CN108752444B

CN108752444B - Chlorophytum comosum root nitrate transport protein CcNPF8.3.2 and coding gene and application thereof

Info

Publication number: CN108752444B
Application number: CN201810635871.9A
Authority: CN
Inventors: 刘昱辉; 朱思慧; 李梅; 谭杰
Original assignee: Biotechnology Research Institute of CAAS
Current assignee: Biotechnology Research Institute of CAAS
Priority date: 2018-06-20
Filing date: 2018-06-20
Publication date: 2020-04-14
Anticipated expiration: 2038-06-20
Also published as: CN108752444A

Abstract

The invention discloses a chloranthus japonicus root nitrate transport protein CcNPF8.3.2 and a coding gene and application thereof.A coding gene CcNPF8.3.2 of the nitrate transport protein is cloned from the root of chloranthus japonicus, and the gene is introduced into △ ynt-Leu double mutation Hansenula polymorpha to obtain a CcNPF8.3.2 yeast.

Description

Chlorophytum comosum root nitrate transport protein CcNPF8.3.2 and coding gene and application thereof

Technical Field

The invention relates to a chlorophytum comosum root nitrate transporter CcNPF8.3.2 and an encoding gene and application thereof.

Background

Nitrogen is a basic nutrient element necessary for plant growth and development, plays an important role in plant growth and development and morphogenesis, and has important significance for life activities and yield formation of crops. Nitrate (NO)₃ ^-) Is the most main nitrogen source of most crops, and the insufficient supply of nitrate can seriously inhibit the growth and development of the crops. Physiological studies have shown that plants absorb NO from soil₃ ^-It is desirable to have a whole set of high-and low-affinity NO3^-The transport system is involved, NO₃ ^-By H across the plasma membrane⁺And (4) gradient driving. Some NO₃ ^-The transport system is constitutively expressed, some are NO-dependent₃ ^-Induced and with NO₃ ^-The assimilation of (A) is negative feedback regulation and control.

A perennial evergreen herb of the family Liliaceae of the genus Thamnolia. The root system of the plant grows in water in a spread mode, and is rapid in growth, developed, dense and thick. Rapidly growing under proper environmental conditions. Only two days were required to grow one lateral root.

In the past decades, Hansenula polymorpha (also known as Pichia angusta) has become a recognized model organism. Widely applied to the research of methanol metabolism, nitrate absorption mechanism and the like. In Hansenula polymorpha, all the genes involved in nitrate metabolism are closely arranged in gene clusters, and about 92% of the DNA fragments of 1040bp in total are coding DNA. Hansenula polymorpha assimilates nitrate and uses nitrate as the sole nitrogen source, it has only one high affinity nitrate transporters (Ynt 1), which can transport nitrate but not chlorate. YNT1 structurally belongs to the NNP (nitrate-nitrate porter) family and is induced by nitrate and nitrite. Hansenula polymorpha genes YNT1, YNR 1 and YNI1 encode nitrate transporter, nitrate reductase (nitrite reductase) and nitrite reductase (nitrite reductase), respectively, the expression of which is induced by nitrate and nitrite and inhibited by salt and glutamate. YNT1 deletion mutation

Can result in the inability of the strain to transport or grow at nitrate concentrations below 500. mu.M.

Disclosure of Invention

The invention aims to provide a chlorophytum comosum root nitrate transporter CcNPF8.3.2 and a coding gene and application thereof.

The invention provides a protein, which is cloned from Chlorophytum comosum and named as CcNPF8.3.2, and is (a1) or (a 2):

(a1) a protein consisting of an amino acid sequence shown in a sequence 5 in a sequence table;

(a2) and (b) the protein which is derived from the sequence 5 and has the nitrate transport function, wherein the amino acid sequence of the sequence 5 is subjected to substitution and/or deletion and/or addition of one or more amino acid residues.

In order to facilitate the purification and detection of the CcNPF8.3.2 protein of (a1), the amino-terminal or carboxy-terminal of the protein consisting of the amino acid sequence shown in sequence No. 5 of the sequence listing is labeled as shown in Table 1.

TABLE 1 sequences of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The CcNPF8.3.2 protein of (a2) above may be synthesized artificially, or it may be obtained by synthesizing the coding gene and then expressing it biologically. The gene encoding the ccnpf8.3.2 protein of (a2) above can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in sequence 4 of the sequence listing, and/or performing missense mutation of one or several base pairs, and/or connecting the coding sequence of the tag shown in table 1 at its 5 'end and/or 3' end.

The gene encoding the CcNPF8.3.2 protein (CcNPF8.3.2 gene) also belongs to the protection scope of the present invention.

