CN110144357A

CN110144357A - Application of the overexpression of gene in the nitrogen content and/or protein content or breeding high-protein plant kind for improving plant

Info

Publication number: CN110144357A
Application number: CN201910350183.2A
Authority: CN
Inventors: 朱四元; 许晓敏; 刘头明; 王延周; 郑霞
Original assignee: Institute of Bast Fiber Crops of CAAS
Current assignee: Institute of Bast Fiber Crops of CAAS
Priority date: 2019-04-28
Filing date: 2019-04-28
Publication date: 2019-08-20

Abstract

The present invention relates to field of biotechnology, in particular to application of the overexpression of gene in the nitrogen content and/or protein content or breeding high-protein plant kind for improving plant.The present invention clones the BnGS1 gene obtained by showing nitrogen content in tobacco and crude protein content is respectively 4.88% and 30.52%, the nitrogen content and crude protein content of wild-type tobacco blade are respectively 2.29% and 14.32%, and the Leaf nitrogen concentration and crude protein content of transgenic plant and wild type reach significant difference.This is also that first passage overexpression in tobacco obtains nitrogen in ramie and nitrogen content reaches the GS gene of significant difference, which has a good application prospect.Ramie protein content is improved in such a way that the relevant gene of molecular breeding cloned proteins is by overexpression, the breeding new high protein variety period is short, and protein content is higher.

Description

The overexpression of gene is high in the nitrogen content and/or protein content that improve plant or breeding Application in protein plant kind

Technical field

The present invention relates to field of biotechnology, in particular to the overexpression of gene is in the nitrogen content and/or egg for improving plant Application in Bai Hanliang or breeding high-protein plant kind.

Background technique

Ramie young stem and leaf is full of nutrition, and crude protein content is high, is good plant feed protein raw materials, and south China is high Albumen high quality forage is most deficient, and Devoting Major Efforts To Developing ramie feed crop has extremely wide prospect, feeding ramie in south China As pasture utilization, we will detect crude protein content to fiber crops first, it is the important finger for measuring a feeding ramie characteristic quality Mark.Traditional ramie high nitrogen-containing kind material screening is carried out by means such as hybridization, and this selection period needs several Year is difficult have the extra high material of nitrogen content in ramee variety material itself, and improving ramie nitrogen content is current feeding ramie Numb breeding needs the significant problem solved, very long by conventional breeding high nitrogen-containing ramee variety material periodicities, is difficult to obtain High nitrogen content kind.

In plant, glutamine synthelase (Glutamine synthetase, GS) is by NH4⁺Glutamine is assimilated into, GS/GOGAT circulation is referred to as with GOGAT (glutamate synthetase).GS includes two large families, GS1 and GS2, wherein the main position GS1 In cytoplasm, GS2 is located in plastid.In plant, generally there are a GS2 gene, multiple GS1 genes.GS gene pairs plant nitrogen benefit Have an impact with rate.In two kinds of the high nitrogen use efficiency of rice and low nitrogen use efficiency, the activity of GS is that the former is significantly higher than the latter. Zheng JS etc. overexpresses BnGS1-2 in tobacco, the results showed that the NO of transgene tobacco₃ ^-And NH₄ ⁺Absorbability enhancing, NR activity dramatically increases, and soluble protein content and nitrogen content are above control group, but difference is not significant.Meanwhile BnGS2-1 and Plant height, fresh weight and the leaf soluble protein content of BnGS2-2 transgene tobacco are significantly increased；Nitrogen content is also improved, but is not had Have and reaches significant difference.

Therefore it provides ramie nitrogen metabolism related gene come improve ramie protein content, shorten breeding high protein variety week Phase has important practical significance.

Summary of the invention

In view of this, the present invention provides the overexpressions of gene in the nitrogen content and/or protein content that improve plant or choosing Educate the application in high-protein plant kind.The present invention is selected using the relevant gene of means clone's ramie nitrogen content of molecular breeding The high New Ramie Variety material of nitrogen content is educated, the time is relatively short, and nitrogen content increase is larger, can obtain ramie high nitrogen-containing faster Kind.

