CN104673829B - β expansin genes GmEXPB2 application - Google Patents

Abstract

The invention discloses β expansin genes GmEXPB2 new opplication, specifically disclosed is new opplications of the β expansin gene GmEXPB2 in terms of legume nitrogen efficiency is improved.Inventor passes through quantitative PCR and chemistry tissue localization method, a β expansin gene GmEXPB2 reached in soybean nodulation formation early stage altimeter is identified, the gene has participated in the development of root nodule former base, cortical cell, parenchyma cell and early stage root nodule micro-pipe tissue；Plant is combined using soybean transgene and the research of whole strain transformed plant shows that overexpression GmEXPB2 significantly increases soybean nodulation quantity and weight, improves N content of crop tissue, ultimately increases the biomass of transfer-gen plant；The research of inventor efficiently and high yield molecular breeding provides genetic resources, and will have important theory and practice meaning to development environment friendly sustainable agriculture for the legume nitrogen including soybean.

Description

β-expansin gene GmEXPB2 application

Technical field

The present invention relates to the new opplication of gene, and in particular to β-expansin gene GmEXPB2 is improving legume nitrogen New opplication in terms of efficiency, belongs to biological technical field.

Background technology

Soybean [Glycine max (L.) Merr], originating from China, is grain important in world wide, oil plant, feed And energy crop, critical role (Palander et al., 2005) is occupied in people's life food configuration.In recent years, with There are serious unbalance, soybean in China and need in the growth of soybean consumption and developing rapidly for aquaculture, domestic soybean supply and demand Contradiction between asking.From 2000, China just becomes maximum in the world now from traditional soybean export state and entered Mouth state (Yang Wenyu etc. 2008).Latest data shows that China's soybean import increased by 8.6% on year-on-year basis up to 63,380,000 tons in 2013, Set a record high-order (Chinese grain information net, http://www.chinagrain.cn/dadou/2014/1/13/ 201411313313124408.shtml).Therefore, the need for meet the domestic consumption to soybean, China is necessary to greatly develop Soybean Industry, solves supply and demand of soybean contradiction, to reduce the dependence to Soybean import.

Soybean in China level is relatively low, and mainly soil nutrient availability of tracing it to its cause is relatively low, and such as nitrogen starved plot is Limit one of principal element of Soybean production.In long-term evolutionary process, plant forms a series of adaptation Low nitrogen stress Mechanism of action.Wherein, nodulation and nitrogen fixation is the important channel for improving legume nitrogen efficiency.Rhizobium infect legume root system institute The syntaxial system of foundation, the sustainable development to agricultural production and ecological environment is significant.Research shows, is inoculated with root nodule Bacterium can improve the nitrogen fixing capacity of legume, improve nitrogen nutrition, promote root growth and improve crop yield (Ferreira et al.,2009).In the soybean main production country such as the U.S., Brazil, Rhizobium Inoculation turns into one of major measure of soybean yield-increasing, greatly Area promotion and application.And legume inoculation technology is applied to agricultural production by China, good benefit (Tang Fuyue is also achieved Deng 2011；Mendes et al.,2003；Sogut 2006；de Freitas et al.,2012).

The formation of root nodule and organ expand closely related with the morphogenesis of cell membrane, and expansin (Expansin) is one Class cell wall expansion protein, plays an important role in terms of inducing cell wall increases and softening, Nodule Growth are developed.

However, the research on expansin in terms of legume nodulation and nitrogen fixation is regulated and controled is very limited, and in soybean In, there is not been reported for the expansin gene of regulation and control nodule formation and development.

The content of the invention

Based on the studies above background, inventor identifies one in soybean by quantitative PCR and chemistry tissue localization method The β that nodule formation early stage altimeter reaches-expansin gene GmEXPB2.It is thin that the gene has participated in root nodule former base, cortex The development of born of the same parents, parenchyma cell and early stage root nodule micro-pipe tissue.

Plant is combined using soybean transgene and the research of whole strain transformed plant shows that overexpression GmEXPB2 is dramatically increased Soybean nodulation quantity and weight, N content of crop tissue is improved, ultimately increase the biomass of transfer-gen plant.

The research of inventor efficiently will provide gene and provide for the legume nitrogen including soybean with high yield molecular breeding Source.

