CN102382804A - Soybean cyst nematode RNA helicase CGH-1, and coding gene and application thereof - Google Patents
Soybean cyst nematode RNA helicase CGH-1, and coding gene and application thereof Download PDFInfo
- Publication number
- CN102382804A CN102382804A CN2011103175555A CN201110317555A CN102382804A CN 102382804 A CN102382804 A CN 102382804A CN 2011103175555 A CN2011103175555 A CN 2011103175555A CN 201110317555 A CN201110317555 A CN 201110317555A CN 102382804 A CN102382804 A CN 102382804A
- Authority
- CN
- China
- Prior art keywords
- sequence
- soybean
- gene
- cyst nematode
- protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 50
- 241000498254 Heterodera glycines Species 0.000 title claims abstract description 16
- 101100495531 Caenorhabditis elegans cgh-1 gene Proteins 0.000 title abstract description 15
- 108090000944 RNA Helicases Proteins 0.000 title abstract 3
- 102000004409 RNA Helicases Human genes 0.000 title abstract 3
- 244000068988 Glycine max Species 0.000 claims abstract description 81
- 235000010469 Glycine max Nutrition 0.000 claims abstract description 80
- 241000196324 Embryophyta Species 0.000 claims abstract description 37
- 239000013604 expression vector Substances 0.000 claims abstract description 19
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 17
- 238000000034 method Methods 0.000 claims abstract description 16
- 230000009261 transgenic effect Effects 0.000 claims abstract description 15
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 8
- 238000011161 development Methods 0.000 claims abstract description 7
- 238000012217 deletion Methods 0.000 claims abstract 2
- 230000037430 deletion Effects 0.000 claims abstract 2
- 238000006467 substitution reaction Methods 0.000 claims abstract 2
- 239000012634 fragment Substances 0.000 claims description 37
- 108020004414 DNA Proteins 0.000 claims description 34
- 239000013612 plasmid Substances 0.000 claims description 27
- 239000002773 nucleotide Substances 0.000 claims description 8
- 125000003729 nucleotide group Chemical group 0.000 claims description 8
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 6
- 241001233957 eudicotyledons Species 0.000 claims description 6
- 241000894006 Bacteria Species 0.000 claims description 5
- 125000000539 amino acid group Chemical group 0.000 claims description 4
- 238000003259 recombinant expression Methods 0.000 claims description 4
- 241000209510 Liliopsida Species 0.000 claims description 3
- 238000010367 cloning Methods 0.000 claims description 3
- 230000000295 complement effect Effects 0.000 claims description 3
- 238000009395 breeding Methods 0.000 claims description 2
- 230000001488 breeding effect Effects 0.000 claims description 2
- 102000053602 DNA Human genes 0.000 claims 13
- 239000013598 vector Substances 0.000 claims 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 abstract description 20
- 102000040650 (ribonucleotides)n+m Human genes 0.000 abstract description 5
- 241000607479 Yersinia pestis Species 0.000 abstract description 5
- 230000000694 effects Effects 0.000 abstract description 2
- 241000238631 Hexapoda Species 0.000 abstract 2
- 108091030071 RNAI Proteins 0.000 abstract 2
- 230000009368 gene silencing by RNA Effects 0.000 abstract 2
- 208000015181 infectious disease Diseases 0.000 abstract 1
- 230000002401 inhibitory effect Effects 0.000 abstract 1
- 102000004190 Enzymes Human genes 0.000 description 15
- 241000589158 Agrobacterium Species 0.000 description 13
- 108090000790 Enzymes Proteins 0.000 description 13
- 239000002299 complementary DNA Substances 0.000 description 13
- 239000000203 mixture Substances 0.000 description 12
- 238000012408 PCR amplification Methods 0.000 description 11
- 230000003071 parasitic effect Effects 0.000 description 11
- 206010011732 Cyst Diseases 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 10
- 208000031513 cyst Diseases 0.000 description 10
- 108091008146 restriction endonucleases Proteins 0.000 description 10
- 101150034254 cgh-1 gene Proteins 0.000 description 8
- 239000007788 liquid Substances 0.000 description 8
- 239000003550 marker Substances 0.000 description 8
- 230000008521 reorganization Effects 0.000 description 8
- 241000589156 Agrobacterium rhizogenes Species 0.000 description 7
- 241000244206 Nematoda Species 0.000 description 7
- 235000018102 proteins Nutrition 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 6
- 230000029087 digestion Effects 0.000 description 6
- 239000003292 glue Substances 0.000 description 6
- 102000002322 Egg Proteins Human genes 0.000 description 5
- 108010000912 Egg Proteins Proteins 0.000 description 5
- 108060004795 Methyltransferase Proteins 0.000 description 5
- 230000003321 amplification Effects 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 230000012010 growth Effects 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 210000004681 ovum Anatomy 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 238000000246 agarose gel electrophoresis Methods 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 108020004999 messenger RNA Proteins 0.000 description 4
- 235000021251 pulses Nutrition 0.000 description 4
- 238000010839 reverse transcription Methods 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 230000009182 swimming Effects 0.000 description 4
- JXCKZXHCJOVIAV-UHFFFAOYSA-N 6-[(5-bromo-4-chloro-1h-indol-3-yl)oxy]-3,4,5-trihydroxyoxane-2-carboxylic acid;cyclohexanamine Chemical compound [NH3+]C1CCCCC1.O1C(C([O-])=O)C(O)C(O)C(O)C1OC1=CNC2=CC=C(Br)C(Cl)=C12 JXCKZXHCJOVIAV-UHFFFAOYSA-N 0.000 description 3
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 3
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 102000003960 Ligases Human genes 0.000 description 3
- 108090000364 Ligases Proteins 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 108091034057 RNA (poly(A)) Proteins 0.000 description 3
