of main diseases insect pest of harm world soybean production, also is the main diseases insect pest of harm soybean in China.Have the area of causing harm extensively, the degree that causes harm is serious, pathogenic strong characteristics, this class disease and pest is extremely difficult to be eradicated soil once infecting, the resistant variety that seed selection at present has adaptability is the most economical effective measures of control soybean Cyst nematode.And the research of enantiopathy gene is the basis of resistant variety seed selection.
Therefore it is significant to clone soybean Cyst nematode resistant gene.At present, the soybean Cyst nematode resistance site of Que Dinging has more than 100 approximately, but but rarely has report according to these site cloned genes.Along with finishing of soybean gene group order-checking, the excavation that the method for employing information biology is carried out gene has become possibility, in addition, no matter be from monocotyledonous or the resistant gene of dicotyledons, some conservative propertys are structurally all arranged, and this is the excavation of the disease-resistant gene condition of providing convenience.
The soybean Cyst nematode has specificity to infecting of soybean, therefore, the candidate gene that excavates can't be verified its resistance in Arabidopis thaliana isotype plant, and soybean is the plant transformed that is difficult to of generally acknowledging, therefore, find a kind of method that can carry out the functional verification of resistance candidate gene fast extremely urgent.
Summary of the invention
Technical problem to be solved by this invention provides a kind of soybean Cyst nematode resistant gene.
Described soybean Cyst nematode resistance nucleotide sequence is as following 1)-4) arbitrary shown in,
1) its nucleotide sequence is shown in SEQ ID NO.1;
2) under stringent condition with 1) nucleotide sequence that the limits dna molecular that can hybridize and can express;
3) with 1) described in nucleotide sequence have 90% above homology, and the mRNA molecule that can express;
4) with 1) described in nucleotide sequence have 90% above homology, and the dna molecular that can express.
Described stringent condition can be at 6 * SSC, in the solution of 0.5%SDS, 65 ℃ of hybridization down, uses 2 * SSC then, and 0.1%SDS and 1 * SSC, 0.1%SDS respectively wash film once.
The recombinant vectors, transgenic cell line or the reorganization bacterium that contain above arbitrary described nucleotide sequence all belong to protection scope of the present invention.
Described recombinant vectors is that above-mentioned encoding gene is inserted the recombinant vectors that pGFPGUS obtains.
The primer of the above arbitrary described nucleotide sequence of amplification is to also belonging to protection scope of the present invention.
The used primer of the amplification described nucleotide sequence of claim 1, its nucleotide sequence is shown in SEQ ID NO.3 and SEQ ID NO.4.
Another technical problem to be solved by this invention provides the method that a kind of soybean Cyst nematode resistant gene is used.
It also is the scope of protection of the invention that the expression of described gene fragment in the plant tissue that does not contain this gene used.
Described plant tissue is root, stem, leaf, flower or seed.
Described plant is monocotyledons or dicotyledons.
Described monocotyledons is a tobacco, and described dicotyledons is Arabidopis thaliana and any cultivation or wild soybean.
Describedly be expressed as expression.
Described expression and/or described the expression excessively are to express under greenhouse experiment by the test soil sample and the solution that contain soybean Cyst nematode cyst, worm's ovum, larva.
Described larva is a second instar larvae, described soil sample is with sick soil and the aseptic fine sand test sick soil with 3: 1 ratio uniform mixing preparation, described solution is every milliliter of suspension that contains 400 worm's ovums at least, and described greenhouse experiment is: illumination mode: day/night=16h/8h; Temperature model: day/night=28 ℃/25 ℃.
The expression analysis result shows that this gene is specifically expressing in disease-resistant parent, and does not express in susceptible parent, and at the expression amount of disease-resistant parent's the root expression amount in the blade.
Adopt Agrobacterium rhizogenes to infect the method for soybean cotyledon node the gene order among the present invention and the CaMV 35S promoter back that links together, induce the kind of sense soybean Cyst nematode to produce hairly root, and in its inductive process, goal gene is changed in the hairly root.The transgenic positive plant is inoculated evaluation.Experiment showed, that after inoculation 15 days, the female borer population in the transgenic positive root obviously is less than the female borer population in the adjoining tree root; Inoculate after 30 days, the cyst number on transgenic positive hairly root surface also is less than contrast, and compared with the control, blade does not have tangible disease symptom.It is certain to show that this gene plays a part in the resistance of soybean to Cyst nematode.
