CN102206651B - 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 resistance 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 the phenomenon such as the denudation so that the area in farmland desertification, arid and saltings increases 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 day by day serious of soybean cyst nematode Heterodera glycines harm to arid, the alkaline edatope of soybean cyst nematode Heterodera glycines happiness.
Soybean cyst nematode Heterodera glycines 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, soil is once infecting extremely difficult elimination of this class disease and pest, the resistant variety that at present seed selection has adaptability is the most economical effective measures of control soybean cyst nematode Heterodera glycines.And the research of enantiopathy gene is the basis of resistant variety seed selection.
Therefore it is significant to clone soybean cyst nematode resistance gene.At present, the soybean cyst nematode Heterodera glycines resistance site of having determined has more than 100 approximately, but but rarely has report according to these sites clone's gene.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 arranged, and this is the excavation of the disease-resistant gene condition of providing convenience.
Soybean cyst nematode Heterodera glycines has specificity to infecting of soybean, therefore, excavation to candidate gene can't in Arabidopis thaliana isotype plant, verify its resistance, and soybean is the plant that is difficult to transform of generally acknowledging, therefore, find a kind of method that can carry out fast the functional verification of resistance candidate gene extremely urgent.
Summary of the invention
Technical problem to be solved by this invention provides a kind of soybean cyst nematode resistance gene.
Described soybean cyst nematode Heterodera glycines resistance nucleotide sequence is such 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 lower hybridization, then uses 2 * SSC, and 0.1%SDS and 1 * SSC, 0.1%SDS respectively wash film once.
The recombinant vectors, transgenic cell line or the recombinant 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 resistance gene is used.
The Expression and Application of described gene fragment in the plant tissue that does not contain this gene also is the scope of protection of the invention.
Described plant tissue is root, stem, leaf, flower or seed.
Described plant is monocotyledons or dicotyledons.
Described monocotyledons is tobacco, and described dicotyledons is Arabidopis thaliana and any cultivation or wild soybean.
Describedly be expressed as expression.
Described expression and/or described excessively the expression are to express under greenhouse experiment by the test soil sample and the solution that contain soybean cyst nematode Heterodera glycines cyst, worm's ovum, larva.
Described larva is second instar larvae, the test sick soil of described soil sample for sick soil and aseptic fine sand evenly are mixed with 3: 1 ratio, described solution is every milliliter of suspension that contains at least 400 worm's ovums, 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.
Gene order among the present invention and the CaMV 35S promoter rear employing Agrobacterium rhizogenes that links together is infected the method for soybean cotyledon node, induce the kind of sense soybean cyst nematode Heterodera glycines to produce hairly root, and in the process that it is induced, goal gene is changed in the hairly root.The transgenic positive plant is carried out inoculated identification.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 is without obvious 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 the phenomenon such as the denudation so that the area in farmland desertification, arid and saltings increases year by year.Because the soybean cyst nematode Heterodera glycines happiness is arid, (humidity 60%, pH:8.0), so the expansion of desertification of land, arid and saltings area has caused the day by day serious of soybean cyst disease to alkaline edatope.Therefore it is significant to clone soybean cyst nematode resistance 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 Heterodera glycines, can obtain soybean cyst nematode Heterodera glycines is had the new soybean varieties of resistance, in Soybean production, 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 Solution on Chemical Reagents in Shanghai factory, and reagent is domestic 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, evaluation of resistance and the economical character correlation analysis of the non-microspecies specificity of soybean cyst nematode Heterodera glycines resistant variety, [J]. 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]. Exploitation of Agriculture in Heilongjiang science, 1993, (01). in.
Deceive the non-specific resistant strain L-10 of farming 37 and this laboratory seed selection as the parent take susceptible variety, 140 F2:6 that make up and the RIL material of F2:7, be documented in Wang Zizhen, evaluation of resistance and the economical character correlation analysis of the non-microspecies specificity of soybean cyst nematode Heterodera glycines resistant variety, [J]. Soybean Science, 2009, (04). in, the public can obtain from Northeast Agricultural University.