- 108091081024 Start codon Proteins 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000003757 reverse transcription PCR Methods 0.000 description 3
- 201000006681 severe congenital neutropenia Diseases 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 244000046052 Phaseolus vulgaris Species 0.000 description 2
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 2
- 239000002585 base Substances 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000002498 deadly effect Effects 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 230000008034 disappearance Effects 0.000 description 2
- 239000008157 edible vegetable oil Substances 0.000 description 2
- 239000003623 enhancer Substances 0.000 description 2
- 238000013467 fragmentation Methods 0.000 description 2
- 238000006062 fragmentation reaction Methods 0.000 description 2
- 101150054900 gus gene Proteins 0.000 description 2
- 230000001744 histochemical effect Effects 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- 230000008676 import Effects 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 230000032696 parturition Effects 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 238000012797 qualification Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 230000005026 transcription initiation Effects 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 108010011170 Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly Proteins 0.000 description 1
- 241000219194 Arabidopsis Species 0.000 description 1
- 108700019444 Arabidopsis CCD7 Proteins 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000244203 Caenorhabditis elegans Species 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 206010058314 Dysplasia Diseases 0.000 description 1
- XZWYTXMRWQJBGX-VXBMVYAYSA-N FLAG peptide Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@@H](N)CC(O)=O)CC1=CC=C(O)C=C1 XZWYTXMRWQJBGX-VXBMVYAYSA-N 0.000 description 1
- 102100022887 GTP-binding nuclear protein Ran Human genes 0.000 description 1
- 101000774835 Heteractis crispa PI-stichotoxin-Hcr2o Proteins 0.000 description 1
- 108010093488 His-His-His-His-His-His Proteins 0.000 description 1
- 101000620756 Homo sapiens GTP-binding nuclear protein Ran Proteins 0.000 description 1
- 108090000144 Human Proteins Proteins 0.000 description 1
- 102000003839 Human Proteins Human genes 0.000 description 1
- 108700005090 Lethal Genes Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 1
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 108010002747 Pfu DNA polymerase Proteins 0.000 description 1
- 101100393821 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) GSP2 gene Proteins 0.000 description 1
- 108020004459 Small interfering RNA Proteins 0.000 description 1
- 208000037065 Subacute sclerosing leukoencephalitis Diseases 0.000 description 1
- 206010042297 Subacute sclerosing panencephalitis Diseases 0.000 description 1
- 108091036066 Three prime untranslated region Proteins 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 1
- 108010028230 Trp-Ser- His-Pro-Gln-Phe-Glu-Lys Proteins 0.000 description 1
- 108090000704 Tubulin Proteins 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 230000036579 abiotic stress Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- QGLZXHRNAYXIBU-WEVVVXLNSA-N aldicarb Chemical compound CNC(=O)O\N=C\C(C)(C)SC QGLZXHRNAYXIBU-WEVVVXLNSA-N 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 238000012197 amplification kit Methods 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000009402 cross-breeding Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 150000002240 furans Chemical class 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 230000003020 moisturizing effect Effects 0.000 description 1
- 238000009335 monocropping Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 238000003976 plant breeding Methods 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001902 propagating effect Effects 0.000 description 1
- 230000003161 proteinsynthetic effect Effects 0.000 description 1
- 238000012113 quantitative test Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
- 125000000858 thiocyanato group Chemical group *SC#N 0.000 description 1
- 238000007862 touchdown PCR Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了一种大豆胞囊线虫RNA解旋酶CGH-1及其编码基因与应用。本发明提供的蛋白质，是如下(a)或(b)：(a)由序列表中序列1所示的氨基酸序列组成的蛋白质；(b)将序列1的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与大豆胞囊线虫发育相关的由序列1衍生的蛋白质。本发明提供了大豆胞囊线虫RNA解旋酶CGH-1及其编码基因，并基于该编码基因设计了RNAi表达载体，将该RNAi表达载体导入植物，可在植物中所述基因的dsRNA，当病虫在摄入转基因植物后，生殖或发育产生严重障碍，从而抑制病虫的侵染和传播。通过本发明的方法可以获得具有很强抗虫活性的转基因植物，为进一步培育广谱抗虫性的大豆新品种奠定基础。The invention discloses a soybean cyst nematode RNA helicase CGH-1 and its coding gene and application. The protein provided by the present invention is the following (a) or (b): (a) a protein consisting of the amino acid sequence shown in Sequence 1 in the Sequence Listing; (b) the amino acid sequence of Sequence 1 after one or more Substitution and/or deletion and/or addition of a group and a protein derived from Sequence 1 associated with soybean cyst nematode development. The present invention provides soybean cyst nematode RNA helicase CGH-1 and its encoding gene, and an RNAi expression vector is designed based on the encoding gene, and the RNAi expression vector is introduced into a plant, and the dsRNA of the gene in the plant can be used as After ingesting the transgenic plants, the pests will have serious obstacles to reproduction or development, thereby inhibiting the infection and spread of the pests. Through the method of the invention, transgenic plants with strong insect-resistant activity can be obtained, which lays the foundation for further cultivating new soybean varieties with broad-spectrum insect-resistant properties.
Description
Technical field
The present invention relates to a kind of soybean SCN rna helicase enzyme CGH-1 and encoding sox and application.