The present invention is advantageous in that：This research is to legume root nodule numbers of the increase including soybean and again Amount, improve N content of crop tissue, increase transfer-gen plant biomass it is significant, to the sustainable agriculture of development environment friendly Industry also has important practice significance, while also providing genetic resources for high yield molecular breeding.

Brief description of the drawings

Fig. 1 is GmEXPB2 Gene Expression Profile Analysis；

Fig. 2 is that soybean transgene is combined the dissection and analysis that GmEXPB2 promoters in plant drive GUS expressive sites；

Fig. 3 is combined the influence of plant dross for excessive, interference GmEXPB2 to soybean transgene；

Fig. 4 contains for excessive, interference GmEXPB2 under high low-phosphorous processing to the compound plant strain growth of soybean transgene and nitrogen/phosphorus The influence of amount；

Fig. 5 is influences of the overexpression GmEXPB2 to Soybean transgenic plant Root morphology；

Fig. 6 is overexpression GmEXPB2 genetically engineered soybean Rhizobium Inoculation USDA110-GFP infection processs；

Fig. 7 is lower influences of the overexpression GmEXPB2 to soybean nodulation of high and low phosphorus processing；

Fig. 8 is overexpression GmEXPB2 to soybean growth and the influence of nitrogen/phosphorus content.

Embodiment

Make specific introduce to the present invention below in conjunction with the drawings and specific embodiments.

First, β-expansin gene GmEXPB2 expression pattern analysis

In the soybean gene group database announced, compared by homologous, predict soybean β-expansin family and be total to There are 9 members, by quantitative PCR technique, determine the gene that GmEXPB2 genes are root nodule Enhanced expressing.GmEXPB2 genes are opened Reading frame length is put for 834bp, the albumen of 278 amino acid is encoded, belongs to β class cell membrane expansins.Existing research table Bright, the gene and nodulation and nitrogen fixation are closely related, and its expression pattern is analyzed first.

Soya seeds HN66 is with 3% (v/v) H₂O₂Surface sterilization one minute, is urged in the quartz sand that 1/2 pancebrin is soaked Bud is sprouted 5 days, and Rhizobium Inoculation is transferred to low nitrogen (nitrogen after 1 hour：530μM NH₄NO₃+KNO₃+Ca(NO₃)₂·4H₂O) seek Handled in nutrient solution.The harvest processing root nodule of the 7th day respectively, the root of the 18th day, stem, leaf, flower and the beanpod and the kind that handle the 29th day Son and extracting RNA.For expression quantity of the comparative analysis GmEXPB2 in different growing stage root nodule, harvest after Rhizobium Inoculation The root system of the 4th day, the root nodule of the 7th, 14,21,30,40 days and the 7th day root system of non-seeded rhizobium, and extract corresponding site RNA, reverse transcription is into cDNA, further with quantitative PCR detection GmEXPB2 expression pattern.The house-keeping gene EF-1a conducts of soybean Internal reference.Primer for quantitative PCR detection gene expression amount is respectively：

The primer of soybean EF-1a genes is：

EF-1a F:5’-TGCAAAGGAGGCTGCTAACT-3’(SEQ ID NO:1)

EF-1a R:5’-CAGCATCACCGTTCTTCAAA-3’(SEQ ID NO:2)

The primer of GmEXPB2 genes is：

GmEXPB2F:5’-TGGTGCTTGTGGTTATGGAAGT-3’(SEQ ID NO:3)

GmEXPB2R:5’-TGAACCACACCCAGGACAGCT-3’(SEQ ID NO:4)

Quantitative PCR response procedures are：CDNA obtained by RNA sample reverse transcription is diluted 50 times and is used as quantitative PCR reaction template. Choose appropriate cDNA stostes and do the template that gradient dilution is standard curve.20 μ L reaction systems are used in experiment, including：10 μ L's 2 × SYBR Green PCR master mix, each 0.6 μ L 10 μM of forward and reverse primers, the cDNA of 2 μ L dilutions is finally used Mili-Q water is mended to 20 μ L.

Quantitative PCR reaction condition is：95 DEG C are denatured 1 minute, and then 94 DEG C are cracked 15 seconds, 60 DEG C of combinations 15 seconds, and 72 DEG C are prolonged Stretch 30 seconds and carry out 40 circulations.