The gene is a DNA molecule as described in any one of (b1) to (b3) below:

(b1) DNA molecule shown in sequence 4 in the sequence table;

(b2) a DNA molecule which hybridizes with the DNA sequence defined in (b1) under stringent conditions and encodes a protein having a nitrate transport function;

(b3) and (b) a DNA molecule which has more than 90% of homology with the DNA sequence defined in (b1) or (b2) and encodes a protein with nitrate transport function.

The stringent conditions can be hybridization and washing with 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS solution at 65 ℃ in DNA or RNA hybridization experiments.

Recombinant expression vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the CcNPF8.3.2 gene also belong to the scope of protection of the invention.

The recombinant expression vector can be specifically an expression vector obtained by replacing a fragment between multiple cloning sites (specifically, sal I and spe I enzyme cutting sites) of the vector pYNR-LEU2 with a double-stranded DNA molecule shown in a sequence 4 of a sequence table.

The invention also protects the application of the CcNPF8.3.2 protein in transporting nitrate.

The invention also protects the application of the CcNPF8.3.2 protein or the CcNPF8.3.2 gene in regulating the absorption rate of biological nitrate.

The invention also protects the application of the CcNPF8.3.2 protein or the CcNPF8.3.2 gene in the regulation of the transport function of biological nitrate.

The invention also protects a method for cultivating transgenic organisms, which is to introduce the CcNPF8.3.2 gene into target organisms to obtain transgenic organisms; the nitrate transport function of the transgenic organism is higher than that of the target organism.

The invention also provides a method for improving the transport function of biological nitrate, which is to increase the expression quantity and/or activity of CcNPF8.3.2 protein in a target organism and improve the transport function of biological nitrate.

The invention also protects the use of the ccnpf8.3.2 protein, or the ccnpf8.3.2 gene, or any of the methods described above, in biological breeding.

The purpose of the biological breeding is to breed organisms with high nitrate transport function.

Any of the above organisms may specifically be a plant, more specifically a chlorophytum comosum.

Any of the above organisms may specifically be yeast, more specifically △ ynt-Leu double mutant Hansenula polymorpha.

The invention clones a coding gene CcNPF8.3.2 of nitrate transport protein from the root of chlorophytum comosum, and introduces the gene into △ ynt-Leu double mutation Hansenula polymorpha to obtain the CcNPF8.3.2 yeast, tests prove that the CcNPF8.3.2 protein has the function of nitrate transport protein, can improve the nitrogen utilization efficiency of plants, and enables the plants to grow rapidly.

Drawings

FIG. 1 is a diagram showing the identification of nitrate transporter genes.

FIG. 2 is a 3' RACE identification map.

FIG. 3 is a 5' RACE identification map.

FIG. 4 is an identification chart of transgenic yeast △ ynt-CcNPF8.3.2.

FIG. 5 shows the functional verification of CcNPF8.3.2 protein.

FIG. 6 is a graph of the nitrate uptake rate of CcNPF8.3.2.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Suspending a chlorophytum comosum: purchased from the flower, bird and fish market.

△ ynt-Leu double mutant Hansenula polymorpha, reference Tobacco Nia2cDNA functional variants a Hansenula polymorpha mutant product, A newexpresson system for the term of plant proteins involved in the biotechnology industry, publicly available from the institute of biotechnology of the Chinese academy of agricultural sciences.

Vector pYNR-LEU 2: reference documents: tobacco Nia2cDNA functional compositions A human polymorphaase mutant production. A new expression system for the student of plant proteins involved in the simulation; the public is available from the institute of biotechnology, academy of agricultural sciences, china.

Wild-type hansenula polymorpha (WT): reference documents: tobacco Nia2cDNA functional complex and human recombinant mutant production. A new expression system for the term of plant proteins involved in the mutation. The public is available from the institute of biotechnology, academy of agricultural sciences, china.

Example 1, CcNPF8.3.2 protein and Gene encoding

Cloning of nitrate transporter gene

1. Total RNA from the roots of Chlorophytum comosum was extracted using the RNAprep Pure Plant kit from TIANGEN, according to the kit instructions, and then reverse-transcribed to obtain cDNA. The reverse transcription system is shown in Table 2. Carrying out reverse transcription at 65 ℃ for 10 min; rapidly cooling to room temperature on ice, adding 1 μ l RNase, extending for 2h at 42 ℃, and terminating the reaction at 70 ℃ for 15 min.

