CN110093355A - It is a kind of participate in legume symbiosis dross legh emoglobin GmLbc2 gene and application - Google Patents
It is a kind of participate in legume symbiosis dross legh emoglobin GmLbc2 gene and application Download PDFInfo
- Publication number
- CN110093355A CN110093355A CN201910390984.1A CN201910390984A CN110093355A CN 110093355 A CN110093355 A CN 110093355A CN 201910390984 A CN201910390984 A CN 201910390984A CN 110093355 A CN110093355 A CN 110093355A
- Authority
- CN
- China
- Prior art keywords
- gene
- gmlbc2
- soybean
- dross
- legh emoglobin
- 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.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biophysics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a kind of legh emoglobin for participating in legume symbiosis drossGmLbc2Gene, length are 438 bp, are located at No. 20 chromosomes of soybean genome.And disclose application of the gene in regulation leguminous plant (crowtoe, Chinese milk vetch, clover, soybean and peanut etc.) nodule number.The present invention is isolated to a Leghemoglobin gene using reverse transcription PCR technology in Soybean RootGmLbc2, and confirm that the gene is a Leghemoglobin gene by the methods of homologous protein Multiple Sequence Alignment and phylogenetic analysis；Confirm that soybean nodulation factor receptor proteins GmNFR1a and legh emoglobin GmLbc2 can interact in yeast and tobacco body for the first time simultaneously；Gene participation is further confirmed by the method for overexpressing genetic transformation and just regulates and controls the dross of leguminous plant.This is to confirm soybean Leghemoglobin gene for the first timeGmLbc2Positive regulation legume symbiosis dross.
Description
Technical field
The present invention relates to field of plant genetic project technology, and in particular to a kind of beans blood for participating in legume symbiosis dross
Lactoferrin GmLbc2 gene and application.
Background technique
A large amount of evidences show that soybean legh emoglobin and nitrogen fixation are closely related, by adjusting the O that dissociates in root nodule2's
Concentration, protection by bacteroid generate vulnerable to O2The azotase of destruction.The content and nitrogenase activity of legh emoglobin in root nodule
Being positively correlated property, after rhizobium infect soybean root system induction legh emoglobin expression, root nodule can normally carry out fixed nitrogen work
With nitrogen source needed for could providing growth for host plant.But early stage root nodule whether is influenced about soybean legh emoglobin
It is formed, is had not been reported at present.
The present invention for the first time connects soybean nodulation factor receptor proteins GmNFR1a and legh emoglobin GmLbc2, card
Real the two can interact in yeast and tobacco body.After overexpressing GmLbc2 gene in leguminous plant crowtoe, knot
Tumor number increased significantly, and then significantly increase leguminous plant nitrogen fixing capacity, therefore the gene has very in agricultural and ecology
Important application value.
Summary of the invention
It is described the object of the present invention is to provide a kind of legh emoglobin GmLbc2 gene for participating in legume symbiosis dross
The sequence of gene is nucleotide sequence shown in SEQ ID NO:1, and length 438bp is located at No. 20 chromosomes of soybean genome；
The protein of the legh emoglobin GmLbc2 gene coding of the present invention for participating in legume symbiosis dross,
Sequence is amino acid sequence shown in SEQ ID NO:2；
The present invention confirms soybean nodulation factor receptor proteins GmNFR1a and legh emoglobin GmLbc2 in yeast and cigarette for the first time
It can interact in cursive script.
It is also another object of the present invention to provide a kind of legh emoglobin GmLbc2 bases for participating in legume symbiosis dross
Because of the application in leguminous plant (crowtoe, Chinese milk vetch, clover, soybean and peanut etc.).Applicant is ground using overexpression technology
Function of the gene in leguminous plant crowtoe in symbiosis process of nodulation is studied carefully.The result shows that compared with adjoining tree, super table
After reaching, nodule number be increased significantly, and have application prospect on legume symbiosis fixed nitrogen.
