CN110438132A

CN110438132A - The tobacco gene NtFIP1 and its cloning process of raising nitrogen utilization efficiency and application

Info

Publication number: CN110438132A
Application number: CN201910757019.3A
Authority: CN
Inventors: 王超; 王大伟; 宋文路; 王勇; 杜昕昕; 权姝璇; 薛丽萍
Original assignee: Jining University
Current assignee: Jining University
Priority date: 2019-08-16
Filing date: 2019-08-16
Publication date: 2019-11-12

Abstract

The invention belongs to molecular biology of plants and gene clone technology field, more particularly to a kind of tobacco gene NtFIP1 and its cloning process for improving nitrogen utilization efficiency, present invention obtains the clones of tobacco gene NtFIP1 coded sequence, and the recombinant expression carrier containing tobacco gene NtFIP1 coded sequence is made, recombinant expression carrier is transformed into arabidopsis fip1 mutant and wild type, obtain heterologous complementary transgenic plant and overexpressing plants, experiments have shown that, tobacco gene NtFIP1 can in heterologous complementary transgenic plant and overexpressing plants high efficient expression, the application of present invention offer tobacco gene NtFIP1, tobacco gene NtFIP1 is applied to plant and obtains transgenic plant to improve the nitrogen utilization efficiency of plant, it can use NtFIP1 regulation tobacco T The content of SNAs is so as to improve cigarette quality.

Description

The tobacco gene NtFIP1 and its cloning process of raising nitrogen utilization efficiency and application

Technical field

The invention belongs to molecular biology of plants and gene clone technology field, and in particular to a kind of raising nitrogen utilization efficiency Tobacco gene NtFIP1 and its cloning process and application.

Background technique

Tobacco is the important industrial crops in China, and it is urgently to be solved strategic as China's tobacco business to improve cigarette quality Problem.Tobacco is happiness nitrate nitrogen (NO₃ ^-) plant, in production since utilization rate of nitrogen fertilizer is low, while the tobacco pair of different genotype Have larger difference in nitrogen utilization efficiency, be easy to cause in tobacco alkaloid type and content difference cause quality of tobacco decline to Influence peasant income.Therefore, tobacco NO is studied₃ ^-The metabolic processes such as absorb, transport, assimilating, utilizing to improve tobacco nitrogen benefit It is the key that solve the above problems with rate.

The height of nitrogen utilization efficiency is determined by Nitrogen absorption efficiency and nitrogen utilization efficiency the two factors.At present in flue-cured tobacco 4 NO are had found in kind K326 (NicotianatobacumL.cv.K326)₃ ^-Transporter gene and 9 NH₄ ⁺Transporter gene.But It is that also not in tobacco, discovery influences the controlling gene that these transporter genes are expressed.The NO of plant absorption₃ ^-In nitrate reductase (NR) and nitrite reductase (NiR) is reduced into NH₄ ⁺, then pass through paddy ammonia phthalein amine synzyme/glutamate synthetase (GS/ GOGAT) circulation is eventually converted to amino acid.Have in arabidopsis largely about influence NO₃ ^-The regulation base of assimilation genes expression Because of the report of function.However, there is presently no the upstream regulating genes that discovery influences the expression of tobacco nitrogen assimilation genes.

The content for reducing nitrogenous harmful substance in tobacco leaf and its product is to promote the important means of cigarette quality.In tobacco leaf tune In system and combustion process, a kind of special nitrosamine of tobacco (TSNAs) with carcinogenesis can be generated, table is more and more studied It is bright, by reducing NO in tobacco body₃ ^-And NO₂ ^-Content to reduce nitrosamine synthesis be also reduce TSNAs content it is effective Means.Further investigation revealed that reducing NO by improving tobacco NR and NiR activity₃ ^-And NO₂ ^-Content can equally reduce The content of TNSAs.

It is nearest the study found that arabidopsis pre-mRNA Polyadenylation factors A tFIP1 is a NO₃ ^-Controlling gene, By influencing the activity regulation plant of NRT1.1 to NO₃ ^-Absorption；By influencing NO₃ ^-Transporter gene NRT1.5, NRT1.8 and same The expression for changing gene NIA, NiR influences NO₃ ^-Distribution in root and overground part.Transcript group analysis shows that FIP1 also will affect Such as nitrogenous secondary metabolites synthesis, Nitrogen Cycling metabolism, abiotic stress and hormon induce physiology course.We send out It is existing, there is the sequence (NtFIP1) of FIP1 very high homology in tobacco gene group, which very likely regulates and controls tobacco to NO₃ ^-It inhales Receipts, transhipment, assimilation and TSNAs content, are worth being furtherd investigate.

Summary of the invention

It is an object of that present invention to provide a kind of tobacco gene NtFIP1 for improving nitrogen utilization efficiency, and provide its clone side Method, obtains the clone of tobacco gene NtFIP1 coded sequence, and another object of the present invention is that tobacco gene NtFIP1 is being mentioned The nitrogen utilization efficiency genetic improvement and improving quality of plant can be improved in application in high transgenic plant.

The tobacco gene NtFIP1, nucleotide sequence such as SEQ ID NO:1 of the present invention for improving nitrogen utilization efficiency It is shown.

The encoded amino acid sequence of tobacco gene NtFIP1 of the present invention is as shown in SEQ ID NO:2.

The cloning process of the tobacco gene NtFIP1 of the present invention for improving nitrogen utilization efficiency, includes the following steps:

(1) tobacco total serum IgE is extracted, reverse transcription synthesizes cDNA；

(2) using cDNA as template, special primer is designed, PCR amplification gene NtFIP1 coding region sequence, PCR amplification are passed through Using Phusion high-fidelity DNA polymerase, wherein PCR amplification the primer sequence is as follows:

NtFIP1 forward primer (F):

5'-CGGGGTACCATGGAAGATGACGACG-3'；

NtFIP1 reverse primer (R):

5'-CCGCTCGAGATTGCTGGTCCATCTCCT-3'；

The reaction condition of PCR amplification are as follows: 98 DEG C of initial denaturations 2min, 98 DEG C of denaturation 20s anneal according to the annealing temperature of primer 20s, 72 DEG C of extension 30s/Kb extend 10min after recycling 30 times, 72 DEG C；

(3) it recycles target fragment in the simultaneously amplified production of purification step (2) and is sequenced, sequencing is correctly tobacco The clone of gene NtFIP1 coded sequence.

The application of the tobacco gene NtFIP1 of the present invention for improving nitrogen utilization efficiency, tobacco gene NtFIP1 is applied Transgenic plant is obtained to improve the nitrogen utilization efficiency of plant in plant, and wherein plant is arabidopsis fip1 mutant, arabidopsis One of Col-0 wild type, tobacco bred K326.