In China, especially in China the Northeast because estrepement, herd excessively, the increasingly sharpening of phenomenon such as the denudation, make the area in farmland desertification, arid and saltings increase year by year.Because the happiness of soybean Cyst nematode is arid, (humidity 60%, pH:8.0), so the expansion of desertification of land, arid and saltings area has caused the serious day by day of soybean cyst disease to alkaline edatope.Therefore it is significant to clone soybean Cyst nematode resistant gene, can not only effectively instruct conventional breeding, but also can provide good genetic resources for the transgenic breeding cause of soybean.Therefore, gene of the present invention is imported in conventional breeding work in the kind of sense soybean Cyst nematode, can obtain the soybean Cyst nematode is had the new soybean varieties of resistance, in soybean produces, have broad application prospects.
Description of drawings
Gene amplification product electrophoresis result synoptic diagram in the disease-resistant gene bunch on Fig. 1 Gml6
1：SRC-J-1；2：SRC-J-2；3：SRC-J-3；4：SRC-J-4；
5：SRC-J-5；6：SRC-J-6；M：Marker?DL2000
The InterProScan of Fig. 2 SRC-J-6 protein structure analyzes synoptic diagram
Fig. 3 SRC-J-6 sxemiquantitative RT-PCR result schematic diagram
1: black agricultural 37 leaves 2: black agricultural 37 root 3:L-10 leaf 4:L-10 roots
The PCR of Fig. 4 SRC-J-6 transfer-gen plant identifies synoptic diagram
1-8: plant 2,3,5,7,9,10,16, CK; M1:DL2000marker
Fig. 5 is for sending out shape root product red colouring synoptic diagram
A: plant 10; B: plant 16; C: negative control; D: blank
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
Dna fragmentation reclaims and adopts vast Imtech to reclaim test kit, and operates by its specification sheets.
All inorganic chemical reagents and organic solvent are available from Shanghai chemical reagent factory, and reagent is homemade analytical pure, and primer is given birth to worker company by Shanghai and synthesized.Ependorf gradient type pcr amplification instrument is available from German Eppendorf company.
Vegetable material:
Soybean (Glycine max (L.) Merrill.) anti-Cyst nematode kind ' L-10 ', be documented in Wang Zizhen, the evaluation of resistance and the economical character correlation analysis of the non-microspecies specificity of soybean Cyst nematode resistant variety, [J]. the soybean science, 2009, (04). in, the public can obtain from Northeast Agricultural University.
Soybean (Glycine max (L.) Merrill.) sense Cyst nematode kind ' black farming 37 ' is documented in Wang Binru, black agricultural 37 seed selections of high yielding soybeans new variety and popularization, [J]. Heilungkiang agricultural sciences, 1993, (01). in.
Non-special resistant strain L-10 with the black farming 37 of susceptible variety and this laboratory seed selection is the parent, 140 F2:6 that make up and the RIL material of F2:7, be documented in Wang Zizhen, the evaluation of resistance and the economical character correlation analysis of the non-microspecies specificity of soybean Cyst nematode resistant variety, [J]. the soybean science, 2009, (04). in, the public can obtain from Northeast Agricultural University.
Soybean (Glycine max (L.) Merrill.) have high transformation efficiency kind ' east farming 50 ', the section of being documented in is sparkling, agriculture bacillus mediated soybean cotyledon node and hypocotyl method for transformation relatively reach optimization, [J]. the soybean science, 2010, (04). in, the public can obtain from Northeast Agricultural University.
Agrobacterium rhizogenes K599 bacterial strain (is preserved and is numbered: ACCC10060), separate by Australian Allen Kerr, and provide by Institute of Crop Science, Chinese Academy of Agricultural Science, be documented in Cho H J, High efficiencyinduction of soybean hairy roots and progagation of the soybean cyst nematode.Planta, 2000, among the 210:195-204..
The soybean Cyst nematode is gathered from soybean continuous cropping plot, Yichun by No. 3 physiological strains.And by soybean biological key lab of the Ministry of Education isolation identification.Be documented in Wang Zizhen, the evaluation of resistance and the economical character correlation analysis of the non-microspecies specificity of soybean Cyst nematode resistant variety, [J]. soybean science, 2009, (04). in, the public can obtain NCPPB2659 from Northeast Agricultural University.
No. 14 physiological strains of soybean Cyst nematode are gathered and test the disease garden from academy of agricultural sciences, Heilongjiang Province grand celebration branch, and learn key lab's isolation identification by the Ministry of Education's soybean biological.Be documented in Wang Zizhen, the evaluation of resistance and the economical character correlation analysis of the non-microspecies specificity of soybean Cyst nematode resistant variety, [J]. soybean science, 2009, (04). in, the public can obtain from Northeast Agricultural University.
PMD-18T vector is available from TAKARA company.
Carrier pGFPGUS is documented in Cho H J, High efficiency induction of soybean hairyroots and progagation ofthe soybean cyst nematode.Planta, and 2000, among the 210:195-204..