Soybean (Glycine max (L.) Merrill.) has kind ' the east farming 50 ' of high transformation efficiency, the section of being documented in is sparkling, agriculture bacillus mediated soybean cotyledon node and Regenerated from Hypocotyl Explants method relatively reach optimization, [J]. Soybean Science, 2010, (04). in, the public can obtain from Northeast Agricultural University.
The Agrobacterium rhyzogenesK599 bacterial strain (is preserved and is numbered: ACCC10060), Allen Kerr by Australia separates, and provided 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, among 2000, the 210:195-204..
No. 3 physiological strains gather from Soybean Cropping plot, Yichun soybean cyst nematode Heterodera glycines.And by soybean biological key lab of the Ministry of Education isolation identification.Be documented in Wang Zizhen, evaluation of resistance and the economical character correlation analysis of the non-microspecies specificity of soybean cyst nematode Heterodera glycines 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 Heterodera glycines gather tests the disease garden from Heilongjiang Academy of Agricultural Sciences grand celebration branch, and learns key lab's isolation identification by the Ministry of Education's soybean biological.Be documented in Wang Zizhen, evaluation of resistance and the economical character correlation analysis of the non-microspecies specificity of soybean cyst nematode Heterodera glycines 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, and High efficiency induction of soybean hairyroots and progagation ofthe soybean cyst nematode.Planta is among 2000, the 210:195-204..
Restriction enzyme and T
4Dna ligases etc. are all available from TAKARA company.
Quantitative test in following examples all arranges repeated experiments three times, results averaged.
Soybean cyst nematode Heterodera glycines resistance nucleotide sequence shown in SEQ ID NO.1, or be lower 1)-3) arbitrary shown in,
1) dna molecular that the nucleotide sequence that limits with SEQ ID NO.1 under stringent condition can be hybridized and can be expressed;
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 lower hybridization, then uses 2 * SSC, 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 embodiment 2 Soybean Resistance Cyst nematode genes
1. gene cloning
1) extraction of the total RNA of plant and reverse transcription: get respectively the L-10 of 12h behind the inoculation nematode, root and the leaf section tissue of black farming 37, 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 operates.
2) clone of disease-resistant candidate gene full length sequence: respectively take the cDNA of the root of the L-10 of above-mentioned reverse transcription, black farming 37 and leaf as template, with the gene specific primer (SEQ ID NO.3 and SEQ ID NO.4) that designs and the method clone goal gene that adopts landing-type PCR.System and the program of reaction are as shown in table 1.After PCR reaction finished, each sample was 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; evaluation and the order-checking of conversion and positive colony: reclaim the purifying that the PCR product is carried out in the test kit explanation according to the low dose of glue of Shanghai Hua Shun company; then; to reclaim fragment and be connected to pMD18-TEasyVector; take each monoclonal bacterium liquid as template, adopt with the used identical primer of clone and carry 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 Soybean Resistance Cyst nematode candidate gene sequence
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.
InterProScan the analysis showed that these 4 genes all include LRR structure and SERINE/THREONINE kinase domain (Fig. 2).Wherein the LRR structure is relevant with extracellular signal identification, kinases structure and the effect that the disease-resistant response member in downstream is had activation.
3. the expression of semi-quantitative RT-PCR analysis candidate gene
Take soybean β-Actin as 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 Heterodera glycines.
