A gene regulating zinc transport in Ceratoides arborescens and its application

Abstract

The invention discloses a gene regulating zinc transport of Ceratoides arborescens and its application; the gene regulating zinc transport of Ceratoides arborescens has one of the following nucleotide sequences: the nucleotide sequence of SEQ NO.1 in the sequence list or SEQ NO.2 in the sequence list; the zinc transport gene encoded by these genes is located in the endoplasmic reticulum of the cell. Yeast complementation experiments show that the zinc transport gene CaMTP1 isolated by the invention has a zinc transport function. The invention successfully isolates and obtains a zinc transport gene CaMTP1 screened in a natural environment for a long time from Ceratoides arborescens, which provides a new direction for plant zinc biofortification using genetic engineering technology and is of great significance for molecular breeding work of nutrient absorption.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a gene for regulating zinc transport in Ceratoides arborescens and an application thereof. Background Art

[0002] Ceratoidesarborescens is a plant of the genus Ceratoides of the Chenopodiaceae family, and is a plant endemic to China. The plant is tall, with luxuriant branches and leaves, a well-developed root system, and the length of the underground part is more than 1.4 times that of the aboveground part; it is rich in nutrients, especially mineral elements and protein, and has strong stress resistance and adaptability. It is a valuable plant resource for improving grasslands, deserts and ecological management, and has broad prospects for development and utilization, and plays a pivotal role. For a long time, a lot of research has been carried out on Ceratoidesarborescens, mainly focusing on introduction and domestication, cultivation technology, ecological characteristics and physiology, but there is no report on the mining of its functional genes.

[0003] There is a huge gap between my country's plant molecular breeding capabilities and those of developed countries. Transport genes with independent intellectual property rights are the focus of competition in the field of plant molecular breeding in the world. For the development and utilization of excellent plant transport genes, current research mainly focuses on model plants such as Arabidopsis or crops such as rice and wheat, and lacks the mining of rich stress resistance genes in wild plants. The present invention uses Illumina's high-throughput sequencing technology to sequence the transcriptome of Ceratoides arborescens, a tree species in arid and semi-arid areas of my country, and obtains a zinc transport gene CaMTP1 that has been screened for a long time in the natural environment. Summary of the invention

[0004] The purpose of the present invention is to clone the zinc transport gene CaMTP1 using molecular cloning technology, and finally obtain an expression vector for gene expression, so that the gene can be introduced into a sensitive yeast mutant, and the function of the gene can be further verified by phenotypic analysis.

[0005] The above object of the present invention is achieved through the following technical solutions:

[0006] The invention provides a zinc transport gene CaMTP1 isolated from Ceratoides arborescens, and the nucleotide sequence of the gene is shown in SEQ NO.1.

[0007] The present invention also provides a protein encoded by a zinc transport gene CaMTP1, characterized in that the amino acid sequence of the protein is shown in SEQ NO. 2, and the amino acid sequence of the protein is a 345 amino acid sequence.

[0008] The invention also provides a recombinant expression vector of the zinc transport gene CaMTP1.

[0009] Preferably, the recombinant expression vector is a recombinant plant expression vector.

[0010] The present invention also provides an application of a zinc transport gene CaMTP1 in improving the ability of plants to absorb, transport or store zinc or iron.

[0011] The present invention also provides an application of a zinc transport gene CaMTP1 that can adapt to high-concentration zinc.

[0012] Preferably, the zinc transport gene CaMTP1 is operably linked to an expression regulatory element to obtain a recombinant plant expression vector; the recombinant plant expression vector is transformed into a recipient plant or plant cell to cultivate a transgenic plant.

[0013] The embodiment of the present invention is to use hydroponic Ceratoides arborescens as experimental material, adopt Illumina Solexa transcriptome high-throughput sequencing method to identify and analyze its transcriptome sequence, and determine Zn 2+ Combined with the type and quantity of proteins, the nucleotide sequence of the zinc transport gene CaMTP1 in Ceratoides arborescens was obtained, and the function was verified through yeast complementation test. The expression position in plants was determined through tobacco subcellular localization.

[0014] The nucleotides mentioned above also include substitution, deletion and insertion mutants as well as allelic variants, splice variants, fragments, derivatives and the like.