Background technology
Soybean originates from China, is the important source of human protein and edible oil, whole world edible oil 31% from soybean.China is soybean consumption big country, and year total quantity consumed surpasses 4,000 ten thousand tons, and domestic soybean gross annual output amount has only 1,700 ten thousand tons of less thaies.Therefore, must rely on a large amount of imports to fill a vacancy.The subject matter of soybean in China is that per unit area yield is low, and 2008 annual per unit area yields have only 114 kilograms/mu, are merely 70% of the average per unit area yield in the world, and to compare gap bigger with advanced countries such as the U.S..Cause the low reason of China's soybean yields except problems such as kind and plantation, disease and pest is a major reason.
Soybean cyst nematode is in the world wide soybean to be produced one of the most serious disease and pest of harm.In China, soybean cyst nematode is the second largest disease that is only second to soybean Mosaic, and it mainly is distributed in northeast and two soybean main producing regions of the Yellow River and Huai He River.Wherein serious with the district of continuous cropping for many years especially with arid and husky alkali Lao Dou district.After soybean receives the sick infringement of SCN, the lighter's underproduction 20%～30%, the severe patient underproduction reaches 70%～80%, and more very the person understands the total crop failure of occurrence of large-area planting site.(Heterodera glycines, Soybean cyst nematode SCN) causes soybean cyst nematode, in the soybean whole growth process, all can take place by SCN.SCN parasitizes on the soybean root, causes the taproot and lateral root dysplasia, and fibrous root increases, and whole root system is the beard and hair shape.On the fibrous root give birth to big or small about 0.5 millimeter convexity of white to yellow-white, be the cyst of nematode.The root nodule of the plant old complaint of being injured seldom or non-nodulating; Aerial growth is slow, and seedling phase cotyledon and true leaf flavescence are big; The growth of grave illness seedling stops, and even dead.The plant that is injured generally shows as short and small, yellow leaf, and bud clusters, and internode shortens, and postpone flowering period, bears pods to lack and maybe can not bear pods.
In view of this, the sick control of SCN is that soybean produces the challenge that is faced for a long time.Though use highly toxic sterilant such as DDT, furans pellet, aldicarb etc. can kill nematode, can cause serious environmental to pollute, thereby disabled or restriction use.The problem of utilizing traditional procedure of breeding cultivation soybean resistant variety to run into is that the program of backcrossing is loaded down with trivial details, and resistance is identified complicated.Through kind of back cross breeding method seed selection, need ten years at least, limited the seed selection of Chinese People's Anti-Japanese Military and Political College's beans SCN new variety widely.
The appearance of RNA perturbation technique (RNAi) makes the high yielding soybeans kind and other crop varieties that obtain to have the anti-SCN of wide spectrum through transgenic technology become possibility.The RNAi phenomenon is found in the plant at first and in non-parasitic nematode-beautiful nematode (Caenorhabditis elegans), is able to and discloses, and in plant, fruit bat and vertebrates, is further confirmed in the animal subsequently.After beautiful nematode contains the intestinal bacteria of dsRNA in absorption; In its enteron aisle, digest, dsRNA wherein can be absorbed and in the iuntercellular transmission, arrive in most tissues and the cell; Cause the specificity degraded of the coded mRNA of target gene, thereby influence the function of goal gene.
Summary of the invention
The purpose of this invention is to provide a kind of soybean SCN rna helicase enzyme CGH-1 and encoding sox and application.
Protein provided by the invention available from soybean SCN (Heterodera glycines, Soybean cyst nematode), is (a) or (b) as follows:
(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 1;
(b) with the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and grow relevant by sequence 1 deutero-protein with the soybean SCN.
In order to make the protein in (a) be convenient to purifying, proteinic N-terminal or C-terminal that can the aminoacid sequence shown in the sequence 1 is formed in by sequence table connect label as shown in table 1.
The sequence of table 1 label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being generally 5) | RRRRR |
| Poly-His | 2-10 (being generally 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag?II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
Above-mentioned (b) but in the protein synthetic, also can synthesize its encoding sox earlier, carry out biology again and express and to obtain.Proteinic encoding sox in above-mentioned (b) can be through the codon with one or several amino-acid residue of disappearance in the dna sequence dna shown in the sequence in the sequence table 2; And/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.
The gene of encoding said proteins also belongs to protection scope of the present invention.
Said gene can be following 1) to 4) in arbitrary described dna molecular:
1) sequence 2 of sequence table is from the dna molecular shown in 5 ' terminal the 499th to 1839 Nucleotide;
2) dna molecular shown in the sequence 2 in the sequence table;
3) under stringent condition with 1) or 2) the dna sequence dna hybridization that limits and the dna molecular of coding soybean SCN development associated protein;
4) with 1) or 2) dna sequence dna that limits has 90% above homology and the dna molecular of the soybean SCN development associated protein of encoding.
Above-mentioned stringent condition can be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, hybridization and wash film under 65 ℃ of conditions.
The recombinant expression vector, expression cassette, transgenic cell line or the reorganization bacterium that contain said gene all belong to protection scope of the present invention.