The purpose of the present invention is achieved through the following technical solutions:
A kind of preparation method for the legh emoglobin GmLbc2 gene participating in legume symbiosis dross, the steps include: to plant
After training the sterilizing of soybean W82 the surface of the seed, hilum is laid in downward on sterile wetting filter paper, and 28 DEG C of dark cultures wait sprouting.It collects new
Fresh soybean root tissue, liquid nitrogen flash freezer.Referring to RNA extracts kit (Invitrogen company, USA) specification, Soybean Root is extracted
Total tissue RNA.The first chain of cDNA is obtained according to TIANGEN company reverse transcription reagent box operating instruction.According to https: //
Soybean GmLbc2 gene (Glyma.20g191200) primers that the website www.soybase.org/search/ is announced
F-GmLbc2 and R-GmLbc2, PCR amplification GmLbc2 target gene.Target fragment is recycled, carrier T is connected, send Nanjing Jin Sirui
Company's sequencing, obtains the overall length of the gene.Leghemoglobin gene GmLbc2 of the invention is located at soybean genome 20 dyeing
Body, CDS section length are 438bp, encode 145 amino acid residues.PCR (polymerase chain can be used
Reaction) technology, amplification obtains GmLbc2 gene of the invention and any interested from genome, mRNA and cDNA
Section of DNA or the section of DNA homologous with it.
A kind of soybean nodulation factor receptor proteins GmNFR1a and legh emoglobin GmLbc2 is mutual in yeast and tobacco body
The detection of operative condition the steps include: that GmLbc2/GmNFR1 α-pk, feminine gender and positive control plasmid are transferred to saccharomycete respectively
Strain Y2HGold, is coated on QDO/X/A culture medium, 30 DEG C of 3~5d of culture.It has been observed that GmNFR1 α-pk and target protein GmLbc2
When cotransformation, bacterium colony is blue on QDO/X/A culture medium, the results showed that the two can interact in yeast.
GmNFR1 α and GmLbc2 gene is building up to respectively on two carriers matched in BiFC, it is then total by transient expression system
Transformation of tobacco, a situation arises for observation fluorescence under laser confocal microscope, it was demonstrated that there is also phases in tobacco body for two albumen
Interaction, and the position to interact is in the cell membrane of Tobacco Epidermis.
A kind of Leghemoglobin gene GmLbc2 participating in legume symbiosis dross is in crowtoe, Chinese milk vetch, clover, big
Application on beans and peanut the steps include: to realize by genetic transformation.The super table of plant of present invention building GmLbc2 gene
It up to carrier, is transferred in Agrobacterium LBA1334 by freeze-thaw method, leguminous plant is then transferred to by the hair root of mediated by agriculture bacillus
It in crowtoe, is identified after obtaining positive plant by GUS, by positive plant kind in husky and vermiculite 1:1 mixing potting, is inoculated with root
Tumor bacterium.The result shows that leguminous plant crowtoe dross number significantly improves, this is to confirm beans in crowtoe for the first time after overexpression
Hemoglobin gene GmLbc2 is just regulating and controlling symbiosis process of nodulation.
Compared with the prior art, the invention has the following beneficial effects:
1. the present invention is isolated to a Leghemoglobin gene GmLbc2 using reverse transcription PCR technology in Soybean Root,
And confirm that the gene is a Leghemoglobin gene by the methods of homologous protein Multiple Sequence Alignment and phylogenetic analysis；
Confirm that soybean nodulation factor receptor proteins GmNFR1a and legh emoglobin GmLbc2 can in yeast and tobacco body for the first time simultaneously
Interaction；Gene participation is further confirmed by the method for overexpressing genetic transformation and just regulates and controls the knot of leguminous plant
Tumor.This is to confirm that soybean Leghemoglobin gene GmLbc2 is just regulating and controlling legume symbiosis dross for the first time.
2. leguminous plant increases the fertility of soil by root nodule symbiotic nitrogen fixation, but in natural situation in leguminous plant root
The Limited Number of root nodule, therefore the number by increasing root nodule, and then increase the nitrogen fixing capacity of leguminous plant to increase soil
Fertility effectively reduces the use of chemical fertilizer etc., suffers from important meaning in agricultural and ecology.The present invention is exactly based on super table
Confirm that the gene just regulates and controls dross up to technology, it is possible thereby to make gene excess in leguminous plant by the means of transgenosis
Expression, can thus greatly increase the number of root nodule, to reinforce nitrogen fixing capacity, this legume fixed nitrogen Mechanism Study with
And there is certain application prospect in agricultural production.