In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:

The present invention provides the overexpressions of gene to plant in the nitrogen content and/or protein content or breeding high protein for improving plant Application in article kind；

The gene has any one in nucleotide sequence as follows:

I, with the nucleotide sequence of BnGS1；Or

II, it modified, replaced, missed or added one or more bases obtain with the nucleotide sequence as shown in I Nucleotide sequence；Or

III and the nucleotide sequence as shown in I are with gained albumen and base after the sequence or translation of at least 80% homology Because of the same or similar nucleotide sequence of the protein function of BnGS1 expression；Or

The complementary series of IV, the sequence as shown in I, II or III.

In some specific embodiments of the invention, the plant is ramie or tobacco.

On the basis of the studies above, the present invention also provides expression vectors, the gene including overexpression and

Carrier is carrier；

The gene has any one in nucleotide sequence as follows:

I, with the nucleotide sequence of BnGS1；Or

The complementary series of IV, the sequence as shown in I, II or III.

The present invention also provides the construction methods of the expression vector, and gene is connect with the carrier is carrier, and conversion is chosen Bacterium extracts plasmid, sequencing；

The gene has any one in nucleotide sequence as follows:

I, with the nucleotide sequence of BnGS1；Or

The complementary series of IV, the sequence as shown in I, II or III.

The present invention also provides the host cells for converting or transfecting the expression vector.

The present invention also provides the expression vectors or the host cell to contain in the nitrogen content and/or albumen for improving plant Application in amount or breeding high-protein plant kind

In some specific embodiments of the invention, the plant is ramie or tobacco.

The present invention also provides the methods for improving nitrogen content in plant, and the expression vector is taken to be transferred to Agrobacterium competence In, culture chooses bacterium, positive bacterium colony is taken to infect plant.

The present invention also provides the methods for improving protein content in plant, and the expression vector is taken to be transferred to Agrobacterium competence In, culture chooses bacterium, positive bacterium colony is taken to infect plant.

The present invention also provides the breeding methods of high-protein plant, and the expression vector is taken to be transferred in Agrobacterium competence, Culture, chooses bacterium, positive bacterium colony is taken to infect plant.

The present invention clones the BnGS1 gene obtained by showing that nitrogen content and crude protein content are distinguished in tobacco For 4.88% and 30.52%, the nitrogen content and crude protein content of wild-type tobacco blade are respectively 2.29% and 14.32%, are turned The Leaf nitrogen concentration and crude protein content of gene plant and wild type reach significant difference.This be also in ramie first passage exist Overexpression obtains nitrogen in tobacco and nitrogen content reaches the GS gene of significant difference, which has a good application prospect.It is logical It crosses the relevant gene of molecular breeding cloned proteins and improves ramie protein content, the new high protein product of breeding by way of overexpression The kind period is short, and protein content is higher.

Detailed description of the invention

In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below There is attached drawing needed in technical description to be briefly described.

Fig. 1 shows BnGS1 gene gel electrophoresis figure；Wherein, Fig. 1 (A) shows the pcr amplification product of BnGS1 gene, and swimming lane is from a left side 1:DNA marker, 2-3:BnGS1 gene are followed successively by the right side；Fig. 1 (B) show TA monoclonal PCR verifying, swimming lane from left to right according to It is secondary be 1,4:DNA marker, 2-3:BnGS1 gene；

Fig. 2 shows the CDS sequence and amino acid sequence (gray scale expression conserved domain) of BnGS1；

Fig. 3 shows BnGS1 structural analysis of protein；

Fig. 4 shows PEZR- (K)-LN-GFP: the unloaded expression in tobacco leaf；Wherein, A, D: light field；B, E: green Color fluorescence；C, F: synthesis；