TABLE 2 reverse transcription System

2. By comparing the amino acid sequences of nitrate transporters such as small-particle physcomitrella, wild rice, arabidopsis thaliana, rice and the like, a pair of degenerate primers is designed according to a conserved region and consists of a primer L5 and a primer L3;

primer L5: 5 '-ACNGAYGTNGARGARGTL-3';

primer L3: 5 '-CATNCCYTTNGGRCAYTC-3'.

3. And (3) carrying out PCR amplification by using the cDNA obtained in the step (1) as a template and adopting a primer L5 and a primer L3 to obtain a PCR amplification product. The PCR reaction system is shown in Table 3. The PCR reaction program is: preheating at 95 deg.C for 5 min; denaturation at 95 ℃ for 30 s; annealing at 62-43 ℃ for 30s (1 ℃ per cycle), extending at 72 ℃ for 30s, denaturing at 95 ℃ for 30s, annealing at 42 ℃ and extending at 72 ℃ for 30s, and performing 20 cycles; 72 ℃ for 10 min.

TABLE 3 PCR reaction System

And (3) detecting the PCR amplification product by 1% agarose gel electrophoresis, connecting the PCR amplification product to a T vector for sequencing, and indicating that: a band with the size of 589bp is obtained by PCR amplification (namely, the electrophoresis result of the nitrate transport protein gene is shown in a figure 1), and the nucleotide sequence of the band is shown as a sequence 1 in a sequence table.

Acquisition of two, 3' RACE

1. Total RNA of the root of a short pearl is extracted by using an RNAprep Pure Plant kit of TIANGEN company according to the kit specification, and cDNA is obtained by reverse transcription. The reverse transcription system is shown in Table 4. The reverse transcription procedure was: 60min at 42 ℃; 70 ℃ for 15 min.

TABLE 4 reverse transcription System

2. Using the cDNA template obtained in the step 1, performing PCR reaction by using a primer Outer (5'-CCAGACAAGTTTTCAGCAATTG-3') and a primer Inner (5'-GCCGCTACAGTTTCTTCAGTTGATAC-3') and TaKaRa LA Taq (Code No. RR002A), wherein the experimental operation is as follows:

(1) outer PCR reaction

The Outer PCR reaction system is shown in Table 5. The Outer PCR reaction program is: preheating at 95 deg.C for 3 min; denaturation at 95 ℃ for 30 s; annealing at 55 ℃ for 30 s; extension at 72 ℃ for 30s for 20 cycles; 72 ℃ for 10 min.

TABLE 5 Outer PCR reaction System

(2) Inner PCR reaction

The Inner PCR reaction system is shown in Table 6. The Inner PCR reaction procedure was: preheating at 95 deg.C for 3 min; denaturation at 95 ℃ for 30 s; annealing at 59 ℃ for 30 s; extension at 72 ℃ for 1min for 30 cycles; 72 ℃ for 10 min.

TABLE 6 Inner PCR reaction System

And (3) detecting the PCR amplification product by 1% agarose gel electrophoresis, connecting the PCR amplification product to a T vector for sequencing, and indicating that: a1230 bp band (i.e. 3' RACE, the electrophoresis result is shown in figure 2) is obtained by PCR amplification, and the nucleotide sequence of the band is shown as a sequence 2 in a sequence table.

Acquisition of the three, 5' RACE

1. Total RNA from the root of a short pearl was extracted using the RNAprep Pure Plant kit from TIANGEN, according to the kit instructions.

2. The solutions shown in table 7 were prepared.

TABLE 7 solution system

3. Mu.l of the total RNA obtained in the step 1 was added to the system obtained in the step 2, and after mixing and centrifugation (14000 Xg), the mixture was reacted at 72 ℃ for 3min and at 42 ℃ for 2min to obtain a reaction solution.

4. After the completion of step 3, the solution was prepared according to Table 8, and simultaneously 1. mu.l of Smarter IIA oligo-nucleotide was added to the system, and after mixing and centrifugation (14000 Xg), the reaction was carried out at 42 ℃ for 90min and at 70 ℃ for 10min to obtain a reaction solution.

TABLE 8 solution system

5. The Outer PCR reaction was carried out using TaKaRa LA Taq (Code No. RR002A) as a primer Outer (5'-CTTCGATTTGAGTGACTGTGCAGAGC-3'), and the reaction system is shown in Table 9. The Outer PCR reaction program is: the first round is carried out at 95 ℃ for 3 min; denaturation at 94 ℃ for 30 s; annealing at 72 ℃ for 30 s; extension at 72 ℃ for 1min for 5 cycles; denaturation at 94 ℃ for 30 s; annealing at 68 ℃ for 30 s; extension at 72 ℃ for 1min for 5 cycles; denaturation at 94 ℃ for 30 s; annealing at 64 ℃ for 30 s; extension at 72 ℃ for 1min for 5 cycles; denaturation at 94 ℃ for 30 s; annealing at 60 ℃ for 30 s; extension at 72 ℃ for 1min for 20 cycles; 72 ℃ for 10 min. And a second round: preheating at 95 deg.C for 3 min; denaturation at 94 ℃ for 30 s; annealing at 60 ℃ for 30 s; extension at 72 ℃ for 1min, 30 cycles, 72 ℃ for 10 min.