Detailed description of the invention
Fig. 1 be it is a kind of using DNAMAN software (open use software) by the protein sequence of GmLbc2 predictive genes with
GmLbc2 homologous protein sequence compares schematic diagram.
Fig. 2 is soybean nodulation factor receptor proteins GmNFR1a and legh emoglobin GmLbc2 in yeast (Fig. 2A) and tobacco
Interaction (Fig. 2 B) detection schematic diagram in vivo.
Fig. 3 is that a kind of overexpression GmLbc2 gene promotes crowtoe plant dross schematic diagram, and wherein Fig. 3 A, Fig. 3 B are respectively
It compares normal plant and overexpresses the dross situation of transgenic plant；Fig. 3 C is control normal plant and overexpression transgenic plant
Single plant be averaged dross number comparison.
Fig. 4 is a kind of overexpression effect that GmLbc2 gene in overexpression transgenic plant is detected using Real Time-PCR
The expression quantity schematic diagram of rate and symbiosis marker gene.
Specific embodiment
Further to illustrate technological means and its effect adopted by the present invention, below in conjunction with attached drawing and of the invention excellent
Embodiment is selected to be described in detail.
Embodiment 1
A kind of preparation method for the Leghemoglobin gene GmLbc2 participating in legume symbiosis dross, the steps include:
After W82 soya seeds surface sterilizing, hilum is laid in downward on sterile wetting filter paper, and 28 DEG C of dark cultures wait sprouting.
Collect fresh soyabean root tissue, liquid nitrogen flash freezer.Referring to RNA extracts kit (Invitrogen company, USA) specification, extract
Soybean tip of a root total tissue RNA.The first chain of cDNA is obtained according to TIANGEN company reverse transcription reagent box operating instruction.According to
Https: soybean GmLbc2 gene (Glyma.20g191200) sequence that the //website www.soybase.org/search/ is announced
Design primer F-GmLbc2 and R-GmLbc2 (table 1), PCR amplification GmLbc2 target gene.Target fragment is recycled, carrier T is connected,
Send Nanjing Jin Sirui company sequence verification.
Primer used in 1 present invention of table
Note: underlined region is restriction enzyme site sequence
The amino acid sequence of gene coding is analyzed, the steps include: to find using the Blastp tool of the website NCBI
The homologous protein of the cultivated soybean (Glycine max) GmLbc2 albumen (NP_001235248.1), including the cultivated soybean
(Glycine max) GmLbc3 (NP_001235423.1), wild soybean (Glycine soja) GsLbc1 (KHN37871.1),
The cultivated soybean GmLba (NP_001235928.1), pigeonpea (Cajanus cajan) CcLb (XP_020222796.1), red bean
(Vigna angularis) VaLb (XP_017409386.1), mung bean (Vigna radiata) VrLb (XP_
014513332.1), M. truncatula (Medicago truncatula) MtLb (XP_003616494.1), crowtoe (Lotus
Japonicus totally 10 kinds of) LjLb (BAE46737.1), Chinese milk vetch (Astragalus sinicus) AsLb (ABB13622.1)
The legh emoglobin homologous protein of different leguminous plants.It is analyzed using the Multiple Sequence Alignment that DNAMAN software carries out homologous protein
(Fig. 1).
A kind of soybean nodulation factor receptor proteins GmNFR1a and legh emoglobin GmLbc2 is mutual in yeast and tobacco body
The detection of operative condition the steps include: the pGADT7-GmLbc2 plasmid that will be obtained and pGBKT7-GmNFR1 α-pk plasmid, yin
Property and positive control plasmid cotransformation into Y2HGold yeast strain, and on QDO/X/A selective medium cultivate screening,
30 DEG C of 3~5d of culture.It has been observed that when GmNFR1 α-pk and target protein GmLbc2 cotransformation, the bacterium on QDO/X/A culture medium
It falls blue, the results showed that the two can occur to interact (Fig. 2A) in yeast.