The present invention also protects the recombinant plasmid (interference plasmid) that contains double chain DNA fragment A and double chain DNA fragment B; Said double chain DNA fragment B and said double chain DNA fragment A are reverse complementary sequence; Said double chain DNA fragment A like the sequence 2 of sequence 2 from shown in 5 ' terminal 523 to 1090 Nucleotide.Said recombinant plasmid also can comprise 3 ' end untranslated zone of foreign gene, promptly comprises the dna fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.Said polyadenylic acid signal can guide polyadenylic acid to join 3 ' end of mRNA precursor.When making up said recombinant plasmid, before its transcription initiation Nucleotide, can add any enhancement type promotor or constitutive promoter, they can use separately or be used in combination with other promotor; When making up said recombinant plasmid; Also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser zones can be ATG initiator codon or neighboring region initiator codon etc.; But must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of said translation wave and initiator codon is widely, can be natural, also can be synthetic.Translation initiation region can be from transcription initiation zone or structure gene.For the ease of transgenic plant being identified and screening; Can process used recombinant plasmid, can produce enzyme or the gene of luminophor, antibiotic marker thing or the anti-chemical reagent marker gene etc. of colour-change with resistance as adding the coding that in plant, to express.Also can not add any selected marker, directly with adverse circumstance screening transformed plant.Said recombinant plasmid can be different MCSs at carrier pZH01 and inserts the recombinant plasmid that said double chain DNA fragment A and said dna fragmentation B obtain respectively.Said recombinant plasmid specifically can be and is inserting the recombinant plasmid that the said double chain DNA fragment B of insertion obtains between said double chain DNA fragment A, Kpn I and the Sac I restriction enzyme site between the Xba of skeleton carrier pZH01 I and the Sal I restriction enzyme site.
The present invention also protects a kind of method of cultivating transgenic plant, is said recombinant plasmid is imported in the purpose plant, obtains the transgenic plant that soybean SCN resistance is higher than said purpose plant.Conventional biological method transformed plant cells or tissues such as said recombinant plasmid can Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity be led through using, agriculture bacillus mediated (like Agrobacterium rhizogenes K599), particle gun, and the plant transformed tissue cultivating become plant.Said purpose plant both can be that monocotyledons also can be a dicotyledons.Said dicotyledons specifically can be soybean, like big pulse family rich No. 1.Said soybean SCN specifically can be No. 4 physiological strains of soybean SCN.
Said albumen, or said gene, or said recombinant expression vector, expression cassette, transgenic cell line or reorganization bacterium, or said recombinant plasmid all can be used for plant breeding, as cultivating Chinese People's Anti-Japanese Military and Political College beans SCN plant.Said purpose plant both can be that monocotyledons also can be a dicotyledons.Said dicotyledons specifically can be soybean, like big pulse family rich No. 1.Said soybean SCN specifically can be No. 4 physiological strains of soybean SCN.
The invention provides soybean SCN rna helicase enzyme CGH-1 and encoding sox thereof; And designed a kind of RNAi expression vector based on this encoding sox, and this RNAi expression vector is imported plant, can be at the dsRNA of gene described in the plant; After sick worm is being taken in transgenic plant; DsRNA gets into enteron aisle, and the siRNA of generation can further get in other the tissue and cell, causes the inactivation of said gene; Make the reproduction of disease worm or grow to produce serious hindrance, thereby suppress infecting and propagating of disease worm.Transgenic plant through method provided by the invention can obtain to have very strong anti-insect activity lay the foundation for further cultivating the new soybean varieties with wide spectrum pest-resistant property.
Description of drawings
Fig. 1 is the electrophoresis result with KOD Plus archaeal dna polymerase amplification SCN cgh-1 gene fragment; 2 is SCN cgh-1 gene fragment; M is a molecular weight marker; Other swimming lane is and the irrelevant sample of this patent.
Fig. 2 reclaims the segmental electrophoresis result that obtains for glue; 3 is SCN cgh-1 gene fragment; M is a molecular weight marker; Other swimming lane is and the irrelevant sample of this patent.
Fig. 3 is the segmental electrophoresis result that 5 '-RACE and 3 '-the RACE amplification obtains; 2:SCN cgh-15 '-RACE; 7,8:SCN cgh-13 '-RACE; M is a molecular weight marker; Other swimming lane is and the irrelevant sample of this patent.
Fig. 4 is the structural representation of SCN cgh-1RNAi expression vector.
Fig. 5 identifies electrophorogram for the PCR of reorganization Agrobacterium bacterium liquid; M:2000bp Marker; Swimming lane 3 is the pcr amplification product (cgh-1 gene fragment) of reorganization Agrobacterium bacterium liquid.
Fig. 6 is the photo of GUS histochemical stain.
Fig. 7 is the electrophorogram of total RNA of each group soybean; 1-5: the total RNA of the root system of experimental group soybean; CK: the total RNA of root system of control group first soybean.
Fig. 8 identifies electrophorogram for the RT-PCR of each group soybean; M:2000bp Marker; 1-5: the pcr amplification product of experimental group soybean; CK: the pcr amplification product of control group first soybean.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment like no specified otherwise, is ordinary method.Used test materials among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.Quantitative test in following examples all is provided with repeated experiments three times, results averaged.
X-Gluc solution (pH 7.0) solvent is a water; Solute and concentration thereof are following: 50mM Na
3PO
4, 10mM Na
2EDTA, 0.1% (volume ratio) Triton X-100,0.1M K
3[Fe (CN)
6], 0.1M K
4[Fe (CN)
6], 0.5g/L X-Gluc and 20% (volume ratio) methyl alcohol.
Soybean SCN (No. 4 physiological strains): the public can obtain with developmental biology institute from Chinese Academy of Sciences's heredity; Reference: Lu Weiguo, Gai Junyi, sample survey and the research of Li Weidong the Yellow River and Huai He River area soybean SCN physiological strain. Scientia Agricultura Sinica, 2006,39 (2): 306-312.