TABLE 9 Outer PCR reaction System

And (3) detecting the PCR amplification product by 1% agarose gel electrophoresis, connecting the PCR amplification product to a T vector for sequencing, and indicating that: a belt with the size of 874bp is obtained by PCR amplification (namely 5' RACE, the electrophoresis result is shown in figure 3), and the nucleotide sequence of the belt is shown as a sequence 3 in a sequence table.

Cloning of four, all genes

According to the nucleotide sequences of the 3 'RACE and the 5' RACE obtained in the second step and the third step, a primer F8-3b-5 (5'-gtcgacATGGGGAGAGCCTG-3') and a primer F8-3b-3 (5'-actagtTCAAGCCGAAGCTCTTTTAG-3') are designed, and the cDNA obtained in the first step (1) is used as a template for PCR amplification to obtain a PCR amplification product. This was ligated into pEasy-T1, singleton picked and sequenced.

The sequencing result shows that: the PCR amplification obtains a 1629bp band, the nucleotide sequence of which is shown as sequence 4 in the sequence table, the gene shown as sequence 4 is named as CcNPF8.3.2 gene, and the amino acid sequence of the protein coded by the CcNPF8.3.2 gene (named as CcNPF8.3.2 protein) is shown as sequence 5 in the sequence table.

Example 2 functional verification of CcNPF8.3.2 protein and its encoding Gene

This example uses △ ynt-Leu double mutant Hansenula polymorpha to verify the function of CcNPF8.3.2 protein and its coding gene.

Construction of transgenic Yeast

1. The fragment between the sal I and spe I enzyme cutting sites of the vector pYNR-LEU2 is replaced by a double-stranded DNA molecule shown in the sequence 4 of the sequence table to obtain a shuttle plasmid pYNR-CcNPF8.3.2.

2. And (3) carrying out enzyme digestion linearization on the shuttle plasmid pYNR-CcNPF8.3.2 obtained in the step 1 by using Bstp1, carrying out 1% agarose gel electrophoresis, and recovering and purifying a target product to obtain a linearized fragment.

3. The specific steps for preparing the transgenic △ ynt-Leu double-mutation Hansenula polymorpha are as follows:

(1) a single colony of △ ynt-Leu double-mutant Hansenula polymorpha was picked, inoculated into 5mLYGNH liquid medium, and cultured overnight at 37 ℃ at 200 r/min.

YGNH liquid medium: 0.17% (mass percent) YNB, 2% (mass percent) glucose, 5mmol/LNH₄Cl and the solvent is water.

(2) Transferring 100 μ L of the culture obtained in step (1) into 100mL YGNH liquid culture medium, culturing at 37 deg.C and 200r/min for 14-16h to make the bacterial liquid concentration reach OD_600nm＝1.3～1.5。

(3) Transferring the bacterial liquid obtained in the step (2) into a precooled 50mL sterile centrifuge tube, centrifuging for 5min at 4 ℃ at 1200r/min, and collecting thalli.

(4) The cells collected in step (3) were resuspended and washed once with 50mL and 25mL of pre-cooled sterile water, respectively.

(5) And (3) resuspending the thalli obtained in the step (4) by using 2mL of ice-precooled 1mol/L sorbitol solution to obtain a bacterial suspension (△ ynt-Leu delta ynt double mutation Hansenula polymorpha competent cells).

(6) And (3) adding 50mg of the linearized fragment obtained in the step (2) into 80 mu L of the bacterial suspension obtained in the step (5), uniformly mixing, and carrying out ice bath for 5 min.

(7) And (4) electrically shocking the bacterial suspension subjected to ice bath treatment in the step (6) at 150v and 130 omega, adding 600 mu L of ice-precooled 1mol/L sorbitol solution to uniformly mix the thalli, and standing and culturing for 1h at 37 ℃.

(8) After the step (7) is finished, the bacterial liquid is coated on YNGH solid culture medium containing 100mg/mL ampicillin resistance, and cultured for 3-4 days at 37 ℃ until monoclonals exist.

YNGH solid culture medium: 0.17% (mass percent) YNB, 2% (mass percent) glucose, NO₃ ^-(0.1011g/200mL) and the solvent is water.