BiFC carrier is constructed respectively according to GmNFR1 α and the GmLbc2 sequence that the website NCBI is announced；It will be sequenced correct
PSCYNE-NFR1 α and pSCYCE (R)-GmLbc2 fluorescence complementary carrier converts Agrobacterium GV3101 respectively, is inverted, 28 DEG C of cultures
2-3d；Picking single bacterium is fallen in 5mL LBK fluid nutrient medium, and 28 DEG C, shaken cultivation 1-2d reaches 0.6 or so to OD600, from
The heart collects thallus, and the OD600 of Agrobacterium is adjusted to 0.5 respectively with Agrobacterium-mediated Transformation buffer, and it is mutual to mix two fluorescence in equal volume
Mend the agrobacterium suspension of carrier.It is placed at room temperature for 1h activation Agrobacterium；Remove the syringe needle of 1mL syringe, draws Agrobacterium mixing
Liquid injects the lower epidermis of tobacco leaf；Tobacco dark culturing 1-2d after conversion；Then laser confocal microscope is used in film-making
Observation fluorescence signal is simultaneously taken pictures.As a result confirm that there is also interactions in tobacco body for two albumen, and the position of interaction is in cigarette
The cell membrane (Fig. 2 B) of careless epidermal cell.
Embodiment 2
A kind of apoptosis inhibiting factor gene participating in legume symbiosis dross is in crowtoe, Chinese milk vetch, clover, big
Application in beans and peanut (leguminous plant) nodule number, the steps include:
In order to study biological function of the GmLbc2 in leguminous plant crowtoe, applicant is visited by the method for overexpression
The rope biological function of GmLbc2 (Fig. 3).It is mainly concerned with building and the genetic transformation of overexpression vector, specific implementation step
It is rapid as follows:
Overexpress the building of fusion vector: design introduces the primers F-OX and R-OX of restriction enzyme site Kpn I and BamH I
(table 1) will be sequenced in correct target gene insertion plant expression vector p1301U (Gus gene is as riddled basins),
P1301U-GmLbc2 recombinant plasmid is constructed, being transferred to agrobacterium rhizogenes A.rhizogenes LBA1334 through freeze-thaw method, (bacterial strain is
The bacterial strain announced in the world, from U.S. Hong Zonglie teach) in for crowtoe hair root convert.Crowtoe hair root conversion side
Method Primary Reference Lotus japonicus Handbook.
Specific implementation step is as follows:
(1) vegetable material culture: 5 days sprouting crowtoe MG20 (a public crowtoe subspecies) seeds in advance.
With sand paper by seed sanding (grinding lightly than general germination seed), it is placed in liquid nitrogen and freezes 1 minute, 75% (mass volume ratio)
Ethyl alcohol impregnates 1 minute, and 5% (effective chlorine density) NaClO impregnates 15 minutes, and aseptic water washing 5-6 times removes remaining NaClO.
After the completion of the surface of the seed disinfection, stays a little sterile water just to flood seed, dark condition lower 4 DEG C of vernalization treatments 1 day, then go to
23 DEG C dark culture 2 days on the solid medium of sucrose are not added in MS, are placed in illumination box (16h illumination, 8h are dark) 23 DEG C
Continue culture 2 days, for use.
(2) bacterial strain and plasmid prepare: bacterial strain uses therefor is A.rhizogenes LBA1334 (Spectinomycin resistance), will contain mesh
Gene plasmid by electrotransformation method import Agrobacterium A.rhizogenes LBA1334, -70 DEG C of preservation bacterial strains after identification
It is spare.It 2 days in advance, goes bail for and deposits strain stroke plate (plate containing plasmid resistance) activation.
(3) conversion is infected: first day: choosing single colonie and be inoculated in 5ml LB culture medium (containing plasmid resistance), 28- 30 afternoon
DEG C shaken cultivation 16-24 hours.Second day: a small amount of bacterium of the previous day inoculation are expanded into culture, the inoculation of 1:100 ratio, 28-30
DEG C shaken cultivation 8 hours or so.The bacterium solution of mass propgation is centrifuged 10 minutes with 6000 revs/min, thallus is collected, uses sterile water
Thallus is resuspended, makes its OD600=0.8 or so.The hypocotyl base portion of seedling is cut off, part cotyledonous is soaked with this resuspended bacterium solution
Bubble 30 minutes, pulls explant out, is blotted with filter paper, is placed in MS and is not added in sucrose culture medium and co-cultures.
(4) it co-cultures: being first protected from light co-cultivation 3-5 days.Then explant is transferred to HRE and 300 μ gmL-1cefotaxime
Culture medium on continue culture 10 days, hair root is grown from hypocotyl incision during this.