Restriction enzyme and T
4Dna ligases etc. are all available from TAKARA company.
Quantitative test in following examples all is provided with repeated experiments three times, results averaged.
The anti-Cyst nematode gene nucleotide series of embodiment 1 soybean
Soybean Cyst nematode resistance nucleotide sequence shown in SEQ ID NO.1, or be down 1)-3) arbitrary shown in,
1) dna molecular that under stringent condition, can hybridize and can express with the nucleotide sequence of SEQ ID NO.1 qualification;
2) have 90% above homology with the described nucleotide sequence of SEQ ID NO.1, and the mRNA molecule that can express;
3) have 90% above homology with the described nucleotide sequence of SEQ ID NO.1, and the dna molecular that can express.
Described stringent condition can be at 6 * SSC, in the solution of 0.5%SDS, 65 ℃ of hybridization down, uses 2 * SSC then, and 0.1%SDS and 1 * SSC, 0.1%SDS respectively wash film once.
The primer of the above arbitrary described dna fragmentation of amplification is to also belonging to protection scope of the present invention, for example: SEQ ID NO.3 and SEQ ID NO.4.
SEQ ID NO.2 belongs to the described nucleotide sequence of SEQ ID NO.1 and has 90% above homology, and the dna molecular that can express.
The acquisition of the anti-Cyst nematode gene of embodiment 2 soybean
1. the clone of gene
1) extraction of the total RNA of plant and reverse transcription: get the L-10 of 12h behind the inoculation nematode, the root and the leaf portion tissue of black farming 37 respectively, extract RNA, total RNA detects through agarose gel electrophoresis, and 18S and 28S RNA banding pattern are clear and legible, and the uv-spectrophotometric note detects OD
260/ OD
280Ratio illustrates that the RNA integrity is good, purity is high between 1.9～2.0.Reverse transcription reaction is with reference to promega ImProm-II
TM(Promega) the reverse transcription test kit is operated.
2) clone of disease-resistant candidate gene full length sequence: be template with the L-10 of above-mentioned reverse transcription, the root of black farming 37 and the cDNA of leaf respectively, use gene specific primer (SEQ ID NO.3 and SEQ ID NO.4) that designs and the method that adopts landing-type PCR clone goal gene.The system and the program of reaction are as shown in table 1.After treating that PCR reaction finishes, each sample is respectively got 8 μ L and is carried out 1% agarose gel electrophoresis analysis, the result as shown in Figure 1, amplification length is consistent with the product length of expection.
Table 1 disease-resistant candidate gene PCR reaction system and program
3) connection of PCR product; the evaluation and the order-checking of conversion and positive colony: the purifying that reclaims test kit explanation carrying out PCR product according to the low dose of glue of Shanghai Hua Shun company; then; to reclaim fragment and be connected to pMD18-TEasyVector; with each monoclonal bacterium liquid is template, adopts with the used identical primer of clone and carries out bacterium liquid PCR.Reaction conditions is identical with clone gene.Each reaction is taken out 8ul and is carried out 1.0% agarose gel electrophoresis detection PCR product with PCR positive colony bacterium liquid, is sent to Shanghai and gives birth to worker's order-checking.
2. the bioinformatic analysis of the anti-Cyst nematode candidate gene sequence of soybean
Utilize http://www.expasy.ch/tools/protparam.html to analyze, show that SRC-J-1 is for having 1048 amino acid whose polypeptide in the gene cluster, theoretical iso-electric point (pI) is 6.02, and the estimation molecular weight is 117003.1Da; SRC-J-2 is for having 775 amino acid whose polypeptide, and theoretical iso-electric point (pI) is 6.23, and the estimation molecular weight is 83400.1Da.SRC-J-4 is for having 932 amino acid whose polypeptide, and theoretical iso-electric point (pI) is 5.57, and the estimation molecular weight is 103426.2Da.SRC-J-6 is for having 520 amino acid whose polypeptide, and theoretical iso-electric point (pI) is 8.99, and the estimation molecular weight is 57363.0Da.
The InterProScan analysis revealed, these 4 genes all include LRR structure and SERINE/THREONINE kinase domain (Fig. 2).Wherein the LRR structure is relevant with the identification of extracellular signal, and the kinases structure has the activated effect with disease-resistant response member to the downstream.