The preparation method of the transgenic plant of NtFIP1 containing tobacco gene, includes the following steps:

S1 constructs pENTR3C-NtFIP1 recombinant plasmid

Digestion connects clone and the cloning vector pENTR3C of tobacco gene NtFIP1 coded sequence: to the tobacco-based of purifying Because KpnI and XhoI restriction enzyme site is added at 5 ' ends and 3 ' ends respectively in being cloned in for NtFIP1 coded sequence, band is obtained after double digestion There is the target gene fragment of cohesive terminus,cohesive termini, then carries out digestion connection with cloning vector pENTR3C, obtain recombinant plasmid pENTR3C-NtFIP1；

S2 constructs recombinant plant expression vector pMDC83-NtFIP1 using Gateway technology

The plant of correct recombinant plasmid pENTR3C-NtFIP1 and 35S promoter and Gateway system compatible will be sequenced Expression vector pMDC83 carries out LR reaction, obtains recombinant plant expression vector pMDC83-NtFIP1；

S3, Agrobacterium infect plant

Agrobacterium GV3101 competent cell is prepared, then converts Agrobacterium with recombinant expression carrier pMDC83-NtFIP1 GV3101 competent cell, the conversion Agrobacterium GV3101 of picking positive colony infects arabidopsis fip1 mutant and wild type is planted Strain, obtains the transgenic plant that can effectively clone tobacco gene NtFIP1 coded sequence.

Wherein: after conversion Agrobacterium infects arabidopsis fip1 mutant and wild type in S3, being selfed respectively by 3 generations, sieve Choosing obtains the heterologous complement strain containing tobacco gene NtFIP1 coded sequence and overexpression system.

Tobacco bred K326 is in the widely applied flue-cured tobacco cultivars in China, and genome is complicated allotetraploid, therefore, It is cloned in tobacco and identifies that participating in improving nitrogen utilization efficiency and the gene of improving quality makes slow progress.Arabidopsis plant genome It is small, be easy to convert, so the present invention predicts NtFIP1 gene and to clone by bioinformatics software, recycle plant table Express that tobacco gene NtFIP1 gene in arabidopsis fip1 mutant and wild type up to carrier, by studying heterologous complementary strain It is the function of identifying tobacco gene NtFIP1 gene with overexpression system Physiology and biochemistry phenotype, is being mentioned for tobacco gene NtFIP1 Theoretical basis is laid in the work of high tobacco nitrogen utilization efficiency genetic improvement and aspect of making better products.

Compared with prior art, the present invention having the advantages that.

(1) present invention obtains the clones of tobacco gene NtFIP1 coded sequence, and are made and contain tobacco gene NtFIP1 The recombinant expression carrier of coded sequence, recombinant expression carrier are transformed into arabidopsis fip1 mutant and wild type, are obtained heterologous Complementary transgenic plant and overexpressing plants, it is demonstrated experimentally that tobacco gene NtFIP1 can in heterologous complementary transgenic plant and High efficient expression in overexpressing plants；

(2) present invention is in various concentration KNO₃It can be by arabidopsis as tobacco gene NtFIP1 under only nitrogen source condition of culture Fip1 mutant restores to wild type phenotype, can improve the nitrogen utilization efficiency of plant to maintain higher chlorophyll content and master Root growth；

(3) tobacco gene NtFIP1 of the present invention can regulate and control NO in plant^3-Content proves to can use NtFIP1 tune The content of tobacco control grass TSNAs is so as to improve cigarette quality；

To sum up, research of the present invention for tobacco gene NtFIP1 function helps to improve tobacco nitrogen utilization efficiency, illustrates NO₃ ^-In the Distribution dynamics of tobacco root and overground part and the content for reducing the harmful substances such as cigarette TSNAs so as to improve cigarette quality. Meanwhile result of study will provide theoretical basis for the molecular breeding work of high nitrogen effect and high quality tobacco.

Detailed description of the invention

Fig. 1 is the clone of purpose tobacco gene NtFIP1 coded sequence, and wherein M swimming lane is DNAmarker；1,2 swimming lanes are equal It is cloned for the coding region sequence of tobacco gene NtFIP1,3 swimming lanes are negative control；

Fig. 2 is the recombinant plant expression vector process for constructing NtFIP1 and being overexpressed；

Fig. 3 is that recombinant cloning vector pENTR3C-NtFIP1 converts the positive bacterium colony after bacillus coli DH 5 alpha competent cell Qualification result；Wherein M swimming lane is DNAmarker, and 1-4 swimming lane is positive bacterium colony, and No. 5 swimming lanes are negative control；

Fig. 4 is that recombinant expression carrier pMDC83-NtFIP1 converts the positive bacterium colony after bacillus coli DH 5 alpha competent cell Qualification result；Wherein M swimming lane is DNAMarker, and 1-4 swimming lane is positive bacterium colony, and No. 5 swimming lanes are negative control；

Fig. 5 is that recombinant expression carrier pMDC8383-NtFIP1 converts the positive bacteria after Agrobacterium GV3101 competent cell Fall qualification result；Wherein M swimming lane is DNAMarker, and 1-4 swimming lane is positive bacterium colony, and No. 5 swimming lanes are negative control；

Fig. 6 is NtFIP1 expression quantity testing result in heterologous complementary plant and overexpression system；

The heterologous complementary plant of Fig. 7 and overexpression system plant shoots phase are in 0.5mM and 5mMKNO₃For the solid of only nitrogen source Growing state on culture medium；

Nitrate nitrogen content testing result in the heterologous complementary plant of Fig. 8 and overexpression system plant.

Specific embodiment

Below with reference to embodiment and Figure of description, the present invention will be further described.

The material and reagent that embodiment is used are as follows:

Material: gram of arabidopsis Col-0 wild type, arabidopsis FIP1 gene T-DNA mutant fip1, Gateway system Grand carrier pENTR3C and plant expression vector pMDC83 (invitrogen), bacillus coli DH 5 alpha (invitrogen), Agrobacterium GV3101(invitrogen)。

Reagent: Phusion high fidelity enzyme kit, TaqDNApolymerase kit (CWBIO), DNA-Marker (CWBIO), KpnI restriction enzyme XhoI restriction enzyme, DNA purification and recovery kit (Tiangeng), small amount plasmid preparation Kit (CWBIO), T4DNA ligase (invitrogen), LR clone enzyme (invitrogen), RNA purification kit (CWBIO), FirstStrandcDNASynthesisKit (ThermoFisher), agarose (Promega), remaining reagent is such as Trizol, chloroform, isopropanol, dehydrated alcohol, sodium hypochlorite, MES, sucrose, agar etc. are that domestic analysis is pure.