[0015] The present invention also provides a technical solution for verifying the function of the new gene CaMTP1 of the present invention.

[0016] The present invention also provides a technical solution for locating the novel gene CaMTP1 of the present invention.

[0017] The technical advantages of the present invention are the positive effects achieved:

[0018] 1. The present invention discovered the zinc transport gene CaMTP1 of Ceratoides arborescens for the first time, laying a foundation for further research on the stress resistance and adaptability of Ceratoides arborescens.

[0019] 2. The present invention successfully isolated a zinc transport gene CaMTP1 from Ceratoides arborescens that was screened in the natural environment for a long time, which provides a new direction for the use of genetic engineering technology for plant zinc biofortification and is of great significance for the molecular breeding work of nutrient absorption.

[0020] 3. The present invention applies the CaMTP1 gene to plant genetic engineering to improve the survival ability of plants under nutrient deficiency or excess. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 :The overall experimental flow chart;

[0022] Figure 2 :The phenotype of the low iron sensitive yeast mutant △fet3 / △fet4 transformed with CaMTP1 gene;

[0023] Figure 3 :The phenotype of the low zinc sensitive yeast mutant △zrt1 / △zrt2 transformed with CaMTP1 gene;

[0024] Figure 4 :The phenotype of the high zinc-sensitive yeast mutant △zrc1 / △cot1 transformed with CaMTP1 gene;

[0025] Figure 5 : Diagram of CaMTP1 subcellular transient expression in tobacco. DETAILED DESCRIPTION

[0026] The present invention is further described below in conjunction with specific embodiments. The advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

[0027] Example 1: Cultivation of Ceratoides arborescens

[0028] Plant the seeds of Ceratoides arborescens in a filter paper culture dish. When the roots grow to 2-3 cm, transplant the germinated seeds into a hydroponic solution, and change the hydroponic solution once a week.

[0029] Example 2: Extraction of RNA from Ceratoides arborescens

[0030] Seedlings with the same growth conditions were selected and moved to CK, 0×Fe, and 10×Zn hydroponic solutions for 48 h, and root RNA was extracted.

[0031] The method for extracting root RNA adopts the TRIzol method, and the specific operation is as follows:

[0032] 1) The mortar, tweezers, centrifuge tubes, and pipette tips used should be sterilized twice in an autoclave at 121°C for 20 min before use. The mortar, tweezers, and centrifuge tubes should be pre-cooled with liquid nitrogen until the liquid nitrogen does not boil and splash.

[0033] 2) Use tweezers to pick up the roots of Ceratoides arborescens and put them into a mortar filled with liquid nitrogen, remove the aerial part of Ceratoides arborescens, and quickly grind them into powder when the liquid nitrogen evaporates to the bottom of the mortar. Grind them 4 times;

[0034] 3) Scoop the powder into a pre-cooled 2 mL centrifuge tube (powder volume is about 100 μL), quickly add 1.5 mL TRIzol extract, vortex to mix, and place at room temperature for at least 5 min;

[0035] 4) Centrifuge at 10,000 g for 30 min at 4°C;

[0036] 5) Transfer 1.2 mL of supernatant to a new centrifuge tube, add 500 μL of chloroform and vortex thoroughly to mix;

[0037] 6) Centrifuge at 10,000 g for 15 min at 4°C;

[0038] 7) Take 800 μL of supernatant into a new centrifuge tube, add an equal volume of isopropanol stored at -20°C, and vortex to mix thoroughly;

[0039] 8) Place in -20℃ refrigerator for 1 hour;

[0040] After RNA of all samples was extracted, they were stored in dry ice and sent to Beijing Berry Genomics Biotech Co., Ltd. for transcriptome sequencing.

[0041] Example 3: Screening of Ceratoides arborescens iron-zinc transporter

[0042] In the gene annotation file of the transcriptome sequencing results, the annotation information of the gene nr database, Pfam database, and KOG database was searched by keywords. The keywords are as follows: Fe2+, Iron, IRT, Zn2+, Zinc, ZIP, YSL, and genes with complete amino acid sequences were retained; the primary screening genes were subjected to BLAST analysis with Arabidopsis thaliana in the Phytozome genome database (https: / / phytozome.jgi.doe.gov / pz / portal.html); Genome Net (