The expression quantity of each sample is calculated with Rotor-Gene Real-Time Analysis Software 6.0.

Fig. 1 is GmEXPB2 Gene Expression Profile Analysis.Wherein：

Fig. 1 (A) is expression of the GmEXPB2 in different tissues；

Fig. 1 (B) is expression of the GmEXPB2 in different development stage root nodule.

Data are the average value and standard error that 3 biology are repeated in figure.

As a result show：

(1) expression quantity highests of the GmEXPB2 in root nodule, root is taken second place, and also there is trace expression in beanpod, but The accumulation of the gene mRNA is can't detect in stem, leaf, flower and seed, referring to Fig. 1 (A).

(2) qRT-PCR detection and analysis is carried out to the root nodule of different development stage, finds roots of the GmEXPB2 after being inoculated with 4 days Enhanced expressing in system, when Nodule Growth was by the 7th day, expression quantity reaches maximum, but the extension of the growth time with root nodule, Its expression quantity is gradually reduced, and when Nodule Growth was by 30 and 40 days, is nearly no detectable the expression of the gene, referring to Fig. 1 (B), Imply that GmEXPB2 participates in the growth course of regulation and control early stage root nodule.

2nd, GmEXPB2 gene promoters clone, vector construction and tissue expression positioning analysis

Conventionally, soybean leaves or root genomic DNA are extracted, using Soybean genomic DNA as template, upstream is used Special primer and downstream special primer amplification GmEXPB2 promoter 2742bp fragments, after PCR fragment recovery sequencing is errorless, pass through PstI and NcoI is carried out after double digestion to reclaiming fragment and purpose carrier, and GmEXPB2 genes are connected into purpose carrier pCAMBIA3301.Wherein,

Upstream specific primer is：

5’-CTGCAGACCTTCCTGACTTCCCGAAT-3’(SEQ ID NO:5)

Downstream special primer is：

5’-CCATGGGTGTAGGAGCCATAATATCACAACC-3’(SEQ ID NO:6)

Conversion method is injected by agriculture bacillus mediated soybean hypocotyl to obtain after transgenic hairy root, main root is cut, and is protected The hairy root come out from callus director is stayed, hairy root was immersed in rhizobium bacterium solution after 1 hour and moves into the training of water planting condition Support, mill water culture nutrient solution is above-mentioned low nitrogen processing.To determine that GmEXPB2 is positioned in the tissue of soybean nodulation different developmental phases, point Not Shou Huo Rhizobium Inoculation processing 4,7,14, the root nodule of 21 and 30 days carry out GUS staining analysis, the tissue positioning of gene expression can Further observed by paraffin section, as a result see Fig. 2.

Referring to Fig. 2, soybean transgene is combined the dissection and analysis that GmEXPB2 promoters in plant drive GUS expressive sites, its In：

Fig. 2 (A)-Fig. 2 (H) and Fig. 2 (L) drives GUS transgenosis root nodule for expression GmEXPB2 promoters；

Fig. 2 (A)-Fig. 2 (D) is the vertical section of soybean nodulation；

Fig. 2 (E)-Fig. 2 (H) and Fig. 2 (L) is that soybean nodulation is crosscutting；

Fig. 2 (I)-Fig. 2 (K) is expression CaMV35S controls；

Fig. 2 (I) is soybean nodulation rip cutting；

Fig. 2 (J) and Fig. 2 (K) is crosscutting for soybean nodulation.

Soybean transgene is combined plant strain growth in containing Poor nitrogen nutrition liquid, wherein：

Fig. 2 (A) and Fig. 2 (E) is 4 days root nodules of growth；

Fig. 2 (B) and Fig. 2 (F) is 7 days root nodules；

Fig. 2 (C), Fig. 2 (G), Fig. 2 (I), Fig. 2 (J) are 14 days root nodules；

Fig. 2 (L) is 21 days root nodules；

Fig. 2 (D) and Fig. 2 (H) is 30 days root nodules of growth.

NVT is that early stage root nodule does up the beginning；NVB is root nodule vascular bundle；Pa represents parenchyma cell；EN represents cortical cell.

Fig. 2 (A) scale is 20 μm, during Fig. 2 (B), Fig. 2 (C), Fig. 2 (E) and Fig. 2 (F) scale are 50 μm, remaining figure Scale is 100 μm.