Fig. 5 shows that single colonie PCR is identified；Wherein Fig. 5 (A) shows that BnDof14 gene, swimming lane are followed successively by 1:DNA from left to right Marker, 2-6:BnDof14 gene；Fig. 5 (B) shows that BnGS1 gene, swimming lane are followed successively by 1,6:DNA marker, 2- from left to right 3:BnGS1 gene；

Fig. 6 shows the PCR identification of Agrobacterium positive transformants bacterium colony；Wherein, Fig. 6 (A) shows BnDof14 gene, swimming lane from a left side to The right side is followed successively by 1:DNA marker, 2-5,8: false positive bacterium colony；6-7:BnDof14 gene masculine bacterium colony；Fig. 6 (B) shows BnGS1 base Cause, swimming lane are followed successively by the positive bacterium colony of 1-4:BnGS1 gene, 6:DNA marker from left to right；

Fig. 7 shows the regeneration of transgene tobacco；Wherein, Fig. 7 (A) shows the callus of tobacco；Fig. 7 (B) shows that young shoot is taken root；Figure 7 (C) show transgene tobacco transplantation of seedlings into soil；

Fig. 8 shows the DNA of transgene tobacco；Swimming lane is followed successively by 1,7:DNA marker, 2-6: transgene tobacco from left to right DNA；

Fig. 9 shows transgene tobacco PCR product；Swimming lane is followed successively by 1-3:BnGS1 gene, 4:DNA marker from left to right；

Figure 10 shows the relative expression quantity of BnGS1 gene in BnGS1 transgenosis and wild-type tobacco；

Figure 11 shows the GUS dyeing of transgenic tobacco leaf；

Figure 12 shows BnGS1 transgene tobacco and wild-type tobacco phenotype；Wherein, Figure 12 (A) shows transgene tobacco and wild The comparison of type tobacco plant height；Figure 12 (B) shows that transgene tobacco and wild-type tobacco leaf color compare；

Figure 13 shows BnGS1 transgene tobacco and wild-type tobacco Leaf nitrogen concentration；

Figure 14 shows BnGS1 transgene tobacco and wild-type tobacco blade crude protein content.

Specific embodiment

The invention discloses the overexpressions of gene to plant in the nitrogen content and/or protein content or breeding high protein for improving plant Application in article kind, those skilled in the art can use for reference present disclosure, be suitably modified realization of process parameters.Especially need to refer to Out, all similar substitutions and modifications are apparent to those skilled in the art, they be considered as include In the present invention.Method and application of the invention is described by preferred embodiment, and related personnel can obviously not take off Method described herein and application are modified or appropriate changes and combinations from the content of present invention, spirit and scope, come real Now and using the technology of the present invention.

The overexpression of gene provided by the invention is planted in the nitrogen content and/or protein content or breeding high protein for improving plant Raw materials used and reagent is available on the market in application in article kind.

Below with reference to embodiment, the present invention is further explained:

1 ramie BnGS1 gene cloning of embodiment and screening TA monoclonal

Agarose gel electrophoresis figure such as Fig. 1 (A) of ramie BnGS1 gene PCR product, PCR condition are as follows: (1) 94 DEG C of pre- change Property the 54 DEG C of renaturation 30s of 4min, (2) 94 DEG C of denaturation 30s, (3), (4) 72 DEG C of extension 1min 30s, (2)-(4) recycle 32 times, (5) 72 DEG C of extension 5min, 4 DEG C of preservations.BnGS1 gene length 1518bp, glue figure show that PCR product is consistent with required target fragment.It will It connect, is transformed into DH5 α, coated plate and 38 DEG C of CMC models are selected white on plate with pMD18-T after the recycling of target fragment glue Spot shakes bacterium.Bacterium solution PCR result such as Fig. 1 (B).

2 ramie BnGS1 gene biological bioinformatics analysis of embodiment

The CDS sequence (as shown in SEQ ID No.1) and amino acid sequence of BnGS1 gene (as shown in SEQ ID No.2) Such as Fig. 2, protein structure such as Fig. 3.The long 1071bp of CDS sequence of BnGS1 gene, the long 356aa of amino acid sequence.Molecular weight of albumen It is 5.60 for 39.19Da, isoelectric point pI.There is 1 conservative structural domain from 3 to 354 site of amino acid 1, which is GS egg White distinctive structural domain.