Rich No. 1 of big pulse family: the public can obtain with developmental biology institute from Chinese Academy of Sciences's heredity; Reference: Qi-Yun Zhou, Ai-Guo Tian, Hong-Feng Zou, Zong-Ming Xie; Gang Lei, Jian Huang, Chun-Mei Wang, Hui-Wen Wang; Jin-Song Zhang, and Shou-Yi Chen.Soybean WRKY-type transcription factor genes, GmWRKY13; GmWRKY21, and GmWRKY54, confer differential tolerance to abiotic stresses in transgenic Arabidopsis plants.Plant Biotechnology Journal; 2008,6,486-503.
Soybean susceptible variety Lee: the public can obtain with developmental biology institute from Chinese Academy of Sciences's heredity; Reference: Lu Weiguo, Yuan Daohua, Li Jinying, Li Haichao, Wen Zixiang opens brightness. the variation of the anti-SCN gene of soybean different generations heritability, Henan agricultural sciences, 2010, (2): 24-27.
Carrier pZH01: the public can obtain with developmental biology institute from Chinese Academy of Sciences's heredity; Reference: Junhuang Zou, Shuying Zhang, Weiping Zhang; Gang Li, Zongxiang Chen, Wenxue Zhai; Xianfeng Zhao; Xuebiao Pan, Qi Xie and Lihuang Zhu.The rice HIGH-TILLERING DWARF1 encoding an ortholog of Arabidopsis MAX3 is required for negative regulation of the outgrowth of axillary buds.The Plant Journal, 2006; 48,687-696.
Agrobacterium rhizogenes K599: the public can obtain with developmental biology institute from Chinese Academy of Sciences's heredity; Reference: Attila Kereszt, Dongxue Li, Arief Indrasumunar; Cuc DT Nguyen; Sureeporn Nontachaiyapoom, Mark Kinkema & Peter M Gresshoff.Agrobacterium rhizogenes-mediated transformation of soybean to study root biology, NATURE PROTOCOLS; 2007,2 (4): 948-952.
The discovery of embodiment 1, soybean SCN SCN cgh-1 albumen and encoding sox thereof
One, the selection of soybean SCN SCN cgh-1 albumen and encoding sox thereof
In the DB of NCBI, by in May, 2009, the correlated series of soybean SCN has 108045, comprising 81873 nucleotide sequences, and 26172 est sequences, 183 mRNA and 252 protein sequences.Alkharouf et al handles 24438 est sequences and 231 genome sequences of soybean SCN; And find with the comparison of the gene order of beautiful nematode, have 8334 gene orders similar between the two, and this is wherein; There are 1508 genes that deadly phenotype is arranged in nematode; Have the gene of deadly phenotype to divide into groups according to the sequence similarity degree to these, be divided into 6 groups, wherein the most similar (E-values 0 and 1E-100) gene has 150.The contriver is than right 150 genes and human sibship, and factor such as considered amino acid sequence length selected 13 candidates' lethal gene to clone, and one of them gene is that the present invention finds and the gene of protection.
Two, the discovery of soybean SCN SCN cgh-1 albumen and encoding sox thereof
1, the TRIZOL method is extracted total RNA from the cyst of soybean SCN (No. 4 physiological strains).
2, be cDNA (with the RACE test kit of Clontech: SMART RACE cDNA Amplification Kit, Cat No.634923) with total RNA reverse transcription.
3, the acquisition of gene fragment
(1) design following primer to (target sequence 628bp) through comparison and beautiful nematode cgh-1 homologous soybean SCN est sequence in the DB of NCBI, and according to est sequence:
cgh-1HG?Nhe?I?NS：5’-CCGCTAGCGTTTCTTCTTCGCGTCCGAT-3’；
cgh-1HG?Xho?I?CAS：5’-GGCTCGAGTAGAAACTGAACAAGACGGTC-3’。
(2) cDNA with step 2 is a template, with step 3 designed primer to carrying out pcr amplification.
Pcr amplification system: cDNA 2ul, two each 2ul of primer, MgSO
4Solution 2ul, KOD Plus archaeal dna polymerase 1ul, KOD Plus dna polymerase buffer liquid 5ul, dNTP 5ul, DMSO 1ul, ddH
2O 30ul.
Pcr amplification program (BIO-RAD iCycler): 94 ℃ of 2min, 35 * (94 ℃ of 15s, 57 ℃ of 30s, 68 ℃ of 2min), 68 ℃ of 5min, 12 ℃ of preservations.
The agarose gel electrophoresis result of pcr amplification product sees Fig. 1.
(3) cut glue with OMEGA Gel Extraction Kit (D2500-01) and reclaim band, the agarose gel electrophoresis figure that reclaims band sees Fig. 2.
(4) the PCR fragment that reclaims with the NEB digestion with restriction enzyme.
Enzyme is cut system: glue reclaims product 43.5ul, Nhe I 1ul, Xho I 1ul, Buffer 25ul, BSA 0.5ul.37 ℃ of enzymes are cut and are spent the night, and use OMEGA Gel Extraction Kit (D2500-01) to cut glue then and reclaim.
(5) with NEB digestion with restriction enzyme carrier pPD129.36.
Enzyme is cut system: pPD129.365ul, Hind III 1ul, Xho I 1ul, Buffer 25ul, BSA 0.5ul.37 ℃ of enzymes are cut and are spent the night, and use OMEGA Gel Extraction Kit (D2500-01) to cut glue then and reclaim.
(6) step (4) is connected with NEB T4 Ligase with the fragment that step (5) reclaims.
Linked system: pPD129.36 (enzyme is cut the back) 1ul, gene fragment (enzyme is cut the back) 12ul, T4 Ligase Buffer1.5ul, T4 Ligase 1ul.