3. sxemiquantitative RT-PCR analyzes the expression of candidate gene
With soybean β-Actin is confidential reference items, adopt the method for sxemiquantitative RT-PCR to detect soybean β-Actin gene at nematicide kind L-10 and the root of the black farming 37 of sense nematode kind and the expression in the leaf texture, the result shows, SRC-J-6 only expresses in nematicide kind L-10, and do not express in the black farming 37 of sense nematode kind, and the expression amount in the L-10 root is higher than the expression amount (Fig. 3) in the blade.Not obvious or the indifference of the differential expression of other several genes between two parents.This illustrates that the expression of this gene in differing materials there are differences and have tissue expression specificity, and this has hinted that also SRC-J-6 may bring into play certain effect in the resistance of soybean Cyst nematode.
The anti-Cyst nematode gene function checking of embodiment 3 soybean
1, the structure of plant expression vector
Clone SRC-J-6 gene order makes up overexpression carrier pGFPGUS-SRC-J-6 to carrier pGFPGUS, and concrete primer sequence is as shown in table 2:
Table 2.SRC-J-6 amplimer sequence
The purifying of goal gene amplification, PCR product connects, and transforms and the evaluation of positive colony and check order the same.The pGFPGUS empty carrier that has the pMD18-T-SRC-J-6 recombinant plasmid of goal gene and be attached thereto is used Xba I and Sac I double digestion respectively, electrophoresis identifies that blended rubber reclaims the big fragment about test kit recovery 1.6kb left and right sides SRC-J-6 segment and carrier 13kb.The small segment that obtains is connected with the big fragment of pGFPGUS carrier, obtains connecting product.Should connect product transformed into escherichia coli DH5 α competent cell, carry out kalamycin resistance screening, with the transformant that obtains in 37 ℃ of overnight incubation of shaking table, extract plasmid according to, by Xba I and Sac I double digestion, whether the checking plasmid successfully constructs the picking positive plasmid again.
2, Agrobacterium rhizogenes transforms and the positive colony evaluation
By freeze-thaw method transforming agrobacterium rhizogenes K599, concrete grammar is as follows:
1. get the plasmid DNA of 2 μ L (being less than 50ng) purifying, join in the centrifuge tube that contains 100 μ L agrobacterium tumefaciens competent cells, gently mixing;
2. ice bath 30min, quick-frozen 2min in liquid nitrogen puts 37 ℃ of water-bath heat shock 5min rapidly, again ice bath 2min rapidly;
3. the liquid culture that adds 500 μ L YEP antibiotic-frees is based on 28 ℃, and 100rpm shaking culture 3～5h gently recovers;
4. with pipettor bacterium liquid is moved to YEP solid medium (50mg/L Kan) surface, evenly coat whole flat board;
5. be inverted cultivation 1～2d for 28 ℃,, get single bacterium colony and extract plasmid with alkaline process until the bacterium colony that grows suitable size, carry out PCR detection and enzyme and cut evaluation, detect with 1% agarose gel electrophoresis, the result shows can cut out the purpose fragment, can carry out next step transformation experiment with it.
3, Agrobacterium rhizogenes mediated method soybean transformation cotyledonary node
The soybean cotyledon node Transformation Program of Agrobacterium rhizogenes mediation:
1) soybean seeds is sprouted: with soybean varieties east farming 50 usefulness disinfection by chlorines 16 hours, be seeded in the vermiculite, cultivate in illumination box, illumination mode is day/night=16h/8h, and temperature model is day/night=28 ℃/25 ℃; Humidity is 75%.
2) cultivation of Agrobacterium rhizogenes: from YEP flat board (Kan
R) go up the single bacterium colony of Agrobacterium that picking contains the pGFPGUS recombinant plasmid, be inoculated in 5mL YEP (50mg/L Kan) liquid nutrient medium, 28 ℃ of 220rpm shaking culture are spent the night; Get the 2mL culture, join in 50mLYEP (50mg/LKan) liquid nutrient medium, 28 ℃ are cultured to OD600 is 1～1.2; The 50mL culture in the centrifugal 10min of 5000rpm, is abandoned supernatant, collect thalline, with the resuspended thalline of equal-volume 50mL aseptic deionized water.
3) infecting of Agrobacterium rhizogenes: get 1 day seedling and be used to infect.Seed just sprouted grow just surface, and cotyledon still manifests yellow (after approximately sowing 3 days) and is defined as 0 day; Syringe with 1ml is drawn with the resuspended thalline of aseptic deionized water, injects at the cotyledonary node place.Carefully pierce through with syringe needle, cotyledon is not bumped, release several bacterium liquid with syringe, and with the syringe needle that speckles with bacterium liquid back and forth several times, bacterium liquid is evenly distributed in the wound in the wound; Blot unnecessary bacterium liquid with aseptic filter paper, the plant after will infecting places incubator to cultivate, the same sprouting condition of culture condition.