The subcellular localization of embodiment 3BnGS1 gene

Pass through double digestion experimental construction PEZR- (K)-LN-BnGS1-GFP carrier.The specific method is as follows:

(1) double digestion is carried out to PEZR- (K)-LN using EcoRI and KpnI, system is as follows:

(2) 37 DEG C of digestion 1h, then glue recycles.

(3) double digestion is carried out using EcoRI and KpnI restriction enzyme BnGS1 gene, system is as follows:

(4) 37 DEG C of digestion 1h, then glue recycles.

(5) the T4DNA ligase of the glue recovery product in (2), (4) is connected, reaction system (10 μ L): Plasmid DNA 1 μ L, T4DNA ligase of 100ng, target fragment 10ng, 10*Buffer, 0.5 μ L, adds ddH₂O to 10 μ L.Under the conditions of 25 DEG C, even Meet 3h.

PEZR- (the K)-LN-BnGS1-GFP carrier built is transformed into Agrobacterium GV3101, will be contained using syringe There is the GV3101 of carrier to be injected into tobacco leaf, 3d is further cultured for after dark culture 2h, then in confocal laser scanning microscope The luciferase expression situation of leaf slice, as a result such as Fig. 4.The display zero load of subcellular localization picture produces in cytoplasm and nucleus Green florescent signal illustrates that zero load is positioned at nucleus and cytoplasm.PEZR- (K)-LN-BnGS1-GFP is only produced in cytoplasm Raw green fluorescence, therefore BnGS1 albumen is mainly distributed in cytoplasm.

The acquisition of 4 transgene tobacco of embodiment

Overexpression vector is constructed by single endonuclease digestion method, the specific method is as follows:

(1) TSAP Thermosensitive Alkaline Phosphatase kit (Promega, the U.S.) is used Single endonuclease digestion is carried out to pBI121, reaction system is following (10 μ L):

(2) 37 DEG C of digestion 2h.

(3) 74 DEG C of inactivation 15min.

(4) single endonuclease digestion is carried out to BnGS1 gene with SpeI, reaction system is following (20 μ L):

(5) 37 DEG C of digestion 1h, then glue recycles.

(6) the T4DNA ligase of the product in (3), (5) is connected, reaction system (10 μ L): Plasmid DNA 100ng, mesh 1 μ L, T4DNA ligase of segment 10ng, 10*Buffer, 2 μ L, add ddH₂O to 10 μ L.Under the conditions of 25 DEG C, 3h is connected.

The overexpression vector connected is transformed into bacillus coli DH 5 alpha competence, method for transformation is as follows:

(1) it takes 100 μ L competence to melt on ice, 10 μ of pBI121 carrier for having target fragment is added under aseptic condition L is mixed gently, and places 25min on ice.

Then centrifuge tube is quickly transferred on ice, stand 2min by (2) 42 DEG C of heat shock 45s.

(3) under aseptic condition, the not antibiotic LB liquid medium of 600 μ L, 37 DEG C, 200rpm are added in centrifuge tube Under the conditions of, shaken cultivation 1h makes thallus recover.

By the thallus of recovery on the plate containing Kan coated plate, cultivate 12h under the conditions of 37 DEG C, picking single colonie shakes bacterium.It is right Bacterium solution carries out PCR verifying, result such as Fig. 5.Fig. 5 illustrates that the single colonie selected contains target fragment.Plasmid is extracted, plasmid is surveyed As a result sequence proves gene and the successful connection of pBI121 carrier.

Agrobacterium-mediated Transformation and positive colony identification:

The plant overexpression vector built is transferred in Agrobacterium GV3101 competence by heat shock method, specifically such as Under:

(1) the Agrobacterium competent cell GV3101 of -70 DEG C of preservations is taken to melt in ice-water bath.