Embodiment 1

The clone of tobacco gene NtFIP1 coded sequence of the present invention obtains in accordance with the following methods:

1, the extraction (RNA isolation kit) of tobacco total serum IgE

The total serum IgE of corresponding tobacco plant is extracted using RNA isolation kit (pillar total RNA extraction reagent box), specific method is such as Under:

(1) about 100mg tobacco bred K326 blade is weighed in 1.5mLEppendorf pipe, is fully ground in liquid nitrogen；

(2) 1mLBufferRLT (10% of material volume less than or equal to BufferRLT volume) is added, fullys shake mixed It is even, it is stored at room temperature 5min；

It (3) is sufficiently removal cell wall residue, albumen, fat, polysaccharide etc., 4 DEG C, 12000rpm is centrifuged 10min, by supernatant It is transferred in new centrifuge tube；

(4) split-phase:

1. plus 0.2mL chloroform, acutely shake 15s, be stored at room temperature 2min；

2. being centrifuged (4 DEG C, 12000rpm, not more than 12000rpm) 10min；

(5) it precipitates, and Polysaccharide removing:

1. taking colourless aqueous phase (about originally the 50% of BufferRLT volume, about 500 μ L) to a new Eppendorf pipe In, it is sure not to be drawn onto middle layer；

2. plus 0.25mL70% ethyl alcohol, be mixed by inversion；

(6) upper step acquired solution is added in the collecting pipe for having been charged into adsorption column (if liquid volume is more than 700 μ L, Shift in two times), 4 DEG C, 12000rpm is centrifuged 20s, outwells the waste liquid in collecting pipe, adsorption column is placed back in collecting pipe；

(7) 700 μ LBufferRW1 are added into adsorption column, 4 DEG C, 12000rpm is centrifuged 20s, outwells useless in collecting pipe Liquid places back in adsorption column in collecting pipe；

(8) 500 μ LBufferRW2 are added into adsorption column, 4 DEG C, 12000rpm is centrifuged 20s, outwells useless in collecting pipe Liquid places back in adsorption column in collecting pipe；

(9) step (8) are repeated；

(10) 4 DEG C, 12000rpm sky is from 2min；

(11) empty adsorption column is placed in several minutes of room temperature, thoroughly to dry；

(12) by adsorption column be placed in one it is new without in RNase centrifuge tube, 30 μ LRNase-freewater, room temperature is added 1min is placed, 4 DEG C, 12000rpm is centrifuged 1min, collects RNA solution, is placed in -70 DEG C of preservations.

2, reverse transcription

1 μ g total serum IgE is taken, adds 0.5 μ L of Oligd (T), supplement RNase-freewater to total volume is 6 μ L, such as 1 institute of table Show.

1 RNA/ primer denaturing soln of table

5min is placed in 65 DEG C of water-baths, following inverse transcription reaction liquid is configured in pipe after being slightly centrifuged, such as table 2.

2 inverse transcription reaction liquid of table

After 42 DEG C of heat preservation 1h, 70 DEG C of processing 15min, cooled on ice.

3, PCR amplification is carried out using Phusion high-fidelity DNA polymerase

(1) 0.2mLPCR dedicated pipe is taken, following component is sequentially added, as shown in table 3:

The ingredient and dosage that table 3 is added

NtFIP1 forward primer (F): 5 '-CGGGGTACCATGGAAGATGACGACG-3 '；

NtFIP1 reverse primer (R): 5 '-CCGCTCGAGATTGCTGGTCCATCTCCT-3 '；

(2) PCR pipe is closed, is put into PCR instrument.Reaction condition are as follows: 98 DEG C of initial denaturations 2min, 98 DEG C of denaturation 20s, according to drawing Annealing temperature annealing 20s, the 72 DEG C of extension 30s/Kb of object, extend 10min after recycling 30 times, 72 DEG C.

(3) after completion of the reaction, the electrophoresis in 1% Ago-Gel checks expanding effect, PCR product Ago-Gel electricity Swimming testing result can be observed at 4122bp as shown in Figure 1, obtain the clone of purpose tobacco gene NtFIP1 coded sequence To clearly purpose band.

Embodiment 2

The heterologous mutual of homozygous tobacco gene NtFIP1 complement Arabidopsis fip1 mutant and wild type is made in the present embodiment It mends strain and overexpression system, method is as follows:

One, pENTR3C-NtFIP1 recombinant plasmid is constructed

1, it PCR amplification NtFIP1 gene C DS and is connect with pENTR3C

NtFIP1 forward primer (F): 5 '-CGGGGTACCATGGAAGATGACGACG-3 '；

NtFIP1 reverse primer (R): 5 '-CCGCTCGAGATTGCTGGTCCATCTCCT-3 '；

Using the cDNA of extraction as template, PCR amplification is carried out by specific primer F and R, and in target gene upstream under Trip introduces KpnI and XhoI restriction enzyme site respectively.With Tiangeng (Beijing) plastic recovery kit recycle PCR product, PCR product with Gateway compatible pENTR3C carries out digestion connection, and operating procedure according to the form below 4 carries out.

Each Ingredient Amount when 4 digestion of table

Ingredient	Dosage
		Target fragment recovery product	0.5-10ng
pENT3C	0.5μL
		Saltsolution	0.5μL
H₂O	Upto3μL

After mixing, 30 minutes are reacted under room temperature (25 DEG C), then ice bath 1 minute, for converting bacillus coli DH 5 alpha, surface LB culture plate containing kanamycin is applied, 37 DEG C are cultivated 12 hours.Then, what the single white colonies coating of picking was new contains card The LB culture plate of that mycin, 37 DEG C are cultivated 12 hours, carry out colony PCR amplification, as shown in Figure 3.Meanwhile it is correct by bacterium is sieved Bacterial strain send Shanghai Sangon Biotech Company to carry out DNA sequencing.DNAMAN software is by the gene coding region the NtFIP1 sequence of sequencing sequence and prediction Column are compared, and the success of cloning vector pENTR3C-NtFIP1 construction of recombinant plasmid is illustrated after comparing correctly, extracts positive plasmid It is spare.The extraction of plasmid uses Beijing Kang Wei company kit, and by specification step carries out.

(1) bacterium solution for taking 1-5mL to be incubated overnight is added in centrifuge tube, and 12000rpm is centrifuged 1min, inhales abandon supernatant as far as possible.

(2) 250 μ LBufferP1 are added in the centrifuge tube of Xiang Liuyou bacterial sediment, it is thorough using pipettor or turbula shaker Bottom suspended bacterial precipitating.Note: if fungus block does not mix thoroughly, it will influence lytic effect, keep extracted amount and purity relatively low.

(3) 250 μ LBufferP2 are added into centrifuge tube, leniently turns upside down and mixes 4-6 times, split thallus sufficiently Solution, bacterium solution should become limpid sticky at this time.5min is not to be exceeded in time used, in case plasmid is destroyed.

(4) 350 μ LBufferN3 are added into centrifuge tube, leniently turns upside down mix 4-6 times immediately, will occur at this time White flock precipitate, 12000rpm are centrifuged 10min, form precipitating in centrifugation bottom of the tube at this time.

(5) column equilibration: 200 μ LBufferPS being added into the collecting pipe for having been charged into adsorption column (SpinColumn), 12000rpm is centrifuged 2min, outwells the waste liquid in collecting pipe, adsorption column is placed back in collecting pipe.

(6) gained supernatant in step 4 is added in the collecting pipe for having been charged into adsorption column, is careful not to that precipitating is sucked out, 12000rpm is centrifuged 1min, outwells the waste liquid in collecting pipe, adsorption column is put back in collecting pipe.

(7) 600 μ LBufferPW, 12000rpm centrifugation 1min are added into collecting pipe, outwell the waste liquid in collecting pipe.

(8) step 7 is repeated.