The checking of embodiment 3 Soybean Resistance Cyst nematode gene functions
1, the structure of plant expression vector
Clone SRC-J-6 gene order makes up Overexpression vector 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.Use respectively Xba I and Sac I double digestion with the pMD18-T-SRC-J-6 recombinant plasmid of goal gene and the pGFPGUS empty carrier that is attached thereto, electrophoresis identifies, and reclaims test kit with glue and reclaim large fragment about 1.6kb left and right sides SRC-J-6 segment and carrier 13kb.The small segment that obtains is connected with pGFPGUS carrier large fragment, obtains connecting product.Should connect product and transform the bacillus coli DH 5 alpha competent cell, carry out the 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 rapidly 37 ℃ of water-bath heat shock 5min, more rapid ice bath 2min;
3. add the liquid culture of 500 μ L YEP antibiotic-frees based on 28 ℃, gently shaking culture 3～5h recovery of 100rpm;
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 ℃, until grow the bacterium colony of suitable size, get single bacterium colony and extract plasmid with alkaline process, 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) Germination of Soybean Seed: 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 the dull and stereotyped (Kan of YEP
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 for infecting.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 touched off, release several bacterium liquid with syringe, and with the syringe needle that speckles with bacterium liquid in the wound back and forth several times, bacterium liquid is evenly distributed 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 rear about about 7 days, the plant of cotyledonary node place root shape thing projection is taken out from vermiculite, in distilled water, cut off along the about 1cm in projection bottom place, over-ground part is inserted root induction is housed in the triangular flask of sterilized water; After root grew, every about 1em of root clip was 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 turn 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 turn the SRC-J-6 gene plant and are the PCR positive (being numbered 10,16) (Fig. 4).
4, the inoculated identification of transfer-gen plant
Be transplanted into respectively in the matrix that contains No. 3 physiological strains of soybean cyst nematode Heterodera glycines detecting with the plant of transgenic positive root and negative control, blank, 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 slightly anxious current scour soil sample, cyst downstream stream flows on the 60 purpose screens, without soil sample, can stop on 20 purpose screens.
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 the propositions such as Riggs are proper experiment soil samples.
3) preparing experiment soil: soil humidity is to affect the important factor that soybean cyst nematode Heterodera glycines is grown, and too high soil humidity meeting is so that oxygen undersupply in the soil, and the hatching of inhibition soybean cyst nematode Heterodera glycines causes the death of soybean cyst nematode Heterodera glycines.To test soil sample mixes according to 3: 1 ratio with vermiculite, can not only make like this soil become soft, be conducive to 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 Heterodera glycines, 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 greatly reducing artificial and to the damage of plant.Plant according to all around spacing 5em kind, 5 of every kind of strain kinds will be planted check variety Lee68 in each box, have reduced the caused error of different tests condition.After emerging, list, stay three consistent young plants of growth conditions.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 count, and measure length for the root tissue of counting, 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 turns 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 accordingly corresponding minimum significantly extreme difference value LSR (table 3).
5% and the 1%SSR value table of table 3Duncan ' s multiple range test
The Difference of the mean number of four groups of data and LSR value in the table are compared, can judge (table 4) to 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 upper table result, we learn: there is utmost point significant difference in the interior average female borer population of the 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 Heterodera glycines has obvious contribution really.
＜110〉Northeast Agricultural University
＜120〉a kind of soybean cyst nematode resistance gene and application thereof
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 1656
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＜213〉soybean (Glycine max (L.) Merrill.)
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＜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 (1)
1. a soybean cyst nematode resistance gene is characterized in that its nucleotide sequence is shown in SEQ ID NO.1.
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 the pMD18-T carrier that is used for the clone, the pGFPGUS, the pBI121 that are used for expression, pCAMBIA carrier, any for the pJawoh18 carrier of RNAi.
4, the transgenic cell line or the recombinant bacterium that contain the described nucleotide sequence of claim 1.
5, 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.
6, the described nucleotides sequence of claim 1 is listed in the application in Chinese People's Anti-Japanese Military and Political College's beans Cyst nematode.
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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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| CN102206651B true CN102206651B (en) | 2013-01-02 |
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| 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 |
| 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 |
| 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 |
| CN110317825B (en) * | 2019-04-29 | 2023-03-31 | 聊城大学 | Agrobacterium rhizogenes-mediated soybean hairy root induced transformation method |
| CN110862996B (en) * | 2019-12-23 | 2020-12-25 | 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 |
| 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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| 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 |
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