CN109053869A

CN109053869A - Soybean nucleoporin gene GmNup96 is adjusting the developmental application of plant root nodule

Info

Publication number: CN109053869A
Application number: CN201810887072.0A
Authority: CN
Inventors: 傅永福; 陈福禄; 董秀春; 黄鹏辉; 纪丹丹
Original assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Priority date: 2018-08-06
Filing date: 2018-08-06
Publication date: 2018-12-21
Anticipated expiration: 2038-08-06
Also published as: CN109053869B

Abstract

The present invention relates to soybean nucleoporin gene GmNup96 to adjust the developmental application of plant root nodule.The amino acid sequence of GmNup96 gene order and its coding albumen is respectively as shown in SEQ ID NO:1 and 2.The test of first passage gene function and verifying of the present invention, silencing soybean nucleoporin gene GmNup96 results in the decline of soybean nodulation number, show that GmNup96 gene plays a significant role in Radical extension and Active Regulation, it is the important gene for participating in process of nodulation in soybean, can be used for the work such as the molecular breeding of soybean.The present invention will provide foundation for effect of research soybean nucleoporin gene during soybean nodulation, provide new technological means to promote soybean growth and improving soybean yields.Meanwhile reference is provided for other legume nodule growth adjustments.

Description

Soybean nucleoporin gene GmNup96 is adjusting the developmental application of plant root nodule

Technical field

The invention belongs to field of plant genetic project technology, specifically, being related to soybean nucleoporin gene GmNup96 Adjusting the developmental application of plant root nodule.

Background technique

Nucleus is the most important organelle of eukaryocyte (Tamura et al.2010).It contains inhereditary material, passes through Controlling gene is expressed to adjust cell activity.There are nuclear pore complex (nuclear pore complex, abbreviation on nuclear membrane NPC).This structure is the unique passage into nucleus, and mediating protein and RNA are transported between cytoplasm and nucleus, The composition for participating in cytoskeleton arrives different cell processes (the Xu and Meier 2008 such as gene expression； Meier and Brkljacic 2009b；D′Angelo and Hetzer 2008).In cell, it is multiple that nuclear pore complex is the largest polyprotein It is fit and be known as nucleoporin (Nucleoporin, abbreviation Nup) (Rout et containing about 30 different albumen al.2000；Cronshaw et al.2002).

Currently, the research about the structure of nuclear pore complex, composition and nucleoporin function is concentrated mainly on yeast and has In several species (such as drosophila, mouse and people) of limit, and (Grossman et al. 2012) is made great progress, But the functional study and regulatory mechanism to plant nuclear pore complex need to further elucidate (Tamura and Hara- Nishimura 2013)。

The function of soybean nucleoporin gene and its application have not been reported.By adjust the gene expression of soybean nucleoporin come Change Radical extension and active adjusting is not also reported.

Summary of the invention

The object of the present invention is to provide soybean nucleoporin gene GmNup96 to adjust the developmental application of plant root nodule.

In order to achieve the object of the present invention, in a first aspect, the present invention, which provides soybean nucleoporin gene GmNup96, is adjusting plant Application in object Radical extension, wherein the protein of GmNup96 gene coding is for following (a) or (b):

(a) protein that the amino acid sequence shown in SEQ ID NO:2 forms；

(b) sequence shown in SEQ ID NO:2 is substituted, lacks or adds one or several amino acid and has same function The protein as derived from (a).

The nucleotide sequence of GmNup96 gene (Nup96 gene) are as follows:

I) nucleotide sequence shown in SEQ ID NO:1；Or

Ii) nucleotide sequence shown in SEQ ID NO:1 be substituted, lack and/or increase one or more nucleotide and Nucleotide sequence with the same function；Or

Iii) hybridize under strict conditions with sequence shown in SEQ ID NO:1 and nucleotide sequence with the same function, The stringent condition be in 0.1 × SSPE containing 0.1%SDS or 0.1 × SSC solution containing 0.1%SDS, it is miscellaneous at 65 DEG C It hands over, and washes film with the solution；Or

Iv) and i), ii) or nucleotide sequence iii) there is 90% or more homology and nucleotide with the same function Sequence.

Soybean nucleoporin gene GmNup96 of the invention is to clone to obtain from soybean day grand No. 1 using PCR amplification method , PCR amplification (SEQ ID NO:3-4) is carried out using 1 pair of specific primer:

GmNup96.XhoI.F:5′-GTATACTCGAGATGGAATGTGACGTTGGAGGTG-3′

GmNup96.EcoRI.R:5′-TGCCTGAATTCCGTAGCAATCTCTGAGAGATA-3′

Application above-mentioned, the regulation include but is not limited to promote plant root nodule development, increase nodule number.

Further, the application includes:

1) making plant includes GmNup96 gene；Alternatively,

2) plant is made to be overexpressed GmNup96 gene.

In the present invention, the plant is monocotyledon or dicotyledon, preferably soybean or arabidopsis.

Second aspect, the present invention provide application of the soybean nucleoporin gene GmNup96 in plant breeding.

Wherein, breeding objective includes but is not limited to promote plant root nodule development, increases nodule number.

The third aspect, the present invention provides a kind of method for reducing soybean nodulation number, by genetic engineering means, to soybean Nucleoporin gene GmNup96 carries out rite-directed mutagenesis, so that the gene lacks functionality or decrease, to reduce soybean nodulation number Mesh；Or

By importing repressor into soybean come silencing GmNup96 gene, to reduce soybean nodulation number.

Wherein, the repressor includes but is not limited to shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA/DNA；

Preferably, make soybean nucleoporin gene using the method for RNA interference (RNAinterference, abbreviation RNAi) The mRNA expression of GmNup96 reduces, to reduce soybean nodulation number.

It is highly preferred that the repressor is by the corresponding GmNup96 of primer GmNup96RNAi-F and GmNup96RNAi-R The RNA of target dna fragment coding on gene, wherein the sequence of the primer GmNup96RNAi-F and GmNup96RNAi-R is such as Under (SEQ ID NO:5-6):

GmNup96RNAi-F:5′-TCAAGAATGGCAGGCGAGAT-3′

GmNup96RNAi-R:5′-GGAATGGGAGCACTGAAAGC-3′

In addition, the present invention provides the biomaterial for containing the GmNup96 gene, the biomaterial includes but is not limited to Expression cassette, expression vector, cloning vector, engineering bacteria or non-reproducible plant part.

The expression vector for carrying the target gene can be turned by using Ti-plasmids, plant viral vector, direct DNA The standard biologics technical method such as change, microinjection, electroporation imports (Weissbach, 1998, Method in plant cell Plant Molecular Biology VIII, Academy Press, New York, the 411-463 pages；Geiserson and Corey, 1998, Plant Molecular Biology, 2^nd Edition)。

The present invention also provides application of the GmNup96 gene in prepare transgenosis plant.

By above-mentioned technical proposal, the present invention at least have following advantages and the utility model has the advantages that

First passage gene function test of the present invention and verifying, silencing soybean nucleoporin gene GmNup96 are resulted in greatly The decline of beans nodule number, shows that GmNup96 gene plays a significant role in Radical extension and Active Regulation, is participated in soybean The important gene of process of nodulation can be used for the work such as the molecular breeding of soybean.The present invention will be research soybean nucleoporin base Because effect during soybean nodulation provides foundation, new technology is provided to promote soybean growth and improving soybean yields Means.

Further, it is overexpressed in arabidopsis using 35S promoter driving GmNup96-GFP fusion protein, as a result table Bright GmNup96 is located on nuclear membrane.Using RNAi technology, GmNup96-RNAi Transgenic soybean plants are obtained, is detected, turns base Because GmNup96mRNA level declines in soybean plant strain；After being inoculated with root nodule, nodule number be can obviously reduce.Thus, adjust soybean core Radical extension and its activity can be changed in porin gene GmNup96mRNA expression, adjusts for the development of other legume nodules Section provides reference.

Detailed description of the invention

Fig. 1 is the Ago-Gel of the PCR amplification primer of soybean nucleoporin gene GmNup96 in the embodiment of the present invention 1 Electrophoresis result.The swimming lane on the right visible special purpose band between 3000bp and 5000bp in figure.

Fig. 2 is the structural schematic diagram of entry vector GEC in the embodiment of the present invention 1.

Fig. 3 is GmNup96 Gene Expression Profile Analysis in the embodiment of the present invention 1.

Fig. 4 is the structural schematic diagram of plant expression vector BDV2 in the embodiment of the present invention 2.

Fig. 5 is the subcellular localization of soybean nucleoporin GmNup96 in the embodiment of the present invention 2.Scale=50 μm.

Fig. 6 is the structural schematic diagram of pGWCm entry vector in the embodiment of the present invention 3.

Fig. 7 is the structural schematic diagram of plant RNA i expression vector pB7GWIWG2 in the embodiment of the present invention 3.

Fig. 8 is GmNup96-RNAi transgenic plant Molecular Detection in the embodiment of the present invention 3.Wherein, WT indicates wild type； P indicates positive plasmid；#2, #3-1, #3-2, #8, #9 indicate the different genetically engineered soybean strains obtained；M is indicated DL5000DNAMarker。

Fig. 9 is GmNup96-RNAi transgenic plant GmNup96mRNA expression in the embodiment of the present invention 3.Wherein, WT Indicate wild type；#2, #3, #8, #9 indicate the different GmNup96-RNAi genetically engineered soybean strains obtained.

Figure 10 is the size and number of GmNup96-RNAi transgenic plant root nodule in the embodiment of the present invention 4.Wherein, a is Root nodule numbers statistics: WT indicates wild type；#2, #3, #8, #9 indicate the different GmNup96-RNAi genetically engineered soybeans obtained Strain, b are the audio-visual picture of GmNup96-RNAi#9 genetically engineered soybean strain and WT wild type root nodule sum.

Specific embodiment

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment According to conventional laboratory conditions, such as Sambrook molecular cloning experiment handbook (Sambrook J&Russell DW, Molecular Cloning:a Laboratory Manual, 2001), or according to the condition of manufacturer's specification suggestion.

Carrier GEC, BDV2 used in the following embodiment are referring to Wang et al.2013.Carrier pGWCm is referring to Chen et al.2006.Carrier pB7GWIWG2 is referring to Karimi et al.2002.

The acquisition of 1 soybean nucleoporin gene GmNup96 of embodiment and its expression pattern

Using 1 couple of primer GmNup96.XhoI.F and GmNup96.EcoRI.R (SEQ ID NO:3-4) from soybean day grand 1 Number cDNAPCR amplification (table 1) and digestion (table 2) connection (table 3) is to entry vector Fu28-MCS-GFP, skeleton is GEC carrier (Fig. 2).Sequencing obtains the GmNup96 coding region sequence that overall length is 3069bp, as shown in Fig. 1, nucleotide sequence such as SEQ ID Shown in NO:1, corresponding amino acid sequence is as shown in SEQ ID NO:2.

Table 1PCR amplification system

PCR response procedures: 94 DEG C of 5min；98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 3min, totally 30 recycle；72℃ 10min。

2 digestion system of table

3 coupled reaction system of table

The method for transformation of bacillus coli DH 5 alpha competent cell includes: LB of (1) preparation containing chloramphenicol or ampicillin Solid medium；(2) taking-up is stored in -80 DEG C of competent cell, after melting in ice bath, 10 μ l connection products is added and gently mix It is even, 30min is placed in ice bath；(3) 42 DEG C of heat shock 45s, are placed in 3min in ice bath immediately after；800 μ l are added without antibiotic LB liquid medium, 37 DEG C of shaking table shaken cultivation 1h (160rpm)；(4) appropriate culture solution is taken uniformly to be applied to selective medium On, 16h are cultivated in 37 DEG C of inversions, with sterilizing toothpick picking 5-10 (or more) bacterium colony, be placed in 200 μ l selected liq LB culture Bacterium culture is shaken in base；(5) PCR detects the positive colony screened, and auspicious rich sequencing company is sent to be sequenced.

Using fluorescence real-time quantitative PCR detection Nup96 gene, each histoorgan is expressed in grand No. a kind of soybean day Amount, as a result as shown in figure 3, the gene has expression in root, stem, leaf and root nodule, the expression quantity highest in root, leaf and root nodule are secondary It, and expression quantity is minimum in stem.

The subcellular localization of 2 soybean nucleoporin GmNup96 of embodiment

The carrier Fu28-GmNup96-GFP that embodiment 1 is obtained is used with another entry vector Fu76-35S The method (Invitrogen) of GateWay recombinant clone is reacted by LR by 35S promoter and GmNup96-GFP genetic recombination To on plant expression vector BDV2 (Fig. 4), and expand in bacillus coli DH 5 alpha numerous.It, will by agrobacterium mediation converted method The 35S:GmNup96-GFP gene that BDV2 is carried is transferred to arabidopsis, obtains transformation of Arabidopsis thaliana plant.The result shows that soybean nucleopore egg White GmNup96 is located on nuclear membrane (Fig. 5).

The acquisition of 3 soybean nucleoporin gene GmNup96-RNAi transgenic plant of embodiment

Using 1 couple of primer GmNup96RNAi-F and GmNup96RNAi-R (SEQ ID NO:5-6) with the acquisition of embodiment 1 Carrier Fu28-GmNup96-GFP be template, carry out PCR amplification.

Target fragment is connected on pGWCm entry vector (Fig. 6) by the method that T-A is cloned, and is sequenced.Using The method (Invitrogen) of GateWay recombinant clone is reacted by LR and clones entry vector pGWC-GmNup96-RNAi To plant RNA i expression vector pB7GWIWG2 (Fig. 7), Agrobacterium EHA105 conversion is completed, identifies positive strain, -80 DEG C of guarantors It deposits.Then grand No. a kind in GmNup96-RNAi carrier soybean transformation day is obtained into GmNup96-RNAi transgenic plant, through dividing Sub- test positive plant (Fig. 8).We are further to T₁It is sampled for plant leaf, extracts total serum IgE and reverse transcription is at cDNA, benefit GmNup96 gene expression amount in different strains is detected with fluorescence real-time quantitative PCR, is found in #2, #3 (#3-1, #3-2), #8, #9 The gene expression amount is implicitly present in different degrees of decline (Fig. 9) in four RNAi transgenic lines, illustrates that these plant are certain For transgenic positive seedling.

Application of the 4 soybean nucleoporin gene GmNup96 of embodiment in regulation nodule number

The statistics that transgenic line carries out root nodule numbers, each Line are stablized to the GmNup96-RNAi that embodiment 3 obtains Statistical magnitude is greater than 6 plants, and biology is in triplicate, and as a result as shown in Figure 10 a, the root nodule numbers of different transgenic lines are equal It is fewer than wild type, while transgenosis #9 strain is subjected to sample mixing, under Figure 10 b intuitively shows that transgenic plant root nodule numbers are certain Drop, therefore illustrate that soybean Nup96 gene may participate in process of nodulation, and gene expression amount declines, and subtracts along with root nodule numbers It is few.

Although above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be modified or is improved, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.

Bibliography

1、Tamura K,Fukao Y,Iwamoto M,Haraguchi T,Hara-Nishimura I(2010) Identification and characterization of nuclear pore complex components in Arabidopsis thaliana.Plant Cell 22(12):4084-4097.doi:10.1105/tpc.110.079947

2、Xu XM,Meier I(2008)The nuclear pore comes to the fore.Trends Plant Sci 13(1):20-27. doi:10.1016/j.tplants.2007.12.001

3、Meier I,Brkljacic J(2009b)The nuclear pore and plant development.Curr Opin Plant Bio1 12(1):87-95. doi:10.1016/j.pbi.2008.09.001

4、D′Angelo MA,Hetzer MW(2008)Structure,dynamics and function of nuclear pore complexes.Trends Cell Biol 18 (10):456-466.doi:10.1016/ j.tcb.2008.07.009

5、Rout MP,Aitchison JD,Suprapto A,Hjertaas K,Zhao Y,Chait BT(2000)The yeast nuclear pore complex:composition, architecture,and transport mechanism.J Cell Biol 148(4):635-651

6、Cronshaw JM,Krutchinsky AN,Zhang W,Chait BT,Matunis MJ(2002) Proteomic analysis of the mammalian nuclear pore complex.J Cell Biol 158(5): 915-927.doi:10.1083/jcb.200206106

7、Grossman E,Medalia O,Zwerger M(2012)Functional architecture of the nuclear pore complex.Annu Rev Biophys 41:557-584.doi:10.1146/annurev-biophys- 050511-102328

8、Tamura K,Hara-Nishimura I(2013)The molecular architecture of the plant nuclear pore complex.J Exp Bot 64 (4):823-832.doi:10.1093/jxb/ers258

9、Chen QJ,Zhou HM,Chen J,Wang XC(2006)Using a modified TA cloning method to create entry clones.Anal Biochem 358(1):120-125.doi:10.1016/ j.ab.2006.08.015

10、Karimi M,Inze D,Depicker A(2002)GATEWAY vectors for Agrobacterium- mediated plant transformation.Trends Plant Sci 7(5):193-195

11、Wang X,Fan C,Zhang X,Zhu J,Fu YF(2013)BioVector,a flexible system for gene specific-expression in plants. BMC Plant Biol 13:198.doi:10.1186/ 1471-2229-13-198。

Sequence table

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Glu Ile Leu Phe Gln Tyr Cys Glu Thr Trp Ser Ser Asp Glu Ser Gln

805 810 815

Gln Gln Phe Ile Glu Asp Leu Gly Ile Pro Thr Asp Trp Met His Glu

820 825 830

Ala Leu Ala Ile Tyr Tyr Asn Tyr Asn Gly Asp His Ser Lys Ala Leu

835 840 845

Asp Gln Phe Leu Gln Cys Ala Asn Trp Gln Lys Ala His Ala Ile Phe

850 855 860

Ile Thr Ser Val Ala His Arg Leu Phe Leu Gln Ala Lys His Ala Glu

865 870 875 880

Ile Trp Arg Ile Ala Thr Ser Met Glu Asp His Lys Ser Glu Ile Glu

885 890 895

Asn Trp Glu Leu Gly Ala Gly Ile Tyr Ile Ser Phe Tyr Leu Met Arg

900 905 910

Asn Ser Leu Gln Asp Asp Thr Asn Ala Met Thr Glu Leu Asp Ser Leu

915 920 925

Glu Ser Lys Asn Ala Ala Cys Gln Asp Phe Val Ser Gln Leu Asn Glu

930 935 940

Ser Leu Ala Val Trp Gly Cys Arg Leu Pro Val Asp Ala Arg Val Val

945 950 955 960

Tyr Ser Arg Met Ala Gly Glu Ile Cys Asp Leu Leu Leu Ser Gly Val

965 970 975

Gly Glu Gly Ala Thr Arg Asp Glu Gln Phe Asn Cys Phe Asp Thr Ala

980 985 990

Phe Ser Ala Pro Ile Pro Glu Asp Gln Arg Ser Gly His Leu Gln Asp

995 1000 1005

Ala Val Tyr Leu Phe Thr Ser Tyr Leu Ser Glu Ile Ala Thr

1010 1015 1020

<210> 3

<211> 33

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

gtatactcga gatggaatgt gacgttggag gtg 33

<210> 4

<211> 32

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

tgcctgaatt ccgtagcaat ctctgagaga ta 32

<210> 5

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

tcaagaatgg caggcgagat 20

<210> 6

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 6

ggaatgggag cactgaaagc 20

Claims

1. soybean nucleoporin gene GmNup96 is adjusting the developmental application of plant root nodule, wherein GmNup96 gene coding Protein be following (a) or (b):

(a) protein that the amino acid sequence shown in SEQ ID NO:2 forms；

(b) sequence shown in SEQ ID NO:2 be substituted, lack or add one or several amino acid and with same function by (a) protein derived from.

2. application according to claim 1, which is characterized in that the regulation includes promoting plant root nodule development, increases root Tumor number.

3. application according to claim 1, which is characterized in that the application includes:

1) making plant includes GmNup96 gene；Alternatively,

2) plant is made to be overexpressed GmNup96 gene.

4. application according to claim 1-3, which is characterized in that the plant is monocotyledon or dicotyledonous Plant, preferably soybean or arabidopsis.

5. application of the soybean nucleoporin gene GmNup96 in plant breeding, wherein the definition of the GmNup96 gene is same Described in claim 1.

6. application according to claim 5, which is characterized in that breeding objective includes promoting plant root nodule development, increases root Tumor number.

7. application according to claim 5, which is characterized in that the plant be monocotyledon or dicotyledon, it is excellent Select soybean or arabidopsis.

8. a kind of method for reducing soybean nodulation number, which is characterized in that by genetic engineering means, to soybean nucleoporin base Because GmNup96 carries out rite-directed mutagenesis, so that the gene lacks functionality or decrease, to reduce soybean nodulation number；Or

By importing repressor into soybean come silencing GmNup96 gene, to reduce soybean nodulation number；

Wherein, the definition of the GmNup96 gene is the same as described in claim 1.

9. according to the method described in claim 8, it is characterized in that, the repressor include shRNA, siRNA, dsRNA, MiRNA, cDNA, antisense RNA/DNA.

10. according to the method described in claim 9, it is characterized in that, the repressor be by primer GmNup96RNAi-F and The RNA of target dna fragment coding on the corresponding GmNup96 gene of GmNup96RNAi-R；

The sequence of the primer GmNup96RNAi-F and GmNup96RNAi-R is as follows:

GmNup96RNAi-F:5′-TCAAGAATGGCAGGCGAGAT-3′

GmNup96RNAi-R:5′-GGAATGGGAGCACTGAAAGC-3′。