(7) connect product heat shock method and transform DH5 α competent cell, be coated with LB flat board (Amp+), 37 ℃ of overnight cultures; After waiting to grow the clone; Choose mono-clonal and identify, obtain positive colony (8) (11), send the order-checking of the English Weihe River prompt base (Shanghai) trade Co., Ltd with the Cloning PCR method.Sequencing result is seen the sequence 3 of sequence table.
4, the segmental acquisition of 5 '-RACE fragment and 3 '-RACE
(1) the PCR Master Mix in the preparation table 2, and gentle vortex mixing avoids producing bubble in the process.
Table 2PCR Master Mix forms
| Composition | Each reaction (ul) | 9 reactions (ul) |
| PCR-Grade?Water | 34.5 | ?310.5 |
| 10X?Advantage?2PCR?Buffer | 5.0 | ?45.0 |
| dNTP?Mix(10mM) | 1.0 | ?9.0 |
| 50X?Advantage?2Polymerase?Mix | 1.0 | ?9.0 |
| Total?Volume | 41.5 | ?373.5 |
(2) on the basis of step (1), the reacted constituent of 5 '-RACE PCR and 3 '-RACE PCR is seen table 3 and table 4.
The reactive component of table 35 '-RACE PCR
| Composition | Each reaction (ul) |
| 5’-RACE-Ready?cDNA | 2.5 |
| UPM(10X) | 5 |
| GSP1 | 1 |
| Master?Mix | 41.5 |
| Total?Volume | 50.0 |
The reactive component of table 43 '-RACE PCR
| Composition | Each reaction (ul) |
| 5’-RACE-Ready?cDNA | 2.5 |
| UPM(10X) | 5 |
| GSP2 | 1 |
| Master?Mix | 41.5 |
| Total?Volume | 50.0 |
(3) use BioRad iCycler PCR appearance working procedure (Touchdown PCR), condition is: 5 * (94 ℃ of 30s, 72 ℃ of 3min), 5 * (94 ℃ of 30s, 70 ℃ of 30s, 72 ℃ of 3min), 27 * (94 ℃ of 30s, 68 ℃ of 30s, 72 ℃ of 3min), and 4 ℃ of for ever.Amplification is seen Fig. 3.
(4) behind the agarose gel electrophoresis, cut glue with OMEGA Gel Extraction Kit (D2500-01) and reclaim.
(5) the pEASY-Blunt Simple Cloning Kit with full formula King Company makes up the clone; Obtain positive colony; And send Invitrogen Corp. (INVITROGEN) order-checking, and the sequencing result of 3 '-RACE is seen the sequence 4 of sequence table, the sequencing result of 5 '-RACE is seen the sequence 5 of sequence table.
With the cgh-1 of albumen called after SCN shown in the sequence 1 of sequence table albumen (forming), be a kind of rna helicase enzyme of ATP dependent form by 446 amino-acid residues.With the proteic encoding sox called after of SCN cgh-1 SCN cgh-1 gene; Its cDNA shown in the sequence 2 of sequence table (2128bp), the long 498bp of 5 ' UTP, coding head of district 1341bp (sequence 2 of sequence table is from 5 ' terminal 499-1839 position Nucleotide), the long 289bp of 3 ' UTR.
One, the structure of SCN cgh-1RNAi expression vector
1, from the cyst of soybean SCN (No. 4 physiological strains), extract total RNA, and reverse transcription is cDNA.
2, with cDNA be template, the primer of forming with Primer-F and Primer-R carries out pcr amplification to (target sequence is that the sequence 2 of sequence table is from 5 ' terminal 523-1090 position Nucleotide, i.e. SCN cgh-1 gene fragment), obtains pcr amplification product.
Primer-F:5 '-CGC
TCT AGA GAG CTCCGT CCG ATG GAA GAT TG-3 ' (underscore mark Xba I enzyme cuts recognition site and Sac I enzyme is cut recognition site);
Primer-R:5 '-ACG C
GT CGA CGG TAC CGG AAG TCC TGG GAA AGG-3 ' (underscore mark Sal I enzyme cuts recognition site and Kpn I enzyme is cut recognition site);
Pcr amplification system (50 μ l): cDNA 1 μ l, 10 * buffer, 5 μ l, dNTP (10mM) 1 μ l, Primer-F1 μ l, Primer-R 1 μ l, Pfu DNA Polymerase (TaKaRa) 1 μ l, ddH
2O 40 μ l.
Pcr amplification condition: 94 ℃ of 3min; 94 ℃ of 30s, 50 ℃ of 30s, 72 ℃ of 30s, 30 circulations; 72 ℃ of 10m.
Pcr amplification product carries out 1% agarose gel electrophoresis, reclaims test kit with gel and reclaims the purpose fragment about 500bp.
3, the structure of SCN cgh-1RNAi expression vector
1. use the purpose fragment of restriction enzyme Sac I and Kpn I double digestion step 2, reclaim enzyme and cut product.
2. use restriction enzyme Sac I and Kpn I double digestion carrier pZH01, reclaim carrier framework (about 11.5Kp).
3. step enzyme is 1. cut product and be connected, obtain the recombinant plasmid first with step carrier framework 2..
4. use the purpose fragment of restriction enzyme XbaI and Sal I double digestion step 2, reclaim enzyme and cut product.
5. use restriction enzyme Xba I and Sal I double digestion step recombinant plasmid first 3., reclaim carrier framework (about 12Kp).