(9) Selecting single colonies, and screening yeast positive clones by using F8-1-5 and F8-1-3 as primers through PCR, wherein the PCR reaction procedure comprises the steps of pre-performing at 95 ℃ for 15min, performing denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, extending at 72 ℃ for 1min for 30 cycles, performing PCR reaction at 72 ℃ for 10min, and performing 1% agarose gel electrophoresis detection on 5-10 mu l of PCR reaction solution to obtain a positive band of 1629bp through PCR amplification (the electrophoresis result is shown in figure 4 and is shown in a sequence 4 of a sequence table through sequencing), wherein the result shows that yeast positive strains of the CcNPF8.3.2 gene are transferred, and the yeast positive strains are uniformly named as △ ynt-CcNPF8.3.2.

4. The shuttle plasmid pYNR-CcNPF8.3.2 was replaced with the vector pYNR-LEU2, and the procedure was followed in

steps

2 and 3 to obtain a yeast with a transferred empty vector.

Second, function verification

The tested strains are △ ynt-Leu double-mutation Hansenula polymorpha, wild type Hansenula polymorpha (WT), △ ynt-CcNPF8.3.2 and yeast with a trans-empty vector.

Selecting single colony of the strain to be detected, inoculating into 10mL YNGH liquid culture medium, culturing at 37 deg.C and 200r/min for 12h, and measuring OD with spectrophotometer_600nmThe absorbance of (a).

The results are shown in FIG. 5, which shows that △ ynt-Leu double mutant Hansenula polymorpha and the yeast with transferred empty vector can not grow in YNGL medium, while the wild type and the transgenic yeast △ ynt-CcNPF8.3.2 can grow normally in YNGL medium, which shows that CcNPF8.3.2 gene can restore the growth of △ ynt-Leu double mutant Hansenula polymorpha with nitrate transporter function.

Third, determination of absorption Rate

Nitrate ions have strong absorption in the ultraviolet region, and the concentration of nitrate can be quantitatively determined by using the absorbance thereof at a wavelength of 220 nm. Nitrate ions do not absorb at 275nm, although organic matter dissolved in the solution will also absorb at 220 nm. Therefore, another measurement was made at 275nm to correct for nitrate nitrogen values. A is corrected to_220nm-A_275nm. According to the literature report, the nitrate absorption efficiency Km of the nitrate transporter is determined to determine whether the protein is a high-affinity or low-affinity transporter. When Km is<1000. mu.M is a high affinity transporter; when Km is>1000. mu.M is a low affinity transporter.

(1) △ ynt-CcNPF8.3.2 single colony was picked and inoculated into 5mL YGNH liquid medium and cultured at 37 ℃ and 200r/min for 12 h.

(2) Inoculating 100 μ l of the culture obtained in step (1) into 100mL YGNH liquid culture medium, culturing at 37 deg.C and 200r/min for 14-16h to make the concentration of bacterial liquid reachTo OD_600nmCentrifugation is carried out at 4 ℃ and 1200r/min for 5min under the condition of 1.3-1.5, and thalli are collected.

(3) Taking 6 50mL centrifuge tubes containing 10mL YG liquid medium, adding 100mg of the thallus obtained in step (2) into each tube, and shake-culturing at 37 deg.C and 200r/min for 2h to keep the yeast in the same growth state in each tube.

(4) After completion of step (3), the cells were centrifuged at 1200 rpm for 5min at 4 ℃ and the supernatant was discarded, and the yeast pellet from each tube was resuspended in 50mL of ice-cold YG liquid medium containing nitrate at 50. mu.M, 100. mu.M, 300. mu.M, 500. mu.M, 1000. mu.M and 1500. mu.M, and washed once.

(5) And (4) repeating the step (4) twice.

(6) After completion of step (5), 10mL of YG liquid medium containing the corresponding nitrate concentration (50. mu.M, 100. mu.M, 300. mu.M, 500. mu.M, 1000. mu.M and 1500. mu.M) was added to each tube, and after shaking culture at 37 ℃ and 200r/min for 30 minutes, the absorbance was measured, an absorption rate curve was plotted, and the Km value was calculated.

The results are shown in FIG. 6. By calculation, the Km value of the CcNPF8.3.2 protein is 1100. mu.M, which is greater than 1000. mu.M, indicating that the CcNPF8.3.2 protein is a low affinity nitrate transporter.

Sequence listing

<110> institute of biotechnology of Chinese academy of agricultural sciences

<120> chlorophytum comosum root nitrate transporter CcNPF8.3.2, and coding gene and application thereof

<160>5

<170>SIPOSequenceListing 1.0

<210>1

<211>589

<212>DNA

<213> Chlorophytum comosum (Thunb.) Baker.)