(9) adsorption column is placed back in collecting pipe, 12000rpm is centrifuged 1min, outwells the waste liquid in collecting pipe.It will inhale Attached column is placed in several minutes of room temperature, thoroughly to dry.

(10) adsorption column is placed in a new centrifuge tube, 50-100 μ is vacantly added dropwise to the intermediate position of adsorbed film LBufferEB is placed at room temperature for several minutes, and 12000rpm is centrifuged 1min, and plasmid solution is collected into centrifuge tube, -20 DEG C of preservations Plasmid.

Two, recombinant plant expression vector pMDC83-NtFIP1 is constructed using Gateway technology

The plant of correct cloning vector pENTR 3C-NtFIP1 and 35S promoter and Gateway system compatible will be sequenced Expression vector pMDC83 carries out LR and is reacted, and obtains the plant of the NtFIP1 Gene Fusion GFP reporter gene of 35S promoter driving Object expression vector.LR reaction system is as follows: cloning vector 1 μ L (about 100ng) carrier, pMDC83 carrier 1 μ L (about 100ng), LR 0.5 μ L of clone enzyme.Above-mentioned substance mixing is placed on (25 DEG C) of room temperature to react 1-2 hours, converts bacillus coli DH 5 alpha.Conversion Bacterial strain surface applies LB culture plate containing kanamycin, and 37 DEG C are cultivated 12 hours.Then, the single white colonies coating of picking is new LB culture plate containing kanamycin, 37 DEG C cultivate 12 hours, carry out colony PCR amplification, as shown in Figure 4.Illustrate to express Carrier pMDC 83-NtFIP1 is constructed successfully, and extraction positive plasmid is spare, and extracting method is as described above.

Three, Agrobacterium infects tobacco gene NtFIP1 complement Arabidopsis fip1 mutant and the heterologous complementary strain of wild type building System and overexpression system

1, the preparation and conversion of Agrobacterium tumefaciems GV3101 competent cell

The preparation method of Agrobacterium competent cell is as follows:

(1) picking single colonie GV3101 is inoculated in 5mLYEP fluid nutrient medium, and 28 DEG C, 200rpm is incubated overnight.

(2) take 2mL culture into 50mL liquid YEP, continuing to cultivate to OD600 is 0.5 or so.

(3) by culture ice bath 30min, 4 DEG C, 5000rpm, it is centrifuged 5min.

(4) it discards supernatant, 10mL0.1mol/L cold NaCl suspension thalline；4 DEG C, 5000rpm, it is centrifuged 5min.

(5) supernatant, the precipitating CaCl cold with 1mL20mmol/L are abandoned₂It suspends, is distributed into 200 μ L/ pipe, after being freezed in liquid nitrogen- 70 DEG C of preservations.

Expression vector converts Agrobacterium and uses freeze-thaw method, and conversion process is as follows:

(1) it from -80 DEG C of taking-up GV3101 competent cells (250 μ L), thaws on ice, 1-2 μ L Plasmid DNA is added immediately.

(2) 5min is stood on ice.

(3) 5min is freezed in liquid nitrogen.

(4) 37 DEG C of water-bath heat shock 5min.

(5) YEP the fluid nutrient medium 1mL, 28 DEG C of shaking table culture 2-4h of antibiotic-free is added.

(6) 4000rpm room temperature is centrifuged 5min, thallus is collected, with 80-100 μ LYEP solution again suspension thalline.

(7) bacterium solution is spread evenly across YEP solid selection medium, 28 DEG C stationary culture 2-3 days.

(8) after growing bacterium colony, picking single colonie is lined again on antibiotic YEP solid plate, and bacterium is carried out after 36h PCR identification is fallen, as a result as shown in Figure 5.

2, the conversion of arabidopsis

The genetic transformation of arabidopsis uses inflorescence dip method, the method is as follows:

(1) cutting the main inflorescence of arabidopsis, to induce the generation of side inflorescence, and side inflorescence is bloomed simultaneously, convenient for turning Change.Material is irrigated into nutrient solution before conversion, watering or nutrient solution are controlled after conversion, in order to which seed is mature in time.

(2) Agrobacterium required for conversion is got out on the day before mentioning, the bacterium solution for taking 2-5mL to be incubated overnight is added to 250mL Culture medium in shaking table culture, can be used for arabidopsis thaliana transformation in order to second day.

(3) the Agrobacterium thallus being incubated overnight is collected, is resuspended in 50-100mL dip dyeing liquid for shell.

(4) by arabidopsis elongation kind pod and opened spend whole cut after, by titbit immersion dip dyeing liquid for shell in, up and down Titbit is shaken, in favor of the entrance of dip dyeing liquid for shell.Immerged time generally continues 1h or so.Arabidopsis after dip dyeing is laid flat into big basin In, film is covered with moisturizing, while outer cover black thin film is protected from light.Arabidopsis taking-up is placed under light after dark processing, to maturation Sowing afterwards.

It is as shown in table 5 below that arabidopsis disseminates formula of liquid:

5 arabidopsis of table disseminates formula of liquid

After configuration, with 1MKOH tune pH value to 5.7.

3. the screening of transgenic line

1, the arabidopsis T0 after infecting is collected for seed, and carry out disinfection processing, the method is as follows:

(1) it takes appropriate seed in 1.5mLEppendorf pipe, indicates relevant information；

(2) 70% ethyl alcohol of configured in advance takes 1mL Yu Guanzhong with pipettor, and suction plays mixing, connects seed fully Ethanol solution is touched, stewing process 5min (pays attention to rinsing the seed that tube wall and pipe cover to guarantee that sterilizing is thorough), it (being careful not to for seed being sucked out) is sucked out in the ethyl alcohol in centrifuge tube with 1mL pipettor afterwards；

(3) it spits warm water washing one time；

(4) 1mL2.6% liquor natrii hypochloritis Yu Guanzhong is taken with pipettor, mixing of turning upside down, stewing process 10min；

(5) it spits warm water washing five times；

(6) 4 DEG C are placed in save for use.

2, the screening of transgenic plant

Collect T1 for seed, sterilize according to the above method, program request on the MS solid medium containing 50 μ g/mL hygromycin, 7d is grown in the dedicated culturing room of plant, condition of culture is 22 DEG C, the long-day (16h illumination/8h is dark).Energy is selected in T2 generation Enough plant for sprouting and growing on hygromycin MS plate, are transferred on vermiculite respectively and cultivate, condition of culture is same as above.

Collect T2 for seed, sterilize according to the above method, program request on the MS solid medium containing 50 μ g/mL hygromycin, 7d is grown in the dedicated culturing room of plant, condition of culture is 22 DEG C, the long-day (16h illumination/8h is dark).Energy is selected in T3 generation It is enough that isolated plant is all sprouted and grow and do not occurred on hygromycin MS plate, obtain heterologous complement strain NtFIP1/ Fip1 and overexpression system NtFIP1-OE.