As a result show：[Fig. 2 (I), Fig. 2 (J) and Fig. 2 (K)], Rhizobium Inoculation 4 days compared with the control of constitutive expression Afterwards, GmEXPB2 is mainly in the soybean root system center pillar corresponding to root nodule happening part and root nodule former base starting position expression [Fig. 2 (A) With Fig. 2 (E)]；When growing 7 days, GUS staining signals are primarily present in the vascular tissue and development shape of root and root nodule connecting portion Into Nodule cortex and parenchyma cell in [Fig. 2 (B) and Fig. 2 (F)]；In the root nodule of growth 14 days, the dimension that root nodule intersects with root system Tubing present two bifurcation states, and GmEXPB2 can with the differentiation of Nodule cortex and parenchyma cell Enhanced expressing [Fig. 2 (C) and Fig. 2 (G)]；When Nodule Growth was by 21 days, GUS dyeing disappears in root nodule vascular tissue, mainly in cortex and thin-walled group In knitting [Fig. 2 (L)]；GUS staining signals [Fig. 2 (D) and Fig. 2 (H)] are nearly no detectable in the root nodule section of growth 30 days.

3rd, excess, interference GmEXPB2 are combined the influence of plant dross to soybean transgene

The soybean hypocotyl injection mediated using Agrobacterium rhyzogenesK599 obtains excessive interference GmEXPB2 transgenosis Compound plant (root is that transgenic hairy root, overground part are non-transgenic).Wherein transgenic hairy root growth length is about Main root can be cut during 10cm, removes the adventitious root grown in non-wound.Follow-up phenotypic evaluation use this strain, CK for pair According to.

Hair root is grown after lateral root, takes root samples to extract RNA, and transgenosis hair root selection markers are hygromycin, can use tide (500 μM of one transgenosis root system progress Rhizobium Inoculation of true and false and reservation and high phosphorus of mycin resistant gene (Hyg) detection hair root KH₂PO₄), low-phosphorous (10 μM of KH₂PO₄) processing sand culture test.The root nodule of harvest growth 7 days, extracts RNA, further with quantitative PCR is detected in the effect of overexpression and interference, quantitative PCR assays using soybean house-keeping gene using EF-1a described above as reference Gene, relative expression quantity is purpose gene GmEXPB2 expression quantity and the ratio of house-keeping gene expression quantity.

Quantitative PCR step：

(1) primer of Hyg genes is：

Hyg F:5’-GCTGTTATGCGGCCATTGTC-3’(SEQ ID NO:7)

Hyg R:5’-GACGTCTGTCGAGAAGTTTC-3’(SEQ ID NO:8)

The primer of soybean EF-1a genes is ibid；

The primer of GmEXPB2 genes is ibid.

(2) reaction system：

2×SYBR Green PCR master mix：10μL

Sense primer (10 μm of ol/L)：0.6μL

Anti-sense primer (10 μm of ol/L)：0.6μL

Mili-Q water：Mend to 20 μ L

The cDNA of dilution：2μL

(3) reaction condition：

Detection and quantitative PCR by resistant gene confirm to obtain effectively different transgenic lines, see Fig. 3 (A) and Fig. 3 (B)。

Harvest different transgenic lines of the Rhizobium Inoculation after 50 days and determine relative physiologic index, including：Root nodule dry weight, root Knurl number, root nodule size etc., wherein root nodule size are the dry weight (root nodule dry weight and the ratio of root nodule number) of single root nodule.Biomass： The root nodule of root is taken after counting, fresh weight is weighed with a ten thousandth balance, all samples are after 105 DEG C of baking ovens finish 30 minutes 75 DEG C of drying are placed in constant weight, dry weight is weighed.

Fig. 3 is combined the influence of plant dross for excessive, interference GmEXPB2 to soybean transgene.Wherein：

Fig. 3 (A) is that PCR detects positive plant；

Fig. 3 (B) is quantitative PCR detection GmEXPB2 expression quantity；

Fig. 3 (C) is root nodule picture；

Fig. 3 (D) is root nodule number；

Fig. 3 (E) is root nodule dry weight；

Fig. 3 (F) is root nodule size.