4) the inducing and transplanting of adventive root: infected the back about about 7 days, the plant of cotyledonary node place root shape thing projection is taken out from vermiculite, in distilled water, cut off, root induction is equipped with in the triangular flask of sterilized water in the over-ground part insertion along the about 1cm in projection bottom place; After treating that root grows, every about 1em of root clip is put in the 1.5ml EP pipe that the GUS staining agent is housed, and 37 ℃ of lucifuges are hatched, and every root repeats for three times.GUS staining agent composition is as follows:
100mmol/L?KH
2PO
4，PH7.0；0.5mmol/L?K
4[Fe(CN)
6]；0.5mmol/L?K
3[Fe(CN)
6]；0.1％Triton?X-100；0.5mg/ml?X-Gluc[X-glucuronide，Molecular?probe]。
Detected result shows: 8 strains are changeed the SRC-J-6 gene plant and are the GUS positive; Extract the DNA that sends out the shape root of GUS reacting positive, adopt the method for PCR to detect the transgenic positive root, identical among reaction conditions and the primer and the sxemiquantitative RT-PCR.Detected result shows: 2 strains are changeed the SRC-J-6 gene plant and are the PCR positive (being numbered 10,16) (Fig. 4).
4, the inoculation of transfer-gen plant is identified
With detection have the plant of transgenic positive root and negative control, blank is transplanted into respectively in the matrix that contains No. 3 physiological strains of soybean Cyst nematode, getting root after 15 days cleans, after bleaching, dyeing, under 20 * opticmicroscope, count, every strain is got 10 roots and is carried out repetition (Fig. 5), and concrete grammar is as follows:
1) mensuration of soil cyst density: the 100g cyst soil sample that weighs up is placed on the 20 purpose screens, and with anxious slightly current scour soil sample, cyst stream downstream flows on the 60 purpose screens, does not have soil sample on 20 purpose screens, can stop.
2) cyst and the impurity of collecting on the 60 order screens is poured in the beaker, static 20min inclines supernatant liquor in beaker or suspension liquid on filter paper, and behind filter paper filtering, numeration ware number goes out the cyst number, keeps a record, and triplicate is averaged.40 above cyst numbers of every 100g soil sample cyst density of propositions such as Riggs are proper experiment soil samples.
3) preparing experiment soil: soil humidity is to influence the important factor that the soybean Cyst nematode is grown, and too high soil humidity can make and suppress the hatching of soybean Cyst nematode by oxygen undersupply in the soil, cause the death of soybean Cyst nematode.To test soil sample mixes according to 3: 1 ratio with vermiculite, can not only make soil become soft like this, help the growth of root, and the water-retentivity of vermiculite is better, generally can adhere to water in 15 days, when ensureing the normal hatching of soybean Cyst nematode, also guarantee the normal growth of soybean plant strain
4) identify the plantation of plant: all plant are planted in the plastics box, can not only the guaranteed conditions uniformity, be good at significantly reducing artificial and to the damage of plant.Plant according to all around spacing 5em kind, every kind of strain system plants 5, all will plant check variety Lee68 in each box, has reduced the caused error of different tests condition.List after waiting to emerge, stay three young plants of growth conditions unanimity.The acquisition of data: after approximately sowing 15d the whole strain of plant is taken out, note the protection of root
5) surflaes of root is rinsed well, is soaked in concentration and is 4min in 3% antiformin (NaOCl) aqueous solution, during stir 2 times.
6) flowing water flushing root 30-45s, and immersion 15min is soaked in root in the C.I. 42685 staining fluid in distilled water, heated and boiled 30s is cooled to room temperature.
7) with the root tissue compressing tablet, 20 * opticmicroscope under counting, and measure the length of the root tissue that is used to count, the female borer population order in the root tissue of Units of Account length.
The collocation method of staining fluid:
1. mother liquor, 0.35g magenta+25ml acetic acid+75ml deionized water;
2. working fluid, 20ml mother liquor+580ml deionized water.
The result shows that No. 10 the interior average female borer population of plant root is 4.3/cm, and No. 16 the interior average female borer population of plant root is 5/cm.The average female borer population that changes in the pGFPGUS negative control root is 7.2/cm.Average female borer population in the agricultural 50 blank plant roots in non-transgenic east is 6.5/cm.The data of gained are carried out Duncan ' s multiple range test, according to data as can be known, have 3 kinds of scopes: 2,3,4; Degree of freedom is 36.The acquisition scope under 0.01,0.05 conspicuous level of tabling look-up is 2,3,4; Degree of freedom is 36 o'clock SSR value and calculates corresponding minimum significantly extreme difference value LSR (table 3) in view of the above.
5% and the 1%SSR value table of table 3Duncan ' s multiple range test
The difference in twos of the mean number of four groups of data and LSR value in the table are compared, can make judgement (table 4) the difference of corresponding population average.