<400>1

ggtgcggatc aatttgatga cacggatcca acagagagag tgaagaaggg ctctttcttc 60

aactggttct acttctccat caacattggt gccctcatat caagcagctt tcttgtgtgg 120

gtacaagaca acatgggttg gggtttgggg tttggtatcc ccacattgtt catgggtttg 180

gctattggaa gcttcttctt gggcaccccg ctttatagat ttcagaaacc tggaggaagc 240

cctctaacca gaatctgcca ggtggtggtt gcctctgttc ggaagtggaa tgtagatgtc 300

cctcgggatg cttctcttct atatgaacta ccagacaagt tttcagcaat tgaagggagt 360

cgaaagattg aacacagtga tgaactcaag tttttagaca aagccgctac agtttcttca 420

gttgatacaa agtccgatag cttatccaac tcgtggcgac tttgcacagt cactcaggtc 480

gaggaactga agatcctggt acgaatgttc ccaatctggg caacaggaat tgtgttctct 540

gcagtctaca gccaaatttc caccatgttc gtcgaactga tcggcgcca 589

<210>2

<211>1230

<212>DNA

<213> Chlorophytum comosum (Thunb.) Baker.)

<400>2

gccgctacag tttcttcagt tgatacaaag tccgatagct tatccaactc gtggcgactt 60

tgcacagtca ctcaggtcga ggaactgaag atcctggtac gaatgttccc aatctgggca 120

acaggaattg tgttctctgc agtctacagc caaatttcca ccatgttcgt cgaacaaggc 180

atggttctca acaccaccat tggcgacttc accatccccc cggcctccct ctcaaccttt 240

gatgtcatca gcgtcgtcat ctgggttcca ctctacgaca ggatcctagt accactagcc 300

cgaaagttca ccggcaaaga gaggggcttc tctgagctgc agaggatggg catcggccta 360

ttcatttcaa tactggccat ggcagccgca gctctagtag agatcaggag attggacatt 420

gcgagagcag aaggtctaat agacaagaag gttgcagtgc ccatgactat cctgtggcaa 480

ataccacagt atttcttggt cggagctgct gagatcttca ctttcatcgg gcagcttgag 540

tttttctatg atcagagtcc tgacgccatg agaagccttt gcagcgcact gtcgcttctg 600

acgactgctc tcgggaactg tttgagctcg tttattctga ccgttgtgac tgtagtgact 660

actcgaggag ggaagactgg ttggattcct gataacttga atgagggcca cttggataac 720

ttcttctggt tgttggctgg actcagttct ctgaacctct tggtttacgt ggtgtgtgct 780

acgaggtata gatctaaaag agcttcggct tgagcaacac aaggagatga gaacgaccaa 840

taccagcaag aaaaacagca acaagcaaaa ttagcctcag ctgagcaaca cacatgtaac 900

aagttgtgcc catttgaatc actcttgctc gactctcttg gtctgaagga tgctttcata 960

ggatttctgc gtttctttag tcaaattaaa tgtatgtata atattcaaga cagaaggttt 1020

ttccgtttac agatagaata attgaacttc taagaactgc ttcttggaga tatataatgt 1080

attcctcgtg atttatgtta atgttcccaa aaaaaaaaaa cctatagtga aatcactagt 1140

ggaggatccg cgaagggcaa ttctgcagat atccatcaca ctggcggccg ctcgagcatg 1200

catctagagg gcccaatcgc cctatacgga 1230

<210>3

<211>874

<212>DNA

<213> Chlorophytum comosum (Thunb.) Baker.)