6. step enzyme is 4. cut product and be connected, obtain recombinant plasmid second, i.e. the SCNcgh-1RNAi expression vector with step carrier framework 5..According to sequencing result; It is following that SCN cgh-1RNAi expression vector is carried out structrual description: skeleton carrier is pZH01; Between the Xba of skeleton carrier I and Sal I restriction enzyme site, insert double chain DNA fragment A (like sequence 2 from shown in 5 ' the terminal 523-1090 position Nucleotide), between the KpnI of skeleton carrier and Sac I restriction enzyme site, inserted double chain DNA fragment B; Double chain DNA fragment B and double chain DNA fragment A are reverse complementary sequence.The structural representation of SCN cgh-1RNAi expression vector is seen Fig. 4.
Two, the conversion of soybean root system is identified with expression
1, with SCN cgh-1RNAi expression vector transforming agrobacterium rhizogenes K599, obtains the Agrobacterium of recombinating.The PCR of reorganization Agrobacterium bacterium liquid identifies that the primer of Primer-F and Primer-R composition (adopt to) electrophorogram sees Fig. 5.Reorganization Agrobacterium bacterium liquid has increased and target fragment band of the same size, explains that SCN cgh-1RNAi expression vector has been integrated into Agrobacterium rhizogenes K599.
With carrier pZH01 transforming agrobacterium rhizogenes K599, obtain contrasting Agrobacterium.
2, soybean root system transforms
(1) conversion of experimental group soybean
HSBC Branch No. 1 soybean seed disinfection, disinfection Zhi sown in stone; 5 days, seedling germination; recombinant Agrobacterium was inoculated seedlings cotyledon node, and then transplant the seedlings, moisturizing growth; 2-3 weeks, the hair root appear; when the hair root 5-10 cm, cut the original root, transplanted into the soil (d6cm × h12cm plastic cup) growth of each plant a plastic cup.
(2) conversion of control group first soybean
To contrast Agrobacterium replaces the reorganization Agrobacterium to carry out step (1) operation.
(3) conversion of control group second soybean
Carry out the operation of step (1), but do not inoculate reorganization Agrobacterium bacterium liquid.
3, identify the expression of hairly root gus gene
In the step 2, after Agrobacterium infected for 4 weeks, the root system of getting experimental group soybean and control group second soybean respectively carried out histochemical stain, identified the expression of gus gene.Concrete steps are following: root system is put into 37 ℃ of dark overnight cultures of X-Gluc solution; At room temperature use 70% (volume ratio) aqueous ethanolic solution to clean 30min then; Then root system is put into water, the blueness under the white background is GUS and expresses the site.
The result sees Fig. 6.It is blue that the root system of control group second soybean does not become, and it is blue that the root of experimental group soybean all becomes, transformation efficiency about 100%.The GUS coloration result shows, adopts the rich technology of recombinant plasmid importing Agrobacterium rhizogenes soybean transformation section effective.
4, RT-PCR analyzes the expression of SCN cgh-1 fragment at soybean root
In the step 2, after Agrobacterium infected for 4 weeks, get the root system of 1 strain control group first soybean and 5 strain experimental group soybean; Extract total RNA (see figure 7); Reverse transcription becomes cDNA, and the cDNA template is all diluted 5 times, carries out the cgh-1 Gene RT-PCR amplification primer of Primer-F and Primer-R composition (adopt to).With tubulin gene adjustment template consumption, when the brightness basically identical of amplified production, can confirm that template concentrations is consistent.
Trizol Reagent is available from Invitrogen company, and the reverse transcription test kit is available from precious biotechnology (Dalian) ltd, TaKaRa Code:D6210A.PCR reaction system (25 μ l): cDNA 2 μ l, Primer-F 1 μ l, Primer-R 1 μ l, 2 * TagPCR Master Mix, 12.5 μ l, ddH
2O 8.5 μ l.Pcr amplification program: 94 ℃ of 4min; 94 ℃ of 40s; 52 ℃ of 40s; 72 ℃ of 50s (30cycles); 72 ℃ of 5min; 10 ℃ of 1h.
Among Fig. 7, the banding pattern of the RNA 28S ribosome-RNA(rRNA) of carrying and 18S ribosome-RNA(rRNA) more clear, no conditions of streaking explains that the RNA that extracts has integrity preferably.
Amplification is as shown in Figure 8, and control group first soybean does not amplify the purpose band, and 5 strain experimental group soybean root systems all amplify the target stripe of 568bp.Show that SCN cgh-1 gene fragment has obtained correctly transcribing in the soybean root system genome.
5, the resistance of soybean SCN is identified
In the step 2, after Agrobacterium infected for 4 weeks, in each plastic cup, add the pathogenic liquid (every milliliter of ovum that contains No. 4 physiological strains of 400 soybean SCNs of having an appointment) of 5ml.Keep the soil moisture at 24-30 ℃ behind the pathogenic liquid of inoculation, calculate the cyst number after 30 days.Experiment is carried out three times and is repeated results averaged.Soybean strain system to the resistance level of soybean SCN generally with parasitic exponential representation.Parasitic index be high anti-kind between 0-9, resists between the 10-30 being, and between the 31-60 middle sense, be susceptible variety greater than 60.
Parasitic index=(on the every gram root of test plant on the every gram root of cyst number/soybean susceptible variety Lee of giving birth to the cyst number of giving birth to) * 100.
Each is organized parasitic exponential statistics and sees table 5.