The result of table 4.Duncan ' s multiple range test
Annotate:
*With
*Represent that respectively significance level is P=0.05 and 0.01; Ns represents that difference is not remarkable, 1: negative control; 2: blank; 3:16 number; 4:10 number.
By last table result, we learn: there is utmost point significant difference in the interior average female borer population of average female borer population in the average female borer population in No. 10 plant roots and the negative control root and blank plant root, and is not remarkable with average female borer population difference in No. 16 plant roots; There is utmost point significant difference in the interior average female borer population of average female borer population in No. 16 plant roots and negative control root, has significant difference with average female borer population in the blank plant root.Thereby the resistance that has confirmed SRC-J-6 gene pairs soybean Cyst nematode has tangible contribution really.
＜110〉Northeast Agricultural University
＜120〉a kind of soybean Cyst nematode resistant gene and application thereof
<160> 6
<170> PatentIn?version?3.5
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＜213〉soybean (Glycine max (L.) Merrill.)
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<211> 2835
<212> DNA
＜213〉soybean (Glycine max (L.) Merrill.)
<400> 2
tggtggagag?ataagtcctt?gtttggctga?tttaaagcat?ttgaattact?tggacttgag 60
cgccaatgta?ttccttggag?aaggtatgtc?aattccttct?ttccttggga?caatgacttc 120
cttgactcac?ctcaacctct?ctcttactgg?attccgtggg?aagattcctc?ctcagattgg 180
gaatctctca?aatttggtgt?atcttgacct?gagttcagat?gttgccaacg?gaacagtacc 240
ctctcagatc?gggaatctct?ctaagcttcg?atatcttgac?ttgagcgcca?attattttga 300
aggtatggca?attccttctt?tcctttgtgc?aatgacctcc?ttgactcacc?tcgacctctc 360
ttatactcta?ttccatggga?agattccatc?tcagattggg?aatctctcca?atttggtgta 420
tcttggcctt?ggaggttatt?ctgatttcga?acctcctctg?tttgctgaaa?atgtagaatg 480
gctatcaagt?atgtggaagc?ttgaatatct?tgatttgagt?aatgcaaacc?tatccaaagc 540
atttcattgg?ctacacactc?tccaatctct?tccttctttg?acccacctat?atttgtcaca 600
ctgcacactc?cctcactata?atgaaccatc?cttgctcaac?ttctcatctc?tgcaaactct 660
cattctttac?aatactagtt?attcccctgc?catttctttt?gtccccaagt?ggatattcaa 720
attgaagaaa?cttgtttctc?ttcaattacg?gggtaataaa?ttccaaggtc?cgattccttg 780
tggtatccga?aacctcacac?ttcttcaaaa?tcttgacttg?tctggaaatt?cattctcatc 840
ttctatacct?gattgcttat?acggtcttca?tcgtctcaag?tctttggacc?taagatccag 900
caacttgcat?gggactattt?ctgatgccct?gggaaatttg?acttctcttg?ttgaacttga 960
tttgtcatac?aatcaacttg?aaggaaccat?tccaacttct?ttgggaaatt?tgacttctct 1020
tgttgcactt?tatttgtcat?ataatcaact?tgaaggaaca?attccgactt?ttttgggaaa 1080
tctccgcaac?tcaagggaga?tagatttaac?atatctcgat?ctctctatta?ataaattcag 1140
tggaaatcca?tttgaaagtc?ttggatcact?ctctaaattg?tcatctcttt?ggattgatgg 1200
caataatttt?caaggagttg?tcaaggaaga?tgatcttgca?aatcttacaa?gcttgacgga 1260
ttttggtgca?tcgggaaaca?atttcacttt?aaaagtgggt?cccaattgga?ttcctaattt 1320
tcaacttacc?tatttggaag?tgacatcatg?gcagttaggt?cccagctttc?cattgtggat 1380
tcagtcacaa?aacaaactta?aatatgttgg?actatctaac?acggggattt?tcgattctat 1440
tcccacttgg?ttctgggaag?cacattctca?ggttttgtat?ttaaacctct?ctcataatca 1500
tatccgtggt?gagcttgtga?ctacaataaa?aaatccaata?tctatccaaa?ctgttgatct 1560
aagcacaaat?cacttatgtg?gtaaattacc?ctatctttca?aatgatgtgt?atgacttaga 1620
cctttcaacc?aattcattct?ctgaatccat?gcaagatttt?ttatgtaaca?atcaggacaa 1680
gccaatgcaa?ttagaatttc?tcaatcttgc?atccaataat?ctgtcaggag?aaatacctga 1740
ttgttggatt?aattggccat?ttctagtgga?agtgaattta?caaagcaacc?attttgttgg 1800
