CN110241121A - The application of soybean E3 ubiquitin ligase GmNLA1 encoding gene - Google Patents
The application of soybean E3 ubiquitin ligase GmNLA1 encoding gene Download PDFInfo
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Abstract
The invention discloses the applications of soybean E3 ubiquitin ligase GmNLA1.Soybean GmNLA1 protein coding gene GmNLA1, nucleotide sequence are as follows: SEQ ID NO.1.Plant the Overexpression vector pMDC83-GmNLA1 and GmNLA1-RNAi of building are transformed into soybean hairy, it is found after handling 15d with 1/2 Hoagland, in GmNLA1-OE transgenic hairy root, the P concentration in GmNLA1-OE transgenic hairy root is significantly reduced.P concentration in GmNLA1-RNAi transgenic hairy root dramatically increases.Generally speaking, GmNLA1 may be by the P concentration in negative regulator soybean transgene hairy come negative regulation soybean phosphorus efficiency.
Description
Technical field
The present invention relates to the applications of soybean E3 ubiquitin ligase GmNLA1 encoding gene, belong to genetic engineering field, specifically
Ground says that soybean E3 ubiquitin ligase GmNLA1 gene influences hairy middle phosphorus concentration of soybean, and then changes phosphorus use efficiency.
Background technique
Phosphorus (P) is one of essential mineral element (L ó pez-Arredondo et in plant growth and breeding
Al.2014), it participates in many metabolic processes, and if energy and cell membrane are formed, nucleic acid is synthesized, photosynthesis and respiration.This
Outside, P content is the 0.05%-0.5% (Vance et al.2003) of plant dry weight.From 1961 to 2013 year, make in the world
Agricultural fertilizer total amount is annual 17500000 tons.The usage amount of unit area phosphate fertilizer in the same period increases about 3 times of (Lu&Tian
2017).Crop yield can be improved in application phosphate fertilizer, but is applied to the P fertilizer 80%-90% of soil by microorganism adsorption, or and metal
Ion forms slightly solubility chelate (Holford 1997).Only orthophosphate ions (H2PO4 -And HPO4 2-) can be direct by plant
It absorbs and utilizes (Hinsinger 2001), lead to the high phosphate fertilizer in soil, but plant phosphorus use efficiency is low.It fixes in the soil
Difficultly soluble phosphatic fertilizer river is entered by rain-out, lead to water eutrophication.Therefore, improving phosphorus efficiency is to solve grain-production
One of with the effective and sustainable development method of water eutrophication.
Soybean GmNLA1 is E3 ubiquitin ligase.So far, it only identifies and belongs in arabidopsis and rice mutant
The limitation of one E3 ubiquitin ligase gene of SPX-RING family, i.e. nitrogen adapts to (NLA).It was found that arabidopsis nla mutant is low
Presenility was shown under the conditions of nitrogen, was since P is poisoned (Kant et al.2011).Under normal P-condition, nla mutant
P content in overground part and root is twice of (Lin et al.2013 of WT；Park et al.2014).And in rice also
Homologous gene OsNLA1 is identified, blade P content dramatically increases in rice osnla1 mutant, and independent of nitrogen (Yue
et al.2017).And there is no the relevant report of GmNLA1 gene function in soybean.Using technique for gene engineering, construct respectively
Overexpression and rna interference vector, discovery GmNLA1 can negative regulation soybean hairy middle phosphorus concentration after soybean transformation hairy.This
A little results will be helpful to understand the molecular mechanism of soybean phosphorus efficiency, while can accelerate the breeding process of soybean phosphorus efficiency kind.
Summary of the invention
It is an object of the invention to the resistance genetic engineering application that open soybean GmNLA1 is E3 ubiquitin ligase, the bases
It importing in soybean hairy because can be used as target gene, being utilized by influencing hairy middle phosphorus concentration of soybean come the phosphorus of regulating and controlling soybean
Efficiency.
The purpose of the present invention can be achieved through the following technical solutions:
Soybean E3 ubiquitin ligase GmNLA1, nucleotide sequence are as follows: SEQ ID NO.1.
Soybean E3 ubiquitin ligase GmNLA1, amino acid sequence are as follows: SEQ ID NO.2.
Application of the soybean E3 ubiquitin ligase gene GmNLA1 in the phosphorus use efficiency for adjusting soybean, the soybean E3
Ubiquitin ligase gene GmNLA1, nucleotide sequence are as follows: SEQ ID NO.1.
Preferably, GmNLA1 is overexpressed in soybean hairy reduces hairy middle phosphorus concentration of overexpression for the application；Or
Person utilizes RNA perturbation technique silencing GmNLA1 in soybean hairy, improves hairy middle phosphorus concentration of interference.