Table 5 is respectively organized parasitic exponential statistics
| The experimental group soybean | Control group first soybean | Control group second soybean | Soybean susceptible variety Lee | |
| Inoculation strain number | 32 | 37 | 5 | 22 |
| Average cyst number/g root | 65 | 111 | 109 | 276 |
| The P value | 0.0002 | |||
| Parasitic index | 23 | 40 | 39 | 100 |
| The IP qualification result | In anti- | Middle sense | Middle sense | |
| Worm's ovum number/g root | 8798 | 18811 | ||
| The P value | ＜0.0001 | |||
| Single cyst worm contains the number of ovum | 128 | 194 | ||
| The P value | 0.122 |
The result of table 5 shows; The number that imports cyst on the every gram root of plant of SCN cgh-1RNAi expression vector significantly reduces, the number that the worm's ovum number significantly lowers, single cyst contains ovum on every gram root also reduces greatly, and promptly the SCNcgh-1RNAi expression vector has suppressed the ability of growing and the prolificacy of SCN.According to parasitic index judgement criteria; Can know by table 5; The parasitic index that contains the experimental group soybean is 23; Parasitic index standard of perfection is anti-in being, because the big rich No. 1 long-term qualification result of pulse family proves the kind (parasitic index about 40) of No. 4 physiological strains of sense soybean SCN in, so changing over to of SCN cgh-1RNAi expression vector significantly improved the resistance of soybean root system to SCN.
Claims (10)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110317555 CN102382804B (en) | 2011-10-18 | 2011-10-18 | Soybean cyst nematode RNA helicase CGH-1, and coding gene and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110317555 CN102382804B (en) | 2011-10-18 | 2011-10-18 | Soybean cyst nematode RNA helicase CGH-1, and coding gene and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102382804A true CN102382804A (en) | 2012-03-21 |
| CN102382804B CN102382804B (en) | 2013-06-12 |
Family
ID=45822638
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110317555 Expired - Fee Related CN102382804B (en) | 2011-10-18 | 2011-10-18 | Soybean cyst nematode RNA helicase CGH-1, and coding gene and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102382804B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109627311A (en) * | 2019-01-31 | 2019-04-16 | 中国农业大学 | Cereal cyst nematode HaHSP4 albumen and its encoding gene and application |
| CN116178515A (en) * | 2022-10-26 | 2023-05-30 | 河南农业大学 | Application of Glyma.19G26700 protein in cultivation of soybean cyst nematode resistant plant |
-
2011
- 2011-10-18 CN CN 201110317555 patent/CN102382804B/en not_active Expired - Fee Related
Non-Patent Citations (2)
| Title |
|---|
| WANG ET AL.: "ADY41281", 《GENE BANK》 * |
| 陈亚洁: "登革2型病毒NS3蛋白NTPase/RNA解旋酶结构域的原核表达及活性研究", 《中华微生物学和免疫学杂质》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109627311A (en) * | 2019-01-31 | 2019-04-16 | 中国农业大学 | Cereal cyst nematode HaHSP4 albumen and its encoding gene and application |
| CN116178515A (en) * | 2022-10-26 | 2023-05-30 | 河南农业大学 | Application of Glyma.19G26700 protein in cultivation of soybean cyst nematode resistant plant |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102382804B (en) | 2013-06-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101906155A (en) | A kind of plant drought resistance, salt tolerance related protein ErNAC7 and its coding gene and application | |
| CN102634522A (en) | Gene for controlling rice fertility, encoded protein and application thereof | |
| CN114437188A (en) | The secretory protein elicitor PlPeL8 of Phytophthora litchi and its application | |
| CN102653763A (en) | Meloidogyne javanica dominant-effect gene (Mj-nulg), related protein and application of Mj-nulg | |
| CN105274101B (en) | The RNAi expression vector and its construction method of colorado potato bug TOR genes and application | |
| CN102382804A (en) | Soybean cyst nematode RNA helicase CGH-1, and coding gene and application thereof | |
| CN101831436A (en) | Method for breeding adverse-resistant plant | |
| CN1308345C (en) | Gossypium barbadense lipoid transition protein and its coding gene and application | |
| CN105349505B (en) | A plant drought and salt tolerance related protein AsSnRK and its encoding gene and application | |
| CN104046633B (en) | Resistance Strain of Cotton nematode gene GhNtR1 and application thereof | |
| CN114591984B (en) | Application of OsAP79 gene in inducing rice to resist brown planthoppers | |
| CN105753952A (en) | Plant drought tolerance related protein Tabzip174 as well as coding gene and application thereof | |
| CN101781654B (en) | Cotton anti-fungal disease related gene GhMPK7 and its application | |
| CN105753955B (en) | A soybean bHLH transcription factor and its encoding gene and application | |
| WO2019027871A1 (en) | Peanut with reduced allergen levels | |
| CN102746391A (en) | Arsenic-resistance related protein PvArrp1, and coding gene and application thereof | |
| CN101704884A (en) | Plant drought resistance and salt tolerance associated protein EeABF6, coding gene and application thereof | |
| CN103849605A (en) | Method for cultivating resistant plant, and special protein and gene thereof | |
| CN103254301A (en) | Plant disease resistance related protein GbMBL1, its coding gene and application | |
| CN108315334B (en) | Transcription factor that increases plant chlorophyll content under high temperature and its application in regulating chlorophyll synthesis in plant petals | |
| CN108517322B (en) | Pinellia palmata trypsin inhibitor gene, protein coded by same and insect-resistant application | |
| CN111718955A (en) | Application of the tomato SlTRXz gene as a repressive target in the defense against M. incognita | |
| CN115838742B (en) | North-south root-knot nematode demethylase Mi-NMAD-1/2 gene and application thereof | |
| CN104611335A (en) | Specific peanut promoter AhRSP and application thereof | |
| CN117777263B (en) | Application of wheat disease resistance-related protein TaMTase in regulating wheat stem rot resistance |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130612 Termination date: 20151018 |
|
| EXPY | Termination of patent right or utility model |