In order to verify the feasibility of the above method, we are to the tobacco gene NtFIP1 complement Arabidopsis for being prepared for homozygosis The heterologous complement strain NtFIP1/fip1 and overexpression system NtFIP1-OE of fip1 mutant and wild type, and have studied tobacco The application of gene NtFIP1 being cloned in crop raising tobacco nitrogen utilization efficiency and quality-improving, as follows:

1, in heterologous complement strain and overexpression system NtFIP1 expression quantity measurement

(1) various concentration KNO₃Culture as heterologous complement strain and overexpression system in only nitrogen source culture medium

In order to detect the expression quantity of NtFIP1 in heterologous complement strain and overexpression system, first by heterologous complement strain The seed disinfection of NtFIP1/fip1 and overexpression system NtFIP1-OE are then transferred into KNO₃For the solid culture of only nitrogen source It is cultivated in plant illumination incubator on base, cultivation temperature is 22 DEG C, long-day (16h illumination/8h is dark) culture 7d.

(2) the expression quantity measurement of NtFIP1

Heterologous complement strain NtFIP1/fip1 and overexpression system are detected using real-time fluorescence quantitative PCR (qRT-PCR) The expression quantity of NtFIP1 in NtFIP1-OE.

The RNA extracted in NtFIP1/fip1 and NtFIP1-OE the cDNA sample obtain after reverse transcription is diluted to 0.5-2ng/ μ L, reaction system are as shown in the table:

Each ingredient and dosage in 6 reaction system of table

QRT-PCR forward primer: 5 '-ATGGAAGATGACGACGAA-3 '；

QRT-PCR reverse primer: 5 '-TTCGTTACCAGTGAT-3 '

PCR pipe is closed, (ABI7500FAST) is put into fluorescence quantitative PCR instrument.Reaction condition are as follows: first 50 DEG C of initial denaturations 20s, then 95 DEG C of initial denaturation 10s, then 95 DEG C of denaturation 15s, 60 DEG C of annealing 1min carry out plate read, since step 3 later Circulation 40 times, solubility curve are 95 DEG C of 15s, 60 DEG C of 1min, 95 DEG C of 30s, 60 DEG C of 15s.

Reaction guarantees the accuracy of result using tublin2 as reference gene, and each processing has biology repetition three times, Three systems repeat.The expression quantity of NtFIP1 is as schemed in heterologous complement strain NtFIP1/fip1 and overexpression system NtFIP1-OE Shown in 6, the results showed that tobacco gene NtFIP1 can in heterologous complement strain and overexpression system high efficient expression.

2, heterologous complement strain and overexpression tie up to the upgrowth situation under Seedling Stage 1/2MS condition of culture

Nitrogen is the most important nutrient for influencing plant growth and development, and nitrogen metabolism will lead to the blade middle period extremely Green element decline and the long shortening of main root.We observe heterologous complement strain and overexpression ties up to Seedling Stage in various concentration KNO₃ As the upgrowth situation under only nitrogen source condition of culture, as a result as shown in Figure 7.After solid culture grows 7d, arabidopsis is wild Type growing state is good, leaf green；Main root growth is normal, and length is about 4cm.However, arabidopsis fip1 mutant is being grown Situation is poor, the obvious chlorosis of blade；Main root length shortens, length about 3cm.The growing state of heterologous complement strain is similar with wild type, Leaf green；Main root growth is normal, and length is about 4cm.The upgrowth situation of overexpression system is better than wild type (Fig. 7).Above-mentioned knot Fruit shows in various concentration KNO₃It can be by arabidopsis fip1 mutant as tobacco gene NtFIP1 under only nitrogen source condition of culture Restore the nitrogen utilization efficiency that plant can be improved to wild type phenotype to maintain higher chlorophyll content and main root to grow.

3, NtFIP1 is to plant NO₃ ^-The influence of content

By reducing NO in tobacco body₃ ^-And NO₂ ^-Synthesis of the content to reduce nitrosamine be that reduce tobacco special The effective means of TSNAs content.The study found that turning containing for TSNAs in the tobacco line of arabidopsis thaliana subspecies nitrate reductase gene NiR Amount is substantially less than wild type.The transgene tobacco strain of overexpression tobacco itself nitrate reductase gene NR reduce blade and The content of TSNAs in flue gas.We have detected in heterologous complement strain NtFIP1 to arabidopsis NO₃ ^-The influence of content.Such as Fig. 8 institute Show, the results showed that NO in fip1 mutant after FIP1 mutation₃ ^-Content significantly reduces, and the heterologous complement strain of NtFIP1 and excessively scale Up to being middle NO₃ ^-For content without significant difference compared with wild type, the above results illustrate that NtFIP1 can regulate and control NO in plant₃ ^-Contain Amount.Therefore, we can use the content of NtFIP1 regulation tobacco TSNAs so as to improve cigarette quality.

Sequence table

<110>Jining institute

<120>a kind of tobacco gene NtFIP1 for improving nitrogen utilization efficiency and its cloning process and application

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 4125

<212> DNA

<213>tobacco (Nicotiana sp)