Sample is preliminary to detect positive plant, EF-1a (gene orders number by qualitative PCR：X56856 it is) the special base of soybean Cause, Hyg is carrier target gene.Relative expression quantity is purpose gene GmEXPB2 expression quantity and the ratio of EF-1a expression quantity.

CK is control strain；OX is GmEXPB2 overexpression strain；RNAi is GmEXPB2 interference strain.It is in figure Average value and standard error that same 4 biology of genetically modified plants are repeated.

Asterisk represents same character in OX or RNAi strains with compareing the significant difference between CK strains：

* the level of signifiance 0.01 is represented<During ρ≤0.05, significant difference；

* represents the level of signifiance 0.001<During ρ≤0.01, the significance of difference between significantly with extremely significantly between；

When * * represent level of signifiance ρ≤0.001, difference is extremely notable；

Ns represents that difference is not notable.

As a result show：Compared with the control, overexpression GmEXPB2 (OX) significantly promotes the growth of root nodule, adds root Knurl number and dry weight, and interfere GmEXPB2 (RNAi) then to significantly suppress the growth of root nodule, reduce root nodule number, reduce root Knurl weight and root nodule size is reduced, illustrate that GmEXPB2 mainly have impact on the formation of root nodule.

4th, excess, interference GmEXPB2 are combined plant strain growth and nitrogen/phosphorus content under high low-phosphorous processing to soybean transgene Influence

Biomass is determined：1 percent balances weigh overground part and root samples fresh weight, and all samples are in 105 DEG C of bakings Case is placed in 75 DEG C of drying to constant weight after finishing 30 minutes, weigh dry weight.

Plant nitrogen, phosphorus are determined：Plant nitrogen and phosphorus content use Continuous Flow Analysis instrument (model SAN++, originate from Holland) Determine, weigh Plant samples 0.2g or so first, add after the 5mL concentrated sulfuric acids are cleared up and to cause 50mL with distilled water constant volume, and by sample Prepare liquid is made in 4 times of dilution.For the measure of plant ammonia nitrogen, using sodium nitroprussiate as catalyst, make the ammonium nitrogen of testing sample with Sodium salicylate and sodium hypochlorite reaction generation blue compound, determine its absorption value at 660nm wavelength；For plant phosphorus content Measure, in the presence of antimonic salt, the phosphato-molybdic heteropolyacid that orthophosphoric acid and ammonium molybdate reaction are generated, by ascorbic acid after heating bath Reduction generation P-Mo blue, then detector colorimetric at 880nm wavelength is flowed into, colorimetric signal is finally inputted into computer, and by Flow Access software result of calculations.

Nutrient content in plant represents that calculation formula is with unit plant nitrogen, phosphorus amount：

Nitrogen content (mg/plant)=nitrogen concentration (mg/g) × plant weights (g/plant)

Phosphorus content (mg/plant)=phosphorus concentration (mg/g) × plant weights (g/plant)

Fig. 4 contains for excessive, interference GmEXPB2 under high low-phosphorous processing to the compound plant strain growth of soybean transgene and nitrogen/phosphorus The influence of amount.Wherein：

Fig. 4 (A) is the phenotypic analysis of high low-phosphorous processing；

Fig. 4 (B) is plant weights；

Fig. 4 (C) is nitrogen content；

Fig. 4 (D) is phosphorus content.

CK is empty vector control；OX is that overexpression GmEXPB2 transgenosis is combined plant；RNAi turns for interference GmEXPB2 Gene is combined plant.

Test data is the average value and standard error that four biology are repeated.

Asterisk represents dry weight, nitrogen and the phosphorus content of GmEXPB2 genes in OX and RNAi strains with compareing difference between strain Property compares (t- inspections)：

* the level of signifiance 0.01 is represented<During ρ≤0.05, significant difference；

* represents the level of signifiance 0.001<During ρ≤0.01, the significance of difference between significantly with extremely significantly between.

As a result show：Overexpression GmEXPB2 significantly promotes the growth of dross soybean, improves soybean transgene and is combined The nitrogen of plant/phosphorus efficiency and biomass, and interfere GmEXPB2 to significantly suppress soybean growth.