gaacttcccc?ccatccatgg?gttccttggc?tgagctgcag?tcattagaaa?ttcgtaacaa 1860
cttgctctcg?ggaatatttc?ctaccagttt?gaagaagact?agccaattga?tatccttgga 1920
tcttggagaa?aataatcttt?caggatgtat?tccaacatgg?gttggagaaa?agctctcaaa 1980
tatgaaaatc?ctccgccttc?gatcaaacag?tttttccggt?cacattccaa?atgaaatatg 2040
tcagatgagt?cttcttcagg?ttttagacct?tgcaaaaaat?aatttttctg?gcaatatacc 2100
cagctgtttc?cgtaacttga?gtgccatgac?actagtgaac?cggagtacat?atccccgtat 2160
ctattctcat?gcaccaaatg?atacatatta?ctcttcggta?tcaggtatag?ttagtgtgct 2220
actatggcta?aaaggaagag?gagatgagta?tcgaaacatt?ctgggtttgg?taacaagtat 2280
tgatctgtca?agtaacaaat?tattaggaga?tattcctaga?gaaatcacag?atctaaatgg 2340
attgaacttt?ttgaacttgt?cccacaacca?attgattggt?cctattccag?aaggtattgg 2400
taatatggga?tcgttacaaa?ccattgatct?ttcaaggaat?caaatttctg?gtgaaatccc 2460
tccaaccatt?tctaatttga?gctttttgag?catgctagac?gtgtcttata?atcatttgaa 2520
gggaaaaatt?ccaacaggaa?ctcaattgca?aacctttgat?gcctccagat?ttattggcaa 2580
caatctatgt?ggtccaccac?tgcccataaa?ctgcagctcc?aatgggaaaa?ctcatagtta 2640
tgaaggaagt?catgggcatg?gagtgaattg?gttttttgtt?agtgcgacaa?ttggatttgt 2700
tgtgggactt?tggatagtga?ttgctccttt?gctgatttgt?agatcatggc?ggcatgccta 2760
ttttcatttc?cttgatcatg?tgtggttcaa?acttcaatct?ttttcctcgt?atagtatcac 2820
tgtttagtgt?tgctt 2835
<210> 3
<211> 25
<212> DNA
<213> 3
<220>
＜223〉be designed for amplification according to gene order
<400> 3
tggtggagag?ataagtcctt?gtttg 25
<210> 4
<211> 30
<212> DNA
<213> 4
<220>
＜223〉be designed for amplification according to gene order
<400> 4
aagcaacact?aaacagtgat?actatacgag 30
<210> 5
<211> 25
<212> DNA
<213> 5
<220>
＜223〉be designed for amplification according to gene order
<400> 5
tggtctagag?ataagtcctt?gtttg 25
<210> 6
<211> 30
<212> DNA
<213> 6
<220>
＜223〉be designed for amplification according to gene order
<400> 6
aagcagagct?caacagtgat?actatacgag 30
Claims (10)
1. a soybean Cyst nematode resistant gene is characterized in that nucleotide sequence is as following 1)-4) arbitrary shown in,
1) its nucleotide sequence is shown in SEQ ID NO.1;
2) under stringent condition with 1) nucleotide sequence that the limits dna molecular that can hybridize and can express;
3) with 1) described in nucleotide sequence have 90% above homology, and the mRNA molecule that can express;
4) with 1) described in nucleotide sequence have 90% above homology, and the dna molecular that can express.
2. the recombinant vectors that contains the described nucleotide sequence of claim 1.
3. recombinant vectors according to claim 2 is characterized in that described carrier is pMD18-T carrier, the pGFPGUS that is used to express, pBI121, pCAMBIA carrier that is used to clone or the pJawoh18 carrier that is used for RNAi.
4. the transgenic cell line or the reorganization bacterium that contain the described nucleotide sequence of claim 1.
5. the amplification described nucleotide sequence of claim 1 used primer, its nucleotide sequence is shown in SEQ ID NO.3 and SEQ ID NO.4.
6. the described nucleotides sequence of claim 1 is listed in the application in expressing in the plant tissue.
7. application according to claim 6 is characterized in that described plant tissue is root, stem, leaf, flower or seed.
8. according to claim 6 or 7 described application, it is characterized in that described plant is monocotyledons or dicotyledons.
9. application according to claim 8, its spy is being that described monocotyledons is a tobacco, described dicotyledons is Arabidopis thaliana and any cultivation or wild soybean.