When constructing plant over-express vector or interference carrier using GmNLA1, it can add and appoint before its transcription initiation nucleotide
A kind of what enhanced promoter or inducible promoter.For the ease of transgenic plant cells or plant are identified and are sieved
Choosing, can process plant expression vector used, and selected marker (gus gene, luciferase are added such as in plant
Gene etc.).Consider from the safety perspective of genetically modified plants, any selected marker can be not added, and is screened by adverse circumstance
Transformed plant.
Soybean GmNLA1 protein coding gene GmNLA1 of the present invention is passing through genetic engineering soybean transformation hairy
Afterwards, it is overexpressed the gene, reduces hairy middle phosphorus concentration；After the gene AF panel gene expression that RNA is mediated, increase hairy
Middle phosphorus concentration.
Carry GmNLA1 of the present invention plant over-express vector and interference carrier can by using Ti-plasmids, Ri plasmid,
The conventional biology methods such as plant viral vector, DNA directly convert, microinjection, conductance, mediated by agriculture bacillus conversion plant cell
Or tissue, and the plant tissue of conversion is cultivated into plant.The plant host being converted is either sorghum, rice, wheat, jade
The monocotyledons such as rice, are also possible to the dicotyledons such as peanut, soybean, rape, tomato, poplar, turfgrass, clover.
Beneficial effect
Soybean GmNLA1 is the gene of a coding E3 ubiquitin ligase, includes SPX and RING domain, belongs to SPX-
RING family gene.SPX-RING family only NLA and PHO2 in arabidopsis and rice isotype crop, which is reported, to participate in planting
Object phosphorus stable state.We have found that the expression quantity of GmNLA1 and hairy middle phosphorus concentration of soybean are negatively correlated in soybean.GmNLA1 exists
There are significant differences for expression quantity in phosphorus efficiency and phosphorus sensitive material.Simultaneously by GmNLA1 gene overexpression and RNA interference hair
Existing GmNLA1 influences phosphorus use efficiency in soybean by influencing the phosphorus concentration in soybean hairy.Therefore, GmNLA1 can be used
In soybean phosphorus efficiency breed breeding.
Detailed description of the invention
The PCR amplification of Fig. 1 GmNLA1 gene.Marker:DL5000
Relative expression quantity of Fig. 2 GmNLA1 in rich No. 1 of section and southern agriculture 1138-2.
The subcellular localization of Fig. 3 GmNLA1.
(a): GFP；(d): GmNLA1-GFP；(b): GFP light field figure；(e): GmNLA1-GFP light field figure；(c): GFP fusion
Figure；(f): GmNLA1-GFP fusion figure.Bars=70 μm of
The hairy phenotype and fresh weight of Fig. 4 .GmNLA1.
(a) phenotype of GmNLA1-OE transgenic hairy root and its unloaded control Control 1 are in 0.5mM KH2PO4Nutrition
15d is grown in liquid.(b) phenotype of GmNLA1-RNAi transgenic hairy root and its unloaded control Control 2 are in 0.5mM
KH2PO415d is grown in nutrient solution.(c) fresh weight of GmNLA1-OE and GmNLA1-RNAi transgenic hairy root and its control
(Control 1/Control 2) is in 0.5mM KH2PO4It is grown 15 days in nutrient solution.
Hairy middle phosphorus concentration of the relative expression quantity of Fig. 5 .GmNLA1 and soybean.
(a) in GmNLA1-OE and GmNLA1-RNAi transgenic hairy root GmNLA1 relative expression quantity.(b)GmNLA1-
P concentration in OE and GmNLA1-RNAi transgenic hairy root and its non-transgenic overground part.GmNLA1-OE: there is pMDC83-
The soybean of GmNLA1 hairy, Control 1: soybean hairy with pMDC83 zero load；GmNLA1-RNAi: have
Soybean hairy of pB7GWIWG2 (II)-GmNLA1RNAi, Control 2: the soybean hair with pB7GWIWG2 (II) zero load
Shape root.Hairy of GmNLA1-OE and GmNLA1-RNAi and its to impinging upon 0.5mM KH2PO4Middle growth 15 days.Three biology are flat
Mean value ± standard error (SE).* with * * respectively on 0.05 and 0.01 probability level it is significant.
Fig. 6 is overexpressed hairy (GmNLA1-OE) PCR detection.M:Marker DL2000, P: positive plasmid, H:
ddH2O, C: hairy negative.
Fig. 7 .RNA interferes hairy (GmNLA1-RNAi) PCR detection.M:Marker DL1000, P: positive plasmid, H:
DdH2O, C: hairy negative.
Specific embodiment
The present invention will be further described with reference to the accompanying drawings and embodiments.
Method therefor is conventional method unless otherwise instructed in following embodiments.