(5) positive transgenic root is identified: the root segment of about 0.5cm long is put into GUS dye liquor at the clip tip of a root, 37 DEG C of dark cultures
Overnight, the root for showing blue is positive transgenic root.
(6) hardening and transplanting: after identification, cutting off non-transformed root, and seedling is placed in the plate equipped with water and (is not covered) refining
It seedling 1 day, is then transplanted in flowerpot in 23 DEG C of cultures of illumination box (16h illumination, 8h are dark).Vermiculite is added in flowerpot in advance
(vermiculite) and husky (sand) matrix for being mixed in 1:1 ratio.Pour a nitrogen-free nutrient solution within every 3 days.
(7) dross counts: Mesorhizobium loti MAFF303099 (is taught from South China Botanical Garden Chinese Academy of Sciences Wu Guojiang
Laboratory is awarded, sees genetic resources table) it after activation, is accessed in liquid TY medium, 28 DEG C of shake culture 24- on YMA plate
36h；Cultured rhizobium are transferred in sterile centrifuge tube, and 4 DEG C, 7000r/min is centrifuged 5min, collect thallus；With sterile
Fahraeus nitrogen-free nutrient solution is washed and is centrifuged 2 times；Nitrogen-free nutrient solution is added, thallus is resuspended, is inoculated in crowtoe seedling root.
It pours nitrogen-free nutrient solution 1 time daily.After being inoculated with 4 weeks, statistics is carried out to plant dross phenotype and is taken pictures.According to individual nodule number and sample
This amount (n) calculates single plant and is averaged dross number, and test is repeated twice, is averaged.
(8) GmLbc2 and dross related gene NIN, ENOD40-1 and ENOD40-2 in table 1 gene expression detection: are utilized
Fluorescent quantitation primer detection complex plant positive Hairy root in each gene transcriptional level, crowtoe UBI gene be used as in
Ginseng.Fluorescence quantitative PCR detection is carried out according to Takara company PrimeScript RT reagent Kit operating instruction, according to phase
To sizing technique (2- ⊿ ⊿ Ct) formula calculated result.Test is repeated 3 times, and test data uses Excel 2007 and SPSS 13.0
Software is analyzed, and draws a diagram (Fig. 4).
The culture medium and agent prescription of above-mentioned steps are as follows:
1) MS culture medium: MS basal salt mixture (Sigma) 4.3g, MS vitamin powder 0.103g,
0.7- 0.8% (mass volume ratio) agar powder, ddH2O is settled to 1000mL, is adjusted to pH 5.8.
2) YMA medium: 10.0g mannitol (or sucrose), 0.4g Yeast Extract, 0.5g K2HPO4, 0.2g
MgSO4·7H2O, 0.1g CaCl2·6H2O, 0.1g NaCl, 4mL Rh liquid microelement, ddH2O is settled to 1000 mL, adjusts
To pH 6.8-7.0,115 DEG C of sterilizing 20min.
3) Rh liquid microelement: 5.0g H3BO3, 5.0g Na2MoO4, ddH2O is settled to 1000mL.
4) Fahraeus nitrogen-free nutrient solution: 0.10g CaCl2·2H2O, 0.12g MgSO4·7H2O, 0.10g KH2PO4,
0.15g Na2HPO4·12H2O, 1mL Gibson liquid microelement, 5mg ironic citrate, ddH2O is settled to 1000mL.
5) Gibson liquid microelement: 2.86g H3BO3, 0.22g ZnSO4·7H2O, 2.03g MnSO4·4H2O, 0.13
g Na2MoO4·2H2O, 0.08g CuSO4·5H2O, ddH2O is settled to 1000mL.
6) GUS dye liquor: 100mM sodium phosphate buffer, pH 7.0,0.1%Triton X-100,
0.1%N- laurylsarcosine, 10mM Na2EDTA, the 1mM potassium ferricyanide (K3Fe(CN)6), 1mM potassium ferrocyanide
(K4Fe(CN)6) and 0.5mg/mL X-GluC.