<400>3

tctaatacga ctcactatag ggcaagcagt ggtatcaacg cagagctaat acgactcact 60

atagggcaag cagtggtatc aacgcagcta atacgactca ctatagggca agcagtggta 120

tcaacgcaga gtctaatacg actcactata gggcaagcag tggtatcaac gcagaggcta 180

atacgactca ctatagggca agcagtggta tcaacgcaga gtctaatacg actcactata 240

gggcaagcag tggtatcaac gcagagtaca tggggagagc ctgcccattc attttaggaa 300

atgaatgttg tggacgtttg gcctattatg gaatatcaac aaaccttgtc acttatctca 360

ctaagaagct acaccaagga aatgttactg ctgcaagaaa tgttactacc tggcaaggta 420

cctgctacct aacccctctg attggtgcag tcttagctga cgcctattgg ggaaggtatt 480

ggacgattgc agtattttca acaatatact tcattggaat gagtacatta attctctctg 540

catcagttcc agcattcaag ccgccttcct gcgtgcaatc agtatgccca gaggcaagtg 600

ctgcacagta tgccatgttc tttttcgggc tatatctcat tgcattagga accggtggca 660

tcaaaccatg tgtttcatcc tttggagccg atcaatttga tgacacggat ccaacagaga 720

gagtgaagaa gggctctttc ttcaactggt tctatttctc catcaacatt ggcgccctca 780

tatcaagcag ctttcttgtg tgggtacaag acaatatggg ttggggtttg gggtttggta 840

tccccacatt gttcatgggt ttggctattg gaag 874

<210>4

<211>1629

<212>DNA

<213> Chlorophytum comosum (Thunb.) Baker.)

<400>4

atggggagagcctgcccttt cattttggga aacgaatgtt gtgaacgttt ggcccattat 60

ggaatttcaa caaatcttgt cacttatctc actaagaagc tacaccaagg aaatgttacc 120

gctgcaagga atgttactac ctggcaaggt acctgctacc tcacccctct gattggtgcc 180

atcttagctg acgcatactg ggggaggtat tggacgagtg cagtgttttc aacaatatac 240

ttcattggaa tgagtacttt aactctctct gcattggttc cagcattcaa gcctccttcc 300

tgcgtgcaat cggtatgccc agaggcaagt gctgcacagt atgccgtatt ctttttcggg 360

ctatatctca tcgcattagg aaccggtggc atcaaaccat gtgtctcatc ctttggagcc 420

gatcaatttg atgacacgga tccaaccgag agagtgaaga agggctcttt cttcaactgg 480

ttctacttct ctgtcaacat tggcgccctc atatcaagca gctttcttgt gtgggtacaa 540

gacaacatgg gttggggttt ggggtttggt atccccacat tgttcatggg tttggctatt 600

ggaagcttct tcttgggcac cccactttat agatttcaga aacctggagg aagccctcta 660

accagaatct gccaggtggt ggttgcctct gttcggaagt ggaatgtaga tgtacctcgg 720

gatgcttctc ttctatatga actaccagag aagttttcag caattgaagg gagtcgaaag 780

attgaacaca gtgatgaact caagttttta gacaaagccg ctacagtttc ttcagttgat 840

acaaagtccg atagcttatc caacccatgg cgactttgca cagtcactca ggtcgaggaa 900

ctgaagatcc tggtacgaat gttcccaatc tgggcaacag gaattgtgtt ctccgcagtc 960

tgcagccaaa tttccaccat gttcgttgaa caaggcatgg ttctcaacac caccattggc 1020

gccttcacca tccccccggc ctccctctca acctttgata tcatcagcat cgtcatctgg 1080

gttccactct acgacaggat cctagtacca ctagcccgaa agttcaccgg caaagagagg 1140

ggcttctctg agctgcagag gatgggcatc ggcctattca tttcaatact ggccatggca 1200

gtcgcagctc tagtagagat caggagattg gacattgcga gagcagaagg tctaatagac 1260

aagaaggttg cagtgcccat gactatcctg tggcaaatac cacagtattt cttggtcgga 1320

gctgctgaga tcttcacttt catcgggcag cttgagttgt tctatgatca gagtcctgac 1380

gccatgagaa gcctttgcag cgcactgtcg cttctgacga ctgctctcgg gaactatttg 1440

agctcgttta ttctgaccgt tgtgactgta gtgactactc gaggagggaa gactggttgg 1500

attcctgata acttgaatga gggccacttg gataacttct tctggttatt ggctggactc 1560

agttctctga acctcttggt ttacgtgctg tgtgctacga ggtatagatc taaaagagct 1620

tcggcttga 1629

<210>5

<211>542

<212>PRT

<213> Chlorophytum comosum (Thunb.) Baker.)