<400> 1

atggaagatg acgacgaatt cggagatctc tataccgatg tgctaacggc gtcgtttcag 60

tctcagcagc cgccagctcc agctccacaa gacaaagccg ccgcctccaa agccgccgcc 120

ggtgcgggcc ccactacccg gccgatcgat ctgaacatca acagcgatga cgaggagata 180

ttatacggag ctcctaatcc gaatttgaat ccaaaattta cagccccgaa ttctattatc 240

actggtaacg aaaaaaccct agccgctttg ccagatgtac aatccgggtc gaacttaccc 300

gaattcaatc taaattttaa tcaaaaagct ggaaaactcg aggatttgag tgatattaat 360

gaatcggatt cgagtgctag ggttttggag aagagtgagg atgtgaaatt acctaaaggg 420

ggttttcaag attcaaattt tatggatgag gataatattg atttcgtagt ggaagagagg 480

gatgataaag atgatatttt gattgagaaa gatcaaaatt taggtgaaag aaacaatgag 540

attctaaagg atgggagtga aaatgtacag aattttgagc cgttgattcc cggcctgtca 600

atcccgggcg tttcaggcgg aggagggaat ggtggagatg gtaatcttga ggatgattgg 660

gatagtgatg atagtgagga tgatttgcag attgtgttga atgacaacac tcatggacca 720

atgggtatgg agagaatggg ggacgacgag gacgatgaag atggagagcc tttggttatt 780

gttgcggata atgatggccc gagccatcca ccaatgataa tggaggagca agaatggggt 840

gaggaagggg gtcctgctgc taatggagag agaaaggaga ttaatgatgc attaaaagtg 900

aatggagcac ctggaggagt ggtggcaaag gttgggtaca acaatcatgg ataccatcat 960

ccgtatcatt cacagtataa gtatgtgaga cctggtgcag ctcctatgcc tggagctcct 1020

ctgttgggtc ctggaggagc acctggtcaa gttcgtcccc ctgtcaatgt gggtccagtt 1080

ggtggacgtg gtagaggtga ttggcgacca acaggaatga aaggtggcta tgggatgtcc 1140

ggttggggag gcagtgcgcc tggccgtggc tttgggattg ggttggaatt cactcttcca 1200

tcgcacaaga ctatatttga agttgacata gatggttttg aggagaaacc ttggagactt 1260

ccaggcattg acgtaacaga tttcttcaac tttggtttga acgaggacat ctggaaagat 1320

tattgcaagc agctggaaca actgcgactt gagtcaacta tgcaaggaag aattcgggta 1380

tatgaaagtg gaagaacaga gcaggaatat gatccagatc ttcccccaga gcttgcagct 1440

gcagctggta ttcaagacat tccatctgaa aatgtgaatc atggtaaaac tgatggaact 1500

tcaagtgacc tagcaagagg atctatacgc atgcgaccgc cactacccac tggaagacct 1560

atacaggttg aaaccggttc tggtgatcgc ctaccatcaa ttgatacccg accaccacgg 1620

cagcgtgact cggatgcaat tattgaaatt gtgtgccagg atgatgacca atactcaggg 1680

aatgacaaga atgaggtgca attgggcaat aattctccaa cagatgattt tagaggcgat 1740

gcaagaggag gccccctgca ggaggattct gatggtttcc agcattctta taaaagccac 1800

aagcaagatc tcagtactag gagatcacag tttatgaacc ctgtaggtga tcgtttgacc 1860

aaaggagatg gagtgggtcc ttttccctcg gaagctcctg gtcagtttgt ttctgattct 1920

agaggacaga cttctgcatg cggcagcaag actaatgttg aaagggaaaa caagggaagt 1980

gcacgcgaag gatcacctga tattagtccc agtggtgata gcagagacag actccaagtt 2040

gataatcaga gagaagaatc agttgaaagt gttgatcata ggcacagtcc tgtccctcct 2100

tcacccacca ctgatagacc tgctcaggag caagatatgg aagatagaga taatatccct 2160

gatcaacctg tgggagctga cacaaacagt gaagttgtga gagaggaaat ggcttttgat 2220

gcaagaagtg atagtgaagc catgaatgat gaatttttac attctaaaaa gcaaaagtta 2280

agttcacgac gtgaacaatc ttctccgcaa gaaactgatg gtggagaaga ctccaaggct 2340

ggaagaagta gcgagaatag caaagcccaa tctggaagca gtagagatta tcgcaaatta 2400

cgggatgatg ttgaggagga agttgttcaa gatggacgtt ctatgcgcat ggataatgcc 2460

aaaaaagcag ttgccaggga cgaggataga gtccgaagaa gagcctacaa tgagaaagaa 2520

gcagaaaagc accgtggggt agtgaaaggg agggaagatt cttactcccg taaaggcgtt 2580

gactctagct cagcccatta cattgaccga cggagggaac gtgaatattc agaggcagta 2640

tggcaaagga gggatgaaga tctaccgggg aggagaacta aagtggaaga accacgaaag 2700

agagagctta ttgatgaaac tggatccaga caccgcagca aggtgcgaga atttgatggg 2760

agtgatagag aagaacgcca tttacataga aagcagctgg agagcattac gttaaggcct 2820

gattatgata aggatatggg agcaaggcaa agagacaggg atgagttgaa gaggtatgat 2880

accttggatg atcgccacaa taaaagaagg aaagaagaga caaaattaag tagggaacat 2940

gctgacaagg aagattcctt ccatccgcat ggagaaaata tggttcatag aaagagagac 3000

agagatgata cctcagatca tcgaaagaga gatgagctat tgagactaag agatgatgag 3060

cagcattata tcaggcacaa agaagatgga gtatttcaga gggagaggag tgacagacag 3120

cgagaacgtg aggagtggta taggcttaaa cagtcccaag aagaggcact atcaaaaagg 3180

gagagagagg aaataagggg cggtatgcgt gctgggcgtg ttgcagagga aaaagcttgg 3240

gccggtcatt ctagagggaa agatgagcac agaaactctg atcagcattt gaaggatgtg 3300

aggcatgctg accatattag aaggagggat cgtgctgaaa atgaaagtcc ttcaaggctg 3360

aggacccgtg aggatgagag aagagcaagg cctgatagag tgagtgcacg cgaagatcgt 3420

gctctccatg ctcctgataa ctccagggtg aatgagaaaa gacataaaga atatttaaag 3480

aaaggtaaag aatttgaggg ggatcataac tcccagattg cttcgaacat gaacgaagac 3540

gaactgaatg gacggagaaa tgagatggtg agcttgaaga gaaaatttga gcaaggaacc 3600

aatgagaaca aggtccaccg gaatcgtcag tcttccagga aacagcagga agaggcatca 3660

tcagatgatg agcaacagga ttccaagagg ggacgttcca agttggaaag gtggacaagc 3720

cacaaggaaa gagactttag tgtcaatgct aaatcatctt ccttgaatgt gaaagacatt 3780

gatgtacaca agagcagtgg tatttcatta gctaacaaaa atccggatga agctttgaag 3840

gcagtcgaag acaatcaaca gcctgcagct aacaataaaa atggaggtgg tccagaaatt 3900

aataatgtag agacaaagca tatggaggat aaacacttgg agacagttga gaagctgaaa 3960

aagcggagtg agcgcttcaa gcttccaatg ccaagtgaaa aagaagctcc agtaagtaaa 4020

aaggcggaag gtgatccaat atcgtctgtc caaagtgaaa tccctccgga ttcggaggtg 4080

aagccagagc gtccagcacg aaggaggaga tggaccagca attaa 4125

<210> 2

<211> 1374

<212> PRT

<213>tobacco (Nicotiana sp)