1) clone of soybean E3 ubiquitin ligase GmNLA1 gene
It is materials object with soybean cultivars south agriculture 1138-2, takes its root, ground with mortar, addition fills lysate
1.5mL EP pipe moves in 1.5mL EP pipe sufficiently after oscillation, extracted total RNA (Total RNA Kit (Tiangeng, Beijing, in
State).Total serum IgE quality, spectrophotometric determination rna content are identified with denaturing formaldehyde gel electrophoresis.Using the total serum IgE of acquisition as template,
Reverse transcription reagent box (the TaKaRa Primer Script provided according to Japanese TaKaRa companyTMRT reagent kit, day
Originally specification) carries out reverse transcription, after obtaining the first chain of cDNA, carries out PCR amplification, PCR program is as follows: 95 DEG C of initial denaturations
3min, 95 DEG C of denaturation 15sec, 60 DEG C of annealing 15sec, 72 DEG C of extension 1min, totally 35 recycle, last 72 DEG C of heat preservations 5min, with
12 DEG C of constant temperature afterwards.Then carry out PCR product rubber tapping purifying, connection and transformation, the sequencing of picking positive monoclonal.It is obtained after sequencing
There must be the CDS sequence for the soybean GmNLA1 gene that the length of complete coding region is 948bp, wherein coding region sequence is shown in SEQ ID
NO.1 is named as GmNLA1, forms (figure by 948bp
1)。
2) the subcellular localization research of GmNLA1
Primer (do not include terminator codon) of the design comprising the complete ORF of GmNLA1 gene, primer sequence is shown in SEQ ID
NO.9 and SEQ ID NO.10, specific PCR process are identical as step 1).Then will not included using XbaI and KpnI double digestion
The complete ORF homologous recombination of the GmNLA1 gene of terminator codon is into expression vector pSuper1300, thus GmNLA1
The complete ORF of gene is merged with the 3 ' ends of the reporter gene GFP on expression vector pSuper1300, forms a 35S-
The mosaic gene of GmNLA1-GFP constructs subcellular localization carrier pSuper1300-GmNLA1.Itself and empty carrier is sharp respectively
Target gene GmNLA1 is transferred to tobacco leaf cell with conversion method for agrobacterium, the results showed that GmNLA1 albumen is located in cell
On film (Fig. 3).
3) relative expression quantity of the GmNLA1 in rich No. 1 of section and southern agriculture 1138-2
Two kinds of material seedling of soybean phosphorus sensitive varieties (Nan Nong 1138-2) and Tolerant to low P kind (rich No. 1 of section) are being contained
0.5mM KH2PO41/2Hoagland nutrient solution in handle 3d, 7d and 12d sampling, saved in -80 DEG C after liquid nitrogen flash freezer.Always
The same step 1) of the extraction of RNA.Using the Tubulin of soybean constitutive expression as internal reference, primer sequence is shown in SEQ ID NO.7
With SEQ ID NO.8, with come from the cultivated soybean phosphorus sensitive varieties (Nan Nong 1138-2) and two kinds of materials of Tolerant to low P kind (rich No. 1 of section)
Expect that the underground part total serum IgE under the conditions of different disposal is template, is reversed to carry out real-time fluorescence quantitative PCR reaction after cDNA
(Real-time RT-PCR), primer sequence are shown in SEQ ID NO.5 and SEQ ID NO.6, detect GmNLA1 gene in different product
Expression quantity changes in kind.
When being transferred to 3d, 7d and 12d in 1/2Hoagland nutrient solution, the relative expression quantity of GmNLA1 is in different P efficiency
Southern agriculture 1138-2 and Ke Feng 1 in have significant difference (Fig. 2).The relative expression quantity of GmNLA1 is significantly low in rich No. 1 of section
Yu Nannong 1138-2.This shows that the expression quantity of GmNLA1 is related to different cultivars phosphorus efficiency.
The genetic engineering application of 2 gene GmNLA1 of embodiment
1) clone of soybean E3 ubiquitin ligase GmNLA1
Using the root total serum IgE of soybean (Glycine max) phosphorus sensitive varieties south agriculture 1138-2 as template, synthesized through reverse transcription
After the first chain of cDNA, PCR amplification is carried out, primer sequence is shown in that SEQ ID NO.3 and SEQ ID NO.4, PCR program is as follows: 95 DEG C
Initial denaturation 3 minutes, 95 DEG C were denaturalized 15 seconds, and 60 DEG C are annealed 15 seconds, and 72 DEG C extend 1 minute, and totally 35 recycle, last 72 DEG C of heat preservations 5
Minute, PCR product is cloned into PUC19-T Vector by subsequent 12 DEG C of constant temperature, and the length with complete coding region is obtained after sequencing
Degree is the CDS sequence of the soybean GmNLA1 gene of 948bp, and wherein coding region sequence is shown in SEQ ID NO.1.