<400>5

Met Gly Arg Ala Cys Pro Phe Ile Leu Gly Asn Glu Cys Cys Glu Arg

1 5 10 15

Leu Ala His Tyr Gly Ile Ser Thr Asn Leu Val Thr Tyr Leu Thr Lys

20 25 30

Lys Leu His Gln Gly Asn Val Thr Ala Ala Arg Asn Val Thr Thr Trp

35 40 45

Gln Gly Thr Cys Tyr Leu Thr Pro Leu Ile Gly Ala Ile Leu Ala Asp

50 55 60

Ala Tyr Trp Gly Arg Tyr Trp Thr Ser Ala Val Phe Ser Thr Ile Tyr

65 70 75 80

Phe Ile Gly Met Ser Thr Leu Thr Leu Ser Ala Leu Val Pro Ala Phe

85 90 95

Lys Pro Pro Ser Cys Val Gln Ser Val Cys Pro Glu Ala Ser Ala Ala

100 105 110

Gln Tyr Ala Val Phe Phe Phe Gly Leu Tyr Leu Ile Ala Leu Gly Thr

115 120 125

Gly Gly Ile Lys Pro Cys Val Ser Ser Phe Gly Ala Asp Gln Phe Asp

130 135 140

Asp Thr Asp Pro Thr Glu Arg Val Lys Lys Gly Ser Phe Phe Asn Trp

145 150 155 160

Phe Tyr Phe Ser Val Asn Ile Gly Ala Leu Ile Ser Ser Ser Phe Leu

165 170 175

Val Trp Val Gln Asp Asn Met Gly Trp Gly Leu Gly Phe Gly Ile Pro

180 185 190

Thr Leu Phe Met Gly Leu Ala Ile Gly Ser Phe Phe Leu Gly Thr Pro

195 200 205

Leu Tyr Arg Phe Gln Lys Pro Gly Gly Ser Pro Leu Thr Arg Ile Cys

210 215 220

Gln Val Val Val Ala Ser Val Arg Lys Trp Asn Val Asp Val Pro Arg

225 230 235 240

Asp Ala Ser Leu Leu Tyr Glu Leu Pro Glu Lys Phe Ser Ala Ile Glu

245 250 255

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

260 265 270

Ala Ala Thr Val Ser Ser Val Asp Thr Lys Ser Asp Ser Leu Ser Asn

275 280 285

Pro Trp Arg Leu Cys Thr Val Thr Gln Val Glu Glu Leu Lys Ile Leu

290 295 300

Val Arg Met Phe Pro Ile Trp Ala Thr Gly Ile Val Phe Ser Ala Val

305 310 315 320

Cys Ser Gln Ile Ser Thr Met Phe Val Glu Gln Gly Met Val Leu Asn

325 330 335

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

340 345 350

Asp Ile Ile Ser Ile Val Ile Trp Val Pro Leu Tyr Asp Arg Ile Leu

355 360 365

Val Pro Leu Ala Arg Lys Phe Thr Gly Lys Glu Arg Gly Phe Ser Glu

370 375 380

Leu Gln Arg Met Gly Ile Gly Leu Phe Ile Ser Ile Leu Ala Met Ala

385 390 395 400

Val Ala Ala Leu Val Glu Ile Arg Arg Leu Asp Ile Ala Arg Ala Glu

405 410 415

Gly Leu Ile Asp Lys Lys Val Ala Val Pro Met Thr Ile Leu Trp Gln

420 425 430

Ile Pro Gln Tyr Phe Leu Val Gly Ala Ala Glu Ile Phe Thr Phe Ile

435 440 445

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

450 455 460

Leu Cys Ser Ala Leu Ser Leu Leu Thr Thr Ala Leu Gly Asn Tyr Leu

465 470 475 480

Ser Ser Phe Ile Leu Thr Val Val Thr Val Val Thr Thr Arg Gly Gly

485 490 495

Lys Thr Gly Trp Ile Pro Asp Asn Leu Asn Glu Gly His Leu Asp Asn

500 505 510

Phe Phe Trp Leu Leu Ala Gly Leu Ser Ser Leu Asn Leu Leu Val Tyr

515 520 525

Val Leu Cys Ala Thr Arg Tyr Arg Ser Lys Arg Ala Ser Ala

530 535 540

Claims

1. A protein has an amino acid sequence shown as a sequence 5 in a sequence table.

2. A gene encoding the protein of claim 1.

3. The gene of claim 2, wherein: the gene is a DNA molecule shown in a sequence 4 in a sequence table.

4. A recombinant expression vector, expression cassette or recombinant bacterium comprising the gene of claim 2 or 3.

5. Use of the protein of claim 1 for transporting nitrate in yeast.

6. Use of the protein of claim 1, or the gene of claim 2 or 3, for modulating the nitrate uptake rate in yeast.

7. Use of the protein of claim 1, or the gene of claim 2 or 3, for modulating nitrate transport function in yeast.

8. A method for producing a transgenic yeast by introducing the gene of claim 2 or 3 into a target yeast to obtain a transgenic yeast; the nitrate transport function of the transgenic yeast is higher than that of the target yeast.

9. A method for improving nitrate transport function in yeast, which comprises increasing the expression level of the protein of claim 1 in a target yeast to improve nitrate transport function in yeast.

10. Use of the protein of claim 1, or the gene of claim 2 or 3, or the method of claim 8 or 9, in yeast breeding.