<400> 2

Met Glu Asp Asp Asp Glu Phe Gly Asp Leu Tyr Thr Asp Val Leu Thr

1 5 10 15

Ala Ser Phe Gln Ser Gln Gln Pro Pro Ala Pro Ala Pro Gln Asp Lys

20 25 30

Ala Ala Ala Ser Lys Ala Ala Ala Gly Ala Gly Pro Thr Thr Arg Pro

35 40 45

Ile Asp Leu Asn Ile Asn Ser Asp Asp Glu Glu Ile Leu Tyr Gly Ala

50 55 60

Pro Asn Pro Asn Leu Asn Pro Lys Phe Thr Ala Pro Asn Ser Ile Ile

65 70 75 80

Thr Gly Asn Glu Lys Thr Leu Ala Ala Leu Pro Asp Val Gln Ser Gly

85 90 95

Ser Asn Leu Pro Glu Phe Asn Leu Asn Phe Asn Gln Lys Ala Gly Lys

100 105 110

Leu Glu Asp Leu Ser Asp Ile Asn Glu Ser Asp Ser Ser Ala Arg Val

115 120 125

Leu Glu Lys Ser Glu Asp Val Lys Leu Pro Lys Gly Gly Phe Gln Asp

130 135 140

Ser Asn Phe Met Asp Glu Asp Asn Ile Asp Phe Val Val Glu Glu Arg

145 150 155 160

Asp Asp Lys Asp Asp Ile Leu Ile Glu Lys Asp Gln Asn Leu Gly Glu

165 170 175

Arg Asn Asn Glu Ile Leu Lys Asp Gly Ser Glu Asn Val Gln Asn Phe

180 185 190

Glu Pro Leu Ile Pro Gly Leu Ser Ile Pro Gly Val Ser Gly Gly Gly

195 200 205

Gly Asn Gly Gly Asp Gly Asn Leu Glu Asp Asp Trp Asp Ser Asp Asp

210 215 220

Ser Glu Asp Asp Leu Gln Ile Val Leu Asn Asp Asn Thr His Gly Pro

225 230 235 240

Met Gly Met Glu Arg Met Gly Asp Asp Glu Asp Asp Glu Asp Gly Glu

245 250 255

Pro Leu Val Ile Val Ala Asp Asn Asp Gly Pro Ser His Pro Pro Met

260 265 270

Ile Met Glu Glu Gln Glu Trp Gly Glu Glu Gly Gly Pro Ala Ala Asn

275 280 285

Gly Glu Arg Lys Glu Ile Asn Asp Ala Leu Lys Val Asn Gly Ala Pro

290 295 300

Gly Gly Val Val Ala Lys Val Gly Tyr Asn Asn His Gly Tyr His His

305 310 315 320

Pro Tyr His Ser Gln Tyr Lys Tyr Val Arg Pro Gly Ala Ala Pro Met

325 330 335

Pro Gly Ala Pro Leu Leu Gly Pro Gly Gly Ala Pro Gly Gln Val Arg

340 345 350

Pro Pro Val Asn Val Gly Pro Val Gly Gly Arg Gly Arg Gly Asp Trp

355 360 365

Arg Pro Thr Gly Met Lys Gly Gly Tyr Gly Met Ser Gly Trp Gly Gly

370 375 380

Ser Ala Pro Gly Arg Gly Phe Gly Ile Gly Leu Glu Phe Thr Leu Pro

385 390 395 400

Ser His Lys Thr Ile Phe Glu Val Asp Ile Asp Gly Phe Glu Glu Lys

405 410 415

Pro Trp Arg Leu Pro Gly Ile Asp Val Thr Asp Phe Phe Asn Phe Gly

420 425 430

Leu Asn Glu Asp Ile Trp Lys Asp Tyr Cys Lys Gln Leu Glu Gln Leu

435 440 445

Arg Leu Glu Ser Thr Met Gln Gly Arg Ile Arg Val Tyr Glu Ser Gly

450 455 460

Arg Thr Glu Gln Glu Tyr Asp Pro Asp Leu Pro Pro Glu Leu Ala Ala

465 470 475 480

Ala Ala Gly Ile Gln Asp Ile Pro Ser Glu Asn Val Asn His Gly Lys

485 490 495

Thr Asp Gly Thr Ser Ser Asp Leu Ala Arg Gly Ser Ile Arg Met Arg

500 505 510

Pro Pro Leu Pro Thr Gly Arg Pro Ile Gln Val Glu Thr Gly Ser Gly

515 520 525

Asp Arg Leu Pro Ser Ile Asp Thr Arg Pro Pro Arg Gln Arg Asp Ser

530 535 540

Asp Ala Ile Ile Glu Ile Val Cys Gln Asp Asp Asp Gln Tyr Ser Gly

545 550 555 560

Asn Asp Lys Asn Glu Val Gln Leu Gly Asn Asn Ser Pro Thr Asp Asp

565 570 575

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

580 585 590

Phe Gln His Ser Tyr Lys Ser His Lys Gln Asp Leu Ser Thr Arg Arg

595 600 605

Ser Gln Phe Met Asn Pro Val Gly Asp Arg Leu Thr Lys Gly Asp Gly

610 615 620

Val Gly Pro Phe Pro Ser Glu Ala Pro Gly Gln Phe Val Ser Asp Ser

625 630 635 640

Arg Gly Gln Thr Ser Ala Cys Gly Ser Lys Thr Asn Val Glu Arg Glu

645 650 655

Asn Lys Gly Ser Ala Arg Glu Gly Ser Pro Asp Ile Ser Pro Ser Gly

660 665 670

Asp Ser Arg Asp Arg Leu Gln Val Asp Asn Gln Arg Glu Glu Ser Val

675 680 685

Glu Ser Val Asp His Arg His Ser Pro Val Pro Pro Ser Pro Thr Thr

690 695 700

Asp Arg Pro Ala Gln Glu Gln Asp Met Glu Asp Arg Asp Asn Ile Pro

705 710 715 720

Asp Gln Pro Val Gly Ala Asp Thr Asn Ser Glu Val Val Arg Glu Glu

725 730 735

Met Ala Phe Asp Ala Arg Ser Asp Ser Glu Ala Met Asn Asp Glu Phe

740 745 750

Leu His Ser Lys Lys Gln Lys Leu Ser Ser Arg Arg Glu Gln Ser Ser

755 760 765

Pro Gln Glu Thr Asp Gly Gly Glu Asp Ser Lys Ala Gly Arg Ser Ser

770 775 780

Glu Asn Ser Lys Ala Gln Ser Gly Ser Ser Arg Asp Tyr Arg Lys Leu

785 790 795 800

Arg Asp Asp Val Glu Glu Glu Val Val Gln Asp Gly Arg Ser Met Arg

805 810 815

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

820 825 830

Arg Arg Ala Tyr Asn Glu Lys Glu Ala Glu Lys His Arg Gly Val Val

835 840 845

Lys Gly Arg Glu Asp Ser Tyr Ser Arg Lys Gly Val Asp Ser Ser Ser

850 855 860

Ala His Tyr Ile Asp Arg Arg Arg Glu Arg Glu Tyr Ser Glu Ala Val

865 870 875 880

Trp Gln Arg Arg Asp Glu Asp Leu Pro Gly Arg Arg Thr Lys Val Glu

885 890 895

Glu Pro Arg Lys Arg Glu Leu Ile Asp Glu Thr Gly Ser Arg His Arg

900 905 910

Ser Lys Val Arg Glu Phe Asp Gly Ser Asp Arg Glu Glu Arg His Leu

915 920 925

His Arg Lys Gln Leu Glu Ser Ile Thr Leu Arg Pro Asp Tyr Asp Lys

930 935 940

Asp Met Gly Ala Arg Gln Arg Asp Arg Asp Glu Leu Lys Arg Tyr Asp

945 950 955 960

Thr Leu Asp Asp Arg His Asn Lys Arg Arg Lys Glu Glu Thr Lys Leu

965 970 975

Ser Arg Glu His Ala Asp Lys Glu Asp Ser Phe His Pro His Gly Glu

980 985 990

Asn Met Val His Arg Lys Arg Asp Arg Asp Asp Thr Ser Asp His Arg

995 1000 1005

Lys Arg Asp Glu Leu Leu Arg Leu Arg Asp Asp Glu Gln His Tyr Ile

1010 1015 1020

Arg His Lys Glu Asp Gly Val Phe Gln Arg Glu Arg Ser Asp Arg Gln

1025 1030 1035 1040

Arg Glu Arg Glu Glu Trp Tyr Arg Leu Lys Gln Ser Gln Glu Glu Ala

1045 1050 1055

Leu Ser Lys Arg Glu Arg Glu Glu Ile Arg Gly Gly Met Arg Ala Gly

1060 1065 1070

Arg Val Ala Glu Glu Lys Ala Trp Ala Gly His Ser Arg Gly Lys Asp

1075 1080 1085

Glu His Arg Asn Ser Asp Gln His Leu Lys Asp Val Arg His Ala Asp

1090 1095 1100

His Ile Arg Arg Arg Asp Arg Ala Glu Asn Glu Ser Pro Ser Arg Leu

1105 1110 1115 1120