2) building of plant expression vector
By GmNLA1 gene order and Invitrogen companyTechnology with ClonaseTM
PDONR221 carrier in II kit carries out BP reaction, and carries out bacterium solution PCR sequence verification, and primer sequence is shown in SEQ ID
NO.11 and SEQ ID NO.12, specific PCR process is identical as step 1), obtains entry clones；By obtained entry clones with
The purpose expression vector pMDC83 of Invitrogen company exploitation carries out recombination exchange, obtains pMDC83-GmNLA1 plant and crosses table
Up to carrier, plant conversion carrier pMDC83 contains 2x 35S strong promoter, can induced strong target gene GmNLA1 in receptor
Expression.Then carrier is transferred in agrobacterium rhizogene strain K599 by freeze-thaw method, while unloaded pMDC83 is also transformed into
In K599, compareed as zero load.
When constructing rna interference vector, one section of interference fragment is expanded first with special primer, primer sequence is shown in SEQ ID
NO.13 and SEQ ID NO.14, PCR product sequence are shown in SEQ ID NO.15, following step and building over-express vector one
Sample only changes pMDC83 carrier into pB7GWIWG2 (II) carrier, the carrier GmNLA1-RNAi built is equally transformed into
In K599.Unloaded pB7GWIWG2 (II) is also transformed into K599 simultaneously, is compareed as zero load.
3) acquisition of transgenosis root hair
The hairy method for transformation of soybean proposed using Kereszt et al. (2007) is contained respectively by what step 2) obtained
PMDC83-GmNLA1 and GmNLA1-RNAi carrier and the agrobacterium rhizogene strain K599 bacterium solution of corresponding unloaded control are injected into 7
Below the cotyledonary node of its soybean seedling, it is placed in constant temperature illumination box, 12h illumination, 12h dark culturing, and keep high humility,
Hairy can grow from injection site after 2-3 weeks, when there is 5-10cm, cut seedling main root, be placed in 1/2Hoagland nutrition
It is cultivated 15 days in liquid, obtains soybean seedling chimera, including non-transgenic overground part and transgenic hairy root.There to be overexpression
Hairy chimera is referred to as GmNLA1-OE, and zero load control is Control 1；To there is the chimera of hairy of interference
Referred to as GmNLA1-RNAi, zero load control are Control 2.To detect whether to be hairy of the positive, specific primer is utilized
PCR detection is carried out to the DNA fragmentation of extraction.It is overexpressed hairy detection primer sequence and sees SEQ ID NO.16 and SEQ ID
NO.17, PCR hairy detection glue figure of the positive are shown in Fig. 6.RNA interferes hairy detection bar primer, is SEQ ID NO.18 and SEQ
ID NO.19, PCR are positive, and Fig. 7 is shown in hairy detection.Real time fluorescent quantitative qPCR discovery is being overexpressed hairy (GmNLA1-OE)
Middle GmNLA1 gene expression amount will be significantly higher than (Fig. 5 a) of Control 1.And (the GmNLA1- in hairy of RNA interference
RNAi GmNLA1 gene expression amount is significantly lower than (Fig. 5 a) of Control 2 in).
When the hairy root long of soybean is to suitable size, with 1/2Hoagland handle after 15d measurement GmNLA1-OE and
P concentration in GmNLA1-RNAi transgenic hairy root.The result shows that in GmNLA1-OE transgenic hairy root, GmNLA1-OE
Middle P concentration is 0.77 times of Control 1, shows that P concentration in GmNLA1-OE transgenic hairy root significantly drops under+P-condition
It is low.But GmNLA1-OE non-transgenic overground part is not significantly different (Fig. 5 b).On the contrary, the P concentration in GmNLA1-RNAi is
1.66 times of transgenosis root Control 2 show that the P concentration under+P-condition in GmNLA1-RNAi transgenic hairy root is significant
Increase.With GmNLA1-OE transgenosis it is hairy, GmNLA1-RNAi non-transgenic overground part is also not significantly different (Fig. 5 b).
These are the result shows that P concentration of the GmNLA1 under+P-condition in negative regulator soybean transgene hairy.
Sequence table
<110>Agricultural University Of Nanjing
<120>application of soybean E3 ubiquitin ligase GmNLA1 encoding gene
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Claims (3)
1. application of the soybean E3 ubiquitin ligase gene GmNLA1 in the phosphorus use efficiency for adjusting soybean, the soybean E3 are general
Element connection enzyme gene GmNLA1, nucleotide sequence are as follows: SEQ ID NO.1.
2. application according to claim 1, which is characterized in that be overexpressed GmNLA1 in soybean hairy, reduce and be overexpressed
Hairy middle phosphorus concentration.
3. application according to claim 1, which is characterized in that utilize RNA perturbation technique silencing in soybean hairy
GmNLA1 improves hairy middle phosphorus concentration of interference.
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