5th, influences of the overexpression GmEXPB2 to Soybean transgenic plant Root morphology

The overexpression GmEXPB2 of full uniformity transgenosis and Wild-type soy are invaded into bubble in 1/4 soybean (dish Beans) in pancebrin to showing money or valuables one carries unintentionally, utilize the filter paper for being impregnated with nutrient solution to carry out vernalization growth.Growth is determined under Stereo microscope respectively The main root root hair zone of 2 days and non-root hair zone length, and the Soybean Root Hairs density of growth 2 days is further compared, each processing has Ten biology are repeated.

Fig. 5 is influences of the overexpression GmEXPB2 to Soybean transgenic plant Root morphology.Wherein：

Fig. 5 (A) is soybean phenotype；

Fig. 5 (B) is root hair phenotype；

Fig. 5 (C) is main root length.

OX1-3 is GmEXPB2 overexpression strain；WT1-2 is wild type.

It is the data of same 10 repetitions of genetically modified plants in figure.

Asterisk represents significant difference of the same character between OX and WT strains：* * represent level of signifiance ρ≤0.001 When, difference is extremely notable.

As a result show：GmEXPB2 has important regulating and controlling effect to soybean main root and growing for root hair, data statistics hair Existing, overexpression GmEXPB2 significantly promotes the growth of soybean root system, adds main root length, root gross density and length.

6th, the influence that overexpression GmEXPB2 infects to genetically engineered soybean

Coil paper vernalization overexpression GmEXPB2 first and Wild-type soy seed, growth soaked soybean seedling root system after 4 days Enter in rhizobium USDA110 (USDA110-GFP is given by Tan Zhiyuan professors laboratory) bacterium solution of GFP marks about 1 hour, then Plant is transferred in sand culture and cultivated, and the developmental state of line and root nodule former base is infected in observation under Laser Scanning Confocal Microscope.

Fig. 6 is overexpression GmEXPB2 genetically engineered soybean Rhizobium Inoculation USDA110-GFP infection processs, wherein：

Fig. 6 (A)-Fig. 6 (F) sticks to root hair zone domain for rhizobium after being inoculated with 2 days；

Fig. 6 (G)-Fig. 6 (L) is formed to infect line (IT) after being inoculated with 3 days；

Fig. 6 (M)-Fig. 6 (R) is IT continued growths after being inoculated with 4 days to root skin confluent monolayer cells.

Fig. 6 (A)-Fig. 6 (C), Fig. 6 (G)-Fig. 6 (I), Fig. 6 (M)-Fig. 6 (O), are wild type；Fig. 6 (D)-Fig. 6 (F), Fig. 6 (J)-Fig. 6 (L), Fig. 6 (P)-Fig. 6 (R), are overexpression GmEXPB2 genetically engineered soybean.

Fig. 6 (A)-Fig. 6 (F) scale is 100 μm, and the scale of other pictures is 50 μm.

As a result show：GmEXPB2 may increase the adhered area of rhizobium, and then promote root nodule by relaxation cell membrane The formation infected and accelerate root nodule of bacterium.

7th, influences of the overexpression GmEXPB2 to genetically engineered soybean dross

The functional analysis of soybean nodulation is tested for overexpression GmEXPB2, the transgenosis of GmEXPB2 overexpressions and Wild-type soy seed (OX1-3：Overexpression GmEXPB2 transgenic line；WT：Wild type), surface sterilization and sprout in 5 days in sterilized sand, the consistent seedling inoculation rihizobium japonicum BXYD3 of growing way is then moved into hydroponic system after mono- hour. Plant strain growth in above-mentioned Poor nitrogen nutrition liquid and carry out high phosphorus (500 μM) and it is low-phosphorous (5 μM) processing.Change weekly one time of nutrition liquid and It is 5.8 or so to keep pH.The soybean nodulation of 25 days carries out root nodule number, dry weight and nitrogenase activity determination analysis after harvest inoculation.

Fig. 7 is influences of the overexpression GmEXPB2 to soybean nodulation under high low-phosphorous processing.Wherein：

Fig. 7 (A) is root nodule picture；

Fig. 7 (B) is root nodule number；

Fig. 7 (C) is root nodule dry weight；

Fig. 7 (D) is nodule nitrogenase activities.

Transgenosis and Wild-type soy are in different phosphorus concentration (high phosphorus：500μM KH₂PO₄；It is low-phosphorous：10μM KH₂PO₄) it is low Grown 25 days in nitrogen nutrition liquid.WT is wild type；OX1-3 is GmEXPB2 overexpression strains.It is same genetically modified plants in figure Average value and standard error that 4 biology is repeated.