(2) under aseptic condition, 1 μ g Plasmid DNA is added into competent cell, mixes gently, ice-water bath stands 5min.

(3) centrifuge tube is placed in quick-frozen 5min in liquid nitrogen, then quickly centrifuge tube is placed in 37 DEG C of water-baths and keeps 5min, It is careful not to shake the water surface at this time；Centrifuge tube is put back in ice-water bath again, ice bath 5min.

(4) add the LB culture medium of 800 μ L, antibiotic-free, shaken cultivation under the conditions of 28 DEG C under aseptic condition in centrifuge tube 2-3h makes thallus recover.

The bacterium solution of recovery is cultivated into 2-3d on the culture medium containing Kan and Rif.Picking single colonie carries out bacterium colony PCR, as a result Such as Fig. 6.Plant overexpression vector successful conversion is into GV3101 thallus as the result is shown.

Agrobacterium infects tobacco leaf:

Agrobacterium positive single colonie is shaken into bacterium, tobacco is infected by leaf disk method.Tobacco leaf after infecting is cultivated in MS0 It is transferred on MS1 culture medium after dark culture 2d on base, continuous light culture, until the long budding (Fig. 7 A) of callus.Bud is grown to It when 2-3cm, is cut, is transferred in MS2, induced bud takes root (Fig. 7 B).MS2 culture medium contains Kan, and positive seedling can be normal Growth, negative seedling can be dead.The seedling of healthy growth is taken out from MS2 culture medium, after rinsing root agar with tap water, kind (Fig. 7 C) is planted in soil.

The identification of transgene tobacco:

The extraction of transgene tobacco DNA:

The DNA for extracting transgene tobacco carries out electrophoresis experiment to DNA with 1% Ago-Gel, the quality of validating DNA, As a result such as Fig. 8.Glue figure, which is shown, does not have protein contamination in the DNA of extraction, also there is no fractures, high-quality, can be used in PCR.

The PCR of transgene tobacco is verified:

Using the DNA of transgene tobacco as template, PCR reaction is carried out by primer of GS1F-1 and GS1R-1.BnGS1 transgenosis The PCR product of tobacco plant such as Fig. 9.BnGS1 gene length 1071bp, Ago-Gel figure show that BnGS1 gene is successfully transferred to cigarette In grass, transgene tobacco seedling is constructed successfully.

The qRT-PCR of transgene tobacco is analyzed:

QRT-PCR analysis is carried out to transgene tobacco, to further determine that the accuracy of transgenic tobacco plant.With cigarette Careless Nlt25 gene is reference gene, BnGS1 gene such as Figure 10 of the relative expression quantity in transgenic plant, as the result is shown BnGS1 It is expressed in transgenic plant, and fails in wild type to detect the mRNA level in-site of BnGS1 allele.

Transgene tobacco GUS dyeing:

Transgene tobacco tender leaf is taken to carry out GUS dyeing, expression of the checking carrier in tobacco leaf, as a result as schemed 11.GUS Coloration experiment shows that BnGS1 gene has stronger expression in transgenic plant.

The analysis of nitrogen content of transgene tobacco:

Flowering period takes the Blade measuring nitrogen content of BnGS1 transgenosis, wild-type tobacco plants respectively.To plant when sampling Strain is observed, as a result such as Figure 12.From picture it can be found that compared with wild type, the plant height of BnGS1 transgenic tobacco plant Significant to reduce, the leaf color of transgenic plant is deeper.The nitrogen content and thick egg of wild type and BnGS1 rotaring gene plant blade Bai Hanliang is respectively such as Figure 13, Figure 14.The nitrogen content and crude protein content of BnGS1 transgenic tobacco leaf are respectively 4.88% He 30.52%, the nitrogen content and crude protein content of wild-type tobacco blade are respectively 2.29% and 14.32%, transgenic plant and The Leaf nitrogen concentration and crude protein content of wild type reach significant difference.