Arg Thr Arg Glu Asp Glu Arg Arg Ala Arg Pro Asp Arg Val Ser Ala

1125 1130 1135

Arg Glu Asp Arg Ala Leu His Ala Pro Asp Asn Ser Arg Val Asn Glu

1140 1145 1150

Lys Arg His Lys Glu Tyr Leu Lys Lys Gly Lys Glu Phe Glu Gly Asp

1155 1160 1165

His Asn Ser Gln Ile Ala Ser Asn Met Asn Glu Asp Glu Leu Asn Gly

1170 1175 1180

Arg Arg Asn Glu Met Val Ser Leu Lys Arg Lys Phe Glu Gln Gly Thr

1185 1190 1195 1200

Asn Glu Asn Lys Val His Arg Asn Arg Gln Ser Ser Arg Lys Gln Gln

1205 1210 1215

Glu Glu Ala Ser Ser Asp Asp Glu Gln Gln Asp Ser Lys Arg Gly Arg

1220 1225 1230

Ser Lys Leu Glu Arg Trp Thr Ser His Lys Glu Arg Asp Phe Ser Val

1235 1240 1245

Asn Ala Lys Ser Ser Ser Leu Asn Val Lys Asp Ile Asp Val His Lys

1250 1255 1260

Ser Ser Gly Ile Ser Leu Ala Asn Lys Asn Pro Asp Glu Ala Leu Lys

1265 1270 1275 1280

Ala Val Glu Asp Asn Gln Gln Pro Ala Ala Asn Asn Lys Asn Gly Gly

1285 1290 1295

Gly Pro Glu Ile Asn Asn Val Glu Thr Lys His Met Glu Asp Lys His

1300 1305 1310

Leu Glu Thr Val Glu Lys Leu Lys Lys Arg Ser Glu Arg Phe Lys Leu

1315 1320 1325

Pro Met Pro Ser Glu Lys Glu Ala Pro Val Ser Lys Lys Ala Glu Gly

1330 1335 1340

Asp Pro Ile Ser Ser Val Gln Ser Glu Ile Pro Pro Asp Ser Glu Val

1345 1350 1355 1360

Lys Pro Glu Arg Pro Ala Arg Arg Arg Arg Trp Thr Ser Asn

1365 1370

Claims

1. a kind of tobacco gene NtFIP1 for improving nitrogen utilization efficiency, it is characterised in that: the nucleotides sequence of tobacco gene NtFIP1 Column are as shown in SEQ ID NO:1.

2. the tobacco gene NtFIP1 according to claim 1 for improving nitrogen utilization efficiency, it is characterised in that: tobacco gene The encoded amino acid sequence of NtFIP1 is as shown in SEQ ID NO:2.

3. a kind of cloning process of the tobacco gene NtFIP1 of any of claims 1 or 2 for improving nitrogen utilization efficiency, feature exist In: include the following steps:

(2) using cDNA as template, special primer is designed, passes through PCR amplification gene NtFIP1 coding region sequence, wherein PCR amplification The primer sequence is as follows:

NtFIP1 forward primer (F):

5'-CGGGGTACCATGGAAGATGACGACG-3'；

NtFIP1 reverse primer (R):

5'-CCGCTCGAGATTGCTGGTCCATCTCCT-3'；

(3) it recycles target fragment in the simultaneously amplified production of purification step (2) and is sequenced, sequencing is correctly tobacco gene The clone of NtFIP1 coded sequence.

4. the cloning process of the tobacco gene NtFIP1 according to claim 3 for improving nitrogen utilization efficiency, it is characterised in that: PCR amplification is carried out using Phusion high-fidelity DNA polymerase in step (2).

5. the cloning process of the tobacco gene NtFIP1 according to claim 3 for improving nitrogen utilization efficiency, it is characterised in that: The reaction condition of PCR amplification are as follows: 98 DEG C of initial denaturations 2min, 98 DEG C of denaturation 20s, according to the annealing temperature of primer anneal 20s, 72 DEG C Extend 30s/Kb, extends 10min after circulation 30 times, 72 DEG C.

6. the application of the tobacco gene NtFIP1 according to claim 1 or 2 for improving nitrogen utilization efficiency, it is characterised in that: Tobacco gene NtFIP1 is applied to plant and obtains transgenic plant to improve the nitrogen utilization efficiency of plant.

7. the application of the tobacco gene NtFIP1 according to claim 6 for improving nitrogen utilization efficiency, it is characterised in that: plant For one of arabidopsis fip1 mutant, arabidopsis Col-0 wild type, tobacco bred K326.

8. the application of the tobacco gene NtFIP1 according to claim 6 for improving nitrogen utilization efficiency, it is characterised in that: turn base Because of the preparation method of plant are as follows:

S1 constructs pENTR3C-NtFIP1 recombinant plasmid

Digestion connects clone and the cloning vector pENTR3C of tobacco gene NtFIP1 coded sequence: to the tobacco gene of purifying KpnI and XhoI restriction enzyme site is added at 5 ' ends and 3 ' ends respectively in being cloned in for NtFIP1 coded sequence, is had after double digestion Then the target gene fragment of cohesive terminus,cohesive termini carries out digestion connection with cloning vector pENTR3C, obtains recombinant plasmid pENTR3C- NtFIP1；

The plant that correct recombinant plasmid pENTR3C-NtFIP1 and 35S promoter and Gateway system compatible is sequenced is expressed Carrier pMDC83 carries out LR reaction, obtains recombinant plant expression vector pMDC83-NtFIP1；

S3, Agrobacterium infect plant

Agrobacterium competent cell is prepared, then converts Agrobacterium competent cell with recombinant expression carrier pMDC83-NtFIP1, The conversion Agrobacterium of picking positive colony infects plant, obtains the transgenosis that can effectively clone tobacco gene NtFIP1 coded sequence Plant, plant are arabidopsis fip1 mutant and wild type.

9. the application of the tobacco gene NtFIP1 according to claim 8 for improving nitrogen utilization efficiency, it is characterised in that: agriculture agriculture Bacillus is Agrobacterium GV3101.

10. the application of the tobacco gene NtFIP1 according to claim 8 for improving nitrogen utilization efficiency, it is characterised in that: S3 It after middle conversion Agrobacterium infects arabidopsis fip1 mutant and wild type, is selfed respectively by 3 generations, screening, which obtains, contains tobacco-based Heterologous complement strain and overexpression system because of NtFIP1 coded sequence.