10. according to claim 6 or 7 described application, it is characterized in that the described expression that was expressed as, under greenhouse experiment, express by the test soil sample and the solution that contain soybean Cyst nematode cyst, worm's ovum, larva.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN 201110106658 CN102206651B (en) | 2011-04-27 | 2011-04-27 | Soybean cyst nematode resistance gene and application thereof |
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| CN 201110106658 CN102206651B (en) | 2011-04-27 | 2011-04-27 | Soybean cyst nematode resistance gene and application thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107338322A (en) * | 2017-08-31 | 2017-11-10 | 吉林省农业科学院 | Soybean cyst nematode Heterodera glycines infect the screening technique of reference gene in lower wild soybean root tissue Real-time PCR Analysis |
| CN107860754A (en) * | 2017-10-30 | 2018-03-30 | 中国农业科学院油料作物研究所 | Soybean browning SCN cyst automatic counting method |
| CN107904244A (en) * | 2017-11-28 | 2018-04-13 | 吉林省农业科学院 | Application of the wild soybean XLOC_023202 genes in soybean plants cyst nematode resistance is improved |
| CN110317825A (en) * | 2019-04-29 | 2019-10-11 | 聊城大学 | A kind of hairy root induction method for transformation of soybean mediated with agrobacterium rhizogenes |
| CN110862996A (en) * | 2019-12-23 | 2020-03-06 | 华中农业大学 | Application of isolated soybean gene in improving soybean cyst nematode resistance |
| CN111073887A (en) * | 2020-01-23 | 2020-04-28 | 西南林业大学 | Extraction method and application of paphiopedilum high-quality total RNA |
| CN111471689A (en) * | 2019-01-23 | 2020-07-31 | 东北农业大学 | Gene for improving resistance of soybean to cyst nematode disease and application thereof |
| CN116445441A (en) * | 2022-11-30 | 2023-07-18 | 东北农业大学 | Soybean glycosyltransferase and encoding gene and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040177417A1 (en) * | 2004-01-30 | 2004-09-09 | Pioneer Hi-Bred International, Inc. | Soybean variety XB29K04 |
| CN1814810A (en) * | 2005-12-16 | 2006-08-09 | 沈阳农业大学 | Molecule label related to soybean resistant germplasm gene and its obtaining method and use |
-
2011
- 2011-04-27 CN CN 201110106658 patent/CN102206651B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040177417A1 (en) * | 2004-01-30 | 2004-09-09 | Pioneer Hi-Bred International, Inc. | Soybean variety XB29K04 |
| CN1814810A (en) * | 2005-12-16 | 2006-08-09 | 沈阳农业大学 | Molecule label related to soybean resistant germplasm gene and its obtaining method and use |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107338322A (en) * | 2017-08-31 | 2017-11-10 | 吉林省农业科学院 | Soybean cyst nematode Heterodera glycines infect the screening technique of reference gene in lower wild soybean root tissue Real-time PCR Analysis |
| CN107860754A (en) * | 2017-10-30 | 2018-03-30 | 中国农业科学院油料作物研究所 | Soybean browning SCN cyst automatic counting method |
| CN107904244A (en) * | 2017-11-28 | 2018-04-13 | 吉林省农业科学院 | Application of the wild soybean XLOC_023202 genes in soybean plants cyst nematode resistance is improved |
| CN107904244B (en) * | 2017-11-28 | 2021-07-02 | 吉林省农业科学院 | Application of wild soybean XLOC _023202 gene in improving resistance of soybean plant cyst nematode |
| CN111471689A (en) * | 2019-01-23 | 2020-07-31 | 东北农业大学 | Gene for improving resistance of soybean to cyst nematode disease and application thereof |
| CN111471689B (en) * | 2019-01-23 | 2022-12-27 | 东北农业大学 | Gene for improving resistance of soybean to cyst nematode disease and application thereof |
| CN110317825A (en) * | 2019-04-29 | 2019-10-11 | 聊城大学 | A kind of hairy root induction method for transformation of soybean mediated with agrobacterium rhizogenes |
| CN110317825B (en) * | 2019-04-29 | 2023-03-31 | 聊城大学 | Agrobacterium rhizogenes-mediated soybean hairy root induced transformation method |
| CN110862996A (en) * | 2019-12-23 | 2020-03-06 | 华中农业大学 | Application of isolated soybean gene in improving soybean cyst nematode resistance |
| CN111073887A (en) * | 2020-01-23 | 2020-04-28 | 西南林业大学 | Extraction method and application of paphiopedilum high-quality total RNA |
| CN116445441A (en) * | 2022-11-30 | 2023-07-18 | 东北农业大学 | Soybean glycosyltransferase and encoding gene and application thereof |
| CN116445441B (en) * | 2022-11-30 | 2023-11-03 | 东北农业大学 | Soybean glycosyltransferase and encoding gene and application thereof |
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| CN102206651B (en) | 2013-01-02 |
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