In conclusion nitrogen and the crude protein content functional verification of ramie BnGS1 gene the result shows that, BnGS1 gene is for the first time Show that nitrogen and crude protein content dramatically increase 2 times or more in tobacco；Molecular breeding can be passed through using BnGS1 gene The high feeding ramee variety of crude protein content of method breeding.

The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered It is considered as protection scope of the present invention.

Sequence table

<110>Hemp Inst., China Academy of Agricultural Sciences

<120>overexpression of gene is in the nitrogen content and/or protein content or breeding high-protein plant kind for improving plant Using

<130> MP1904417

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1071

<212> DNA

<213> BnGS1

<400> 1

atgtctctcc tcaacgatct cctcaacatt aacctgtcag acaccacaga gaagatcatc 60

gccgagtaca tatggatcgg tggatctggc atggacctaa gaagcaaagc caggactttg 120

aatggaccag tttcagatcc atcaaagctt cccaaatgga actacgacgg ttcaagcaca 180

gaccaagctc ccggggacga cagtgaagtc atcttatacc ctcaagcggt gtttaaggat 240

ccattcagga gaggaaacaa tatcctggtg atgtgtgacg cttatacgcc agctggcgag 300

ccgattccga cgaacaaacg acacaatgct gcgaagattt tcagtcaccc tgatgtggtc 360

gctgaggagc cttggtatgg cattgagcaa gaatacactc tccttcagaa ggatgttaac 420

tggccacttg gttggcccgt cggcggcttc ccggggcccc agggtcctta ctactgtgcc 480

gccggggcag acaaggcttt cggtcgcgac atagtgaacg cgcactacaa ggcttgcctc 540

tacgccggag tcaacatcag cggaatcaac ggcgaagtca tgcctggcca atgggagttt 600

caagtgggtc cttctgtcgg cattagctcc ggtgatcaag tctggattgc tcgctacatt 660

cttgagagga tcactgaaat tgcaggagtt gttctttcat ttgatcctaa gccgatcccg 720

ggtgactgga acggcgccgg tgcccacact aactacagca ccaagtcgat gagaaatgaa 780

ggcgggatcg acgtgatcaa gaaggccatc gagaaattag ggttgcgcca caaggagcac 840

atcgccgcct atggagaagg caacgagagg agactgaccg gtcgtcacga gaccgctgac 900

atcaacacct tctcttgggg tgtagcaaat cgtggagcct ctgttcgcgt tggtcgtgac 960

accgaaaagg aaggcaaagg ctacttcgag gaccgaaggc ctgcctcgaa catggatcct 1020

tacgttgtga cgtcgatgat tgcggagacc accatccttt ggaagccttg a 1071

<210> 2

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<212> PRT

<213> BnGS1

<400> 2

Met Ser Leu Leu Asn Asp Leu Leu Asn Ile Asn Leu Ser Asp Thr Thr

1 5 10 15

Glu Lys Ile Ile Ala Glu Tyr Ile Trp Ile Gly Gly Ser Gly Met Asp

20 25 30

Leu Arg Ser Lys Ala Arg Thr Leu Asn Gly Pro Val Ser Asp Pro Ser

35 40 45

Lys Leu Pro Lys Trp Asn Tyr Asp Gly Ser Ser Thr Asp Gln Ala Pro

50 55 60

Gly Asp Asp Ser Glu Val Ile Leu Tyr Pro Gln Ala Val Phe Lys Asp

65 70 75 80

Pro Phe Arg Arg Gly Asn Asn Ile Leu Val Met Cys Asp Ala Tyr Thr

85 90 95

Pro Ala Gly Glu Pro Ile Pro Thr Asn Lys Arg His Asn Ala Ala Lys

100 105 110

Ile Phe Ser His Pro Asp Val Val Ala Glu Glu Pro Trp Tyr Gly Ile

115 120 125

Glu Gln Glu Tyr Thr Leu Leu Gln Lys Asp Val Asn Trp Pro Leu Gly

130 135 140

Trp Pro Val Gly Gly Phe Pro Gly Pro Gln Gly Pro Tyr Tyr Cys Ala

145 150 155 160

Ala Gly Ala Asp Lys Ala Phe Gly Arg Asp Ile Val Asn Ala His Tyr

165 170 175

Lys Ala Cys Leu Tyr Ala Gly Val Asn Ile Ser Gly Ile Asn Gly Glu

180 185 190

Val Met Pro Gly Gln Trp Glu Phe Gln Val Gly Pro Ser Val Gly Ile

195 200 205

Ser Ser Gly Asp Gln Val Trp Ile Ala Arg Tyr Ile Leu Glu Arg Ile

210 215 220

Thr Glu Ile Ala Gly Val Val Leu Ser Phe Asp Pro Lys Pro Ile Pro

225 230 235 240

Gly Asp Trp Asn Gly Ala Gly Ala His Thr Asn Tyr Ser Thr Lys Ser

245 250 255

Met Arg Asn Glu Gly Gly Ile Asp Val Ile Lys Lys Ala Ile Glu Lys

260 265 270

Leu Gly Leu Arg His Lys Glu His Ile Ala Ala Tyr Gly Glu Gly Asn

275 280 285

Glu Arg Arg Leu Thr Gly Arg His Glu Thr Ala Asp Ile Asn Thr Phe

290 295 300

Ser Trp Gly Val Ala Asn Arg Gly Ala Ser Val Arg Val Gly Arg Asp

305 310 315 320

Thr Glu Lys Glu Gly Lys Gly Tyr Phe Glu Asp Arg Arg Pro Ala Ser

325 330 335

Asn Met Asp Pro Tyr Val Val Thr Ser Met Ile Ala Glu Thr Thr Ile

340 345 350

Leu Trp Lys Pro

355

Claims

1. the overexpression of gene answering in the nitrogen content and/or protein content or breeding high-protein plant kind for improving plant With；

The gene has any one in nucleotide sequence as follows:

I, with the nucleotide sequence of BnGS1；Or

II, it modified with the nucleotide sequence as shown in I, replace, miss or add the nucleosides that one or more bases obtain Acid sequence；Or

III and the nucleotide sequence as shown in I are with gained albumen and gene after the sequence or translation of at least 80% homology The same or similar nucleotide sequence of the protein function of BnGS1 expression；Or

The complementary series of IV, the sequence as shown in I, II or III.

2. application as described in claim 1, which is characterized in that the plant is ramie or tobacco.

3. expression vector, which is characterized in that gene and carrier is carrier including overexpression；

The gene has any one in nucleotide sequence as follows:

I, with the nucleotide sequence of BnGS1；Or

The complementary series of IV, the sequence as shown in I, II or III.

4. the construction method of expression vector as claimed in claim 3, which is characterized in that gene is connect with the carrier is carrier, Bacterium is chosen in conversion, extracts plasmid, sequencing；

The gene has any one in nucleotide sequence as follows:

I, with the nucleotide sequence of BnGS1；Or

The complementary series of IV, the sequence as shown in I, II or III.

5. conversion or the host cell for transfecting expression vector as claimed in claim 3.

6. expression vector as claimed in claim 3 or host cell as claimed in claim 5 in the nitrogen content for improving plant and/or Application in protein content or breeding high-protein plant kind.

7. application as claimed in claim 6, which is characterized in that the plant is ramie or tobacco.

8. the method for improving nitrogen content in plant, which is characterized in that expression vector as claimed in claim 3 is taken to be transferred to Agrobacterium sense By in state, culture chooses bacterium, positive bacterium colony is taken to infect plant.

9. the method for improving protein content in plant, which is characterized in that expression vector as claimed in claim 3 is taken to be transferred to Agrobacterium In competence, culture chooses bacterium, positive bacterium colony is taken to infect plant.

10. the breeding method of high-protein plant, which is characterized in that expression vector as claimed in claim 3 is taken to be transferred to Agrobacterium sense By in state, culture chooses bacterium, positive bacterium colony is taken to infect plant.