Asterisk represents significant difference of the same character between OX and WT strains：

* the level of signifiance 0.01 is represented<During ρ≤0.05, significant difference；

* represents the level of signifiance 0.001<During ρ≤0.01, the significance of difference between significantly with extremely significantly between；

When * * represent level of signifiance ρ≤0.001, difference is extremely notable；

Ns represents that difference is not notable.

As a result show：Overexpression GmEXPB2 significantly promotes soybean nodulation, adds the root nodule under the conditions of high and low phosphorus Nodule nitrogenase activities under the conditions of quantity, root nodule dry weight and high phosphorus.

8th, overexpression GmEXPB2 is to soybean growth and the influence of nitrogen/phosphorus content

Biomass and nitrogen, phosphorus determination method are ibid.

Fig. 8 is overexpression GmEXPB2 to soybean growth and the influence of nitrogen/phosphorus content.Wherein,

Fig. 8 (A) is plant phenotype；

Fig. 8 (B) is plant weights；

Fig. 8 (C) is nitrogen content；

Fig. 8 (D) is phosphorus content.

The transgenosis and Wild-type soy of Rhizobium Inoculation processing are in different phosphorus concentration (high phosphorus：500μM KH₂PO₄；It is low-phosphorous： 10μM KH₂PO₄) the lower growth of Poor nitrogen nutrition liquid processing 25 days.WT is wild type；OX1-3 is GmEXPB2 overexpression strains. It is average value and standard error that same 4 biology of genetically modified plants are repeated in figure.

Asterisk represents significant difference of the same character between OX and WT strains：

* the level of signifiance 0.01 is represented<During ρ≤0.05, significant difference；

* represents the level of signifiance 0.001<During ρ≤0.01, the significance of difference between significantly with extremely significantly between；

When * * represent level of signifiance ρ≤0.001, difference is extremely notable；

Ns represents that difference is not notable.

As a result show：Overexpression GmEXPB2 promotes soybean growth by promoting soybean nodulation fixed nitrogen, it is high, Under the conditions of low-phosphorous, the content of nitrogen and phosphorous and biomass of plant are significantly improved.

In summary, β-expansin gene GmEXPB2 has the New function of regulating and controlling soybean nodulation and nitrogen fixation.In the processing of low nitrogen Under the conditions of Rhizobium Inoculation, the overexpression soybean transgene of gene of the present invention be combined the root nodule numbers of plant, weight, Nitrogen/phosphorus content and biomass are all remarkably higher than control strain, GmEXPB2 cultivate efficient nodulation and nitrogen fixation transgenic leguminous plants, Improve the fertilizer utilization efficiency of crops and there is positive application value in terms of improving crop yield.

It should be noted that the invention is not limited in any way for above-described embodiment, all use equivalent substitutions or equivalent change The technical scheme that the mode changed is obtained, all falls within protection scope of the present invention.

Claims (6)

1. applications of the β-expansin gene GmEXPB2 in terms of soybean nitrogen efficiency is improved.

2. application according to claim 1, it is characterised in that the β-expansin gene GmEXPB2 is in soybean nodulation Mesoderm growing early stage is expressed, and is positioned at root nodule former base, Nodule cortex and parenchyma cell.

3. application according to claim 1, it is characterised in that compared with Wild-type soy, overexpression GmEXPB2's turns Transgenic soybean significantly increases root system root hair zone length and root gross density, and then expands connecing for rhizobium and host's root system indirectly Contacting surface is accumulated.

4. application according to claim 1, it is characterised in that compared with Wild-type soy, overexpression GmEXPB2's turns Transgenic soybean, which is significantly increased, infects line number amount, accelerates the formation of root nodule former base.

5. application according to claim 1, it is characterised in that compared with Wild-type soy, overexpression GmEXPB2's turns Transgenic soybean significantly increases root nodule numbers, weight and nodule nitrogenase activities.

6. application according to claim 1, it is characterised in that compared with Wild-type soy, overexpression GmEXPB2's turns Transgenic soybean significantly promotes plant strain growth, adds the content of nitrogen and phosphorous and biomass.