7) HRE culture medium: the SH-A salt of 20X, the UM-C vitamin of 20X, 10g sucrose, 3mL 1M MES, 0.7-0.8%
(mass volume ratio) agar powder, ddH2O is settled to 1000mL, is adjusted to pH 5.8.110 DEG C of sterilizing 30min.
The SH-A salt of 20X:
FeSO is dissolved respectively with 100mL sterile water4·7H2O and NaEDTA, other salt are dissolved in 700mL sterile water, then
Mixing is settled to 1000mL, is packed as the every pipe of 50mL, -20 DEG C of preservations.
The UM-C vitamin of 20X:
ddH2O is settled to 1000mL, is packed as the every pipe of 50mL, -20 DEG C of preservations.
MES (2- [N-Morpholino] ethane-sulfonic acid), 1M storing liquid:
29.28g MES (Sigma) is claimed to use ddH2O is settled to 150mL, is adjusted to pH 5.8, is packed as the every pipe of 6mL, and -20
DEG C save.
Embodiment 3
Embodiment 3 is substantially the same manner as Example 2, the difference is that,
Plant culture materials are to shift to an earlier date 5 days sprouting alfalfa seeds in specific implementation step (1)；Rhizobium connect in step (7)
Kind be rhizobium melioti Sinorhizobium meliloti 1021 (bacterial strain is the bacterial strain announced in the world, Juan etc.,
Comparison of Developmental and Stress-Induced Nodule Senescence in Medicago
Truncatula. Plant Physiol, 152:1574-1584,2010).
Embodiment 4
Embodiment 4 is substantially the same manner as Example 2, the difference is that,
Plant culture materials are to shift to an earlier date 5 days sprouting Chinese Milk Vetch Seeds in specific implementation step (1)；Rhizobium in step (7)
Inoculation is that (bacterial strain is the bacterial strain announced in the world, Li et al. A nodule- to Rhizobium of Milk Vetch Rhizobium huakuii
specific plant cysteine proteinase,AsNODF32,is involved in nodule senescence
and nitrogen fixation activity of the green manure legume Astragalus
sinicus.New phytologist 180:185-192 2008)。
Embodiment 5
Embodiment 5 is substantially the same manner as Example 2, the difference is that,
Plant culture materials are to shift to an earlier date 5 days germinated soybean seeds in specific implementation step (1)；Rhizobium connect in step (7)
Kind is that (bacterial strain is the bacterial strain announced in the world, Chen Changbin etc., group to rihizobium japonicum Rhizobium fredii HN01lux
Facilitation of the molding nifA to rihizobium japonicum (Rhizobium fredii) HN01lux nodulation and nitrogen fixation efficiency, Science Bulletin,
44 (5)1999)。
Embodiment 6
Embodiment 6 is substantially the same manner as Example 2, the difference is that,
Plant culture materials are to shift to an earlier date 5 days Germinating Peanut Seeds in specific implementation step (1)；Rhizobium connect in step (7)
Kind is that (bacterial strain is the bacterial strain announced in the world to peanut rhizobium Spr, beautiful etc. in scape, and the research of celB gene marker method is acid
Native peanut is inoculated with and applies molybdenum effect, plant nutrient and fertilizer journal, 12 (2): 250-253, and 2006).
The pulse family (Leguminosae sp.) is dicotyledon arbor, shrub, undershrub or draft, it is upright or
It climbs up by holding on to, the root nodule plant of Chang Youneng fixed nitrogen.Type genus: Faba P.Miller.About 650 belong to, and 18000 kinds, are widely distributed in the whole world.
There are 172 categories in China, and 1485 kinds, 13 subspecies, 153 mutation, 16 modifications；Each provinces and regions have point.The section has important economic significance,
It is one of starch in human food, protein, oil and important sources of vegetables.Leguminous plant of the present invention is specially hundred
It is arteries and veins root (Lotus japonicus), clover (Medicago truncatula), Chinese milk vetch (Astragalus sinicus), big
Beans (Soybean) and peanut (Peanut) etc..
The above description is merely a specific embodiment, but scope of protection of the present invention is not limited thereto, ability
Other modifications or equivalent replacement that domain those of ordinary skill makes technical solution of the present invention, without departing from skill of the present invention
The spirit and scope of art scheme, are intended to be within the scope of the claims of the invention.
Sequence table
<110>Xinyang Normal College
<120>a kind of legh emoglobin GmLbc2 gene for participating in legume symbiosis dross and application
<141> 2019-05-11
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 438
<212> DNA
<213> Glycine max
<400> 1
atgggtgctt tcactgagaa gcaagaggct ttggtgagta gctcattcga agcattcaag 60
gcaaacattc ctcaatacag cgttgtgttc tacacttcga tactggagaa agcacccgca 120
gcaaaggact tgttctcgtt tctatctaat ggagtagatc ctagtaatcc taagctcacg 180
ggccatgctg aaaagctttt tggattggtg cgtgactcag ctggtcaact taaagcaaat 240
ggaacagtag tggctgatgc cgcacttggt tctatccatg cccaaaaagc aatcactgat 300
cctcagttcg tggtggttaa agaagcactg ctgaaaacaa taaaggaggc agttggggac 360
aaatggagtg atgaattgag cagtgcttgg gaagtagcct atgatgaatt ggcagcagct 420
attaagaagg cattttag 438
<210> 2
<211> 145
<212> PRT
<213> Glycine max
<400> 2
Met Gly Ala Phe Thr Glu Lys Gln Glu Ala Leu Val Ser Ser Ser Phe
1 5 10 15
Glu Ala Phe Lys Ala Asn Ile Pro Gln Tyr Ser Val Val Phe Tyr Thr
20 25 30
Ser Ile Leu Glu Lys Ala Pro Ala Ala Lys Asp Leu Phe Ser Phe Leu
35 40 45
Ser Asn Gly Val Asp Pro Ser Asn Pro Lys Leu Thr Gly His Ala Glu
50 55 60
Lys Leu Phe Gly Leu Val Arg Asp Ser Ala Gly Gln Leu Lys Ala Asn
65 70 75 80
Gly Thr Val Val Ala Asp Ala Ala Leu Gly Ser Ile His Ala Gln Lys
85 90 95
Ala Ile Thr Asp Pro Gln Phe Val Val Val Lys Glu Ala Leu Leu Lys
100 105 110
Thr Ile Lys Glu Ala Val Gly Asp Lys Trp Ser Asp Glu Leu Ser Ser
115 120 125
Ala Trp Glu Val Ala Tyr Asp Glu Leu Ala Ala Ala Ile Lys Lys Ala
130 135 140
Phe
145
Claims (5)
1. a kind of legh emoglobin for participating in legume symbiosis drossGmLbc2Gene, which is characterized in that the sequence of the gene
It is classified as nucleotide sequence shown in SEQ ID NO:1.
2. the legh emoglobin according to claim 1 for participating in legume symbiosis drossGmLbc2The albumen of gene coding
Matter, which is characterized in that the sequence of the protein is amino acid sequence shown in SEQ ID NO:2.
3. the legh emoglobin described in claim 1 for participating in legume symbiosis drossGmLbc2The genetic engineering of gene is answered
With.
4. the legh emoglobin according to claim 1 for participating in legume symbiosis drossGmLbc2Gene is in leguminous plant
In application.
5. the legh emoglobin according to claim 4 for participating in legume symbiosis drossGmLbc2Gene is in regulation pulse family
Application in plant root nodule number.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910390984.1A CN110093355A (en) | 2019-05-11 | 2019-05-11 | It is a kind of participate in legume symbiosis dross legh emoglobin GmLbc2 gene and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910390984.1A CN110093355A (en) | 2019-05-11 | 2019-05-11 | It is a kind of participate in legume symbiosis dross legh emoglobin GmLbc2 gene and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110093355A true CN110093355A (en) | 2019-08-06 |
Family
ID=67447756
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910390984.1A Pending CN110093355A (en) | 2019-05-11 | 2019-05-11 | It is a kind of participate in legume symbiosis dross legh emoglobin GmLbc2 gene and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110093355A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114316031A (en) * | 2022-01-17 | 2022-04-12 | 杭州瑞丰生物科技有限公司 | Method for producing hemopexin |
| CN116507728A (en) * | 2020-10-28 | 2023-07-28 | 先锋国际良种公司 | Leghemoglobin in soybeans |
-
2019
- 2019-05-11 CN CN201910390984.1A patent/CN110093355A/en active Pending
Non-Patent Citations (8)
| Title |
|---|
| KE D X ET AL: "ROP6 is involved in root hair deformation induced by Nod factors in LoLotus", 《PLANT PHYSIOL BIOCHEM》 * |
| 于海鹏: "大豆血红蛋白基因转化根瘤菌工程菌株的构建", 《中国优秀博硕士学位论文全文数据库(硕士) 农业科技辑》 * |
| 侯云龙等: "大豆根瘤共生固氮分子机制研究进展", 《农业与技术》 * |
| 无: "Glycine max leghemoglobin C2（LBC2），mRNA", 《NCBI》 * |
| 李珊珊等: "导入nodD-lba串联基因的根瘤菌工程菌株的构建", 《生物技术通报》 * |
| 柯丹霞等: "大豆半胱氨酸蛋白酶抑制剂基因GmCYS2 的功能鉴定", 《作物学报》 * |
| 柯丹霞等: "百脉根Rac1 基因启动子的克隆与表达分析", 《信阳师范学院学报(自然科学版)》 * |
| 洪锐民等: "豆科植物结瘤固氮研究进展", 《草业科学》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116507728A (en) * | 2020-10-28 | 2023-07-28 | 先锋国际良种公司 | Leghemoglobin in soybeans |
| CN114316031A (en) * | 2022-01-17 | 2022-04-12 | 杭州瑞丰生物科技有限公司 | Method for producing hemopexin |
| CN114316031B (en) * | 2022-01-17 | 2024-04-19 | 杭州瑞丰生物科技有限公司 | Method for producing heme binding protein |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109456982B (en) | Application of rice OsMYB6 gene and encoding protein thereof in drought resistance and salt resistance | |
| CN102485897B (en) | Method for changing petal colors by using cotton gene GbF3H | |
| CN112626080B (en) | R gene for controlling soybean-rhizobium matching property, protein and application thereof | |
| CN110699361B (en) | Rice salt stress resistance related gene Os16 and encoding protein and application thereof | |
| CN104480118B (en) | LRR-RLK (leucine-rich repeat receptor-like kinase) gene in arachis hypogaea.L and application thereof to bacterial wilt resistance of tobaccos | |
| CN108949806A (en) | Transgenic plant cells and the method for producing genetically modified plants | |
| CN106868019A (en) | Control rice tillering gene OsHT1 and its application | |
| CN107435047A (en) | In a kind of plant phosphorus signal network Tolerant to low P key gene GmPHR25 and its with application | |
| CN110117320A (en) | Cotton GhCAL-D07 gene is promoting the application in flowering of plant | |
| CN108913702A (en) | For improving method, specific primer and the kit of tobacco black shank resistance | |
| CN110066774A (en) | Corn receptoroid kinase gene ZmRLK7 and its application | |
| CN110093355A (en) | It is a kind of participate in legume symbiosis dross legh emoglobin GmLbc2 gene and application | |
| CN110283829B (en) | Intermediate complex subunit OsMEDdicator 16 gene for regulating rice blast resistance | |
| CN103333901A (en) | Liriodendron hybrid LhWOX1 gene and application thereof | |
| CN109608530A (en) | A kind of low-phosphorous responsive genes of soybean and its albumen and application for promoting lateral root to be formed | |
| CN110079536A (en) | It is a kind of participate in legume symbiosis dross LjROP4 gene and application | |
| CN109734784A (en) | Application of the SlDALR1 gene in enhancing tomato bacterial leaf spot resistance | |
| CN109837297A (en) | GhAGD13 gene relevant to resistance to verticillium wilt and its application | |
| CN108220303B (en) | Soybean BI-1 gene and application | |
| CN108467867A (en) | HD-Zip I classes transcription factor GmHDL57 genes and application | |
| CN103789325A (en) | Cotton cell wall extensin gene GbEXPATR and application | |
| CN107602694B (en) | Trichoderma reesei metallothionein TrCRD2S, encoding gene and its application in heavy metal resistance plant is cultivated | |
| CN106480073A (en) | Oryza meyeriana Distributed in Yunnan Province MeXB3 gene and its application | |
| CN108085319B (en) | Plant tillering angle related protein and its coding gene and application | |
| CN108220302B (en) | Soybean GmCYS20 gene and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190806 |
|
| RJ01 | Rejection of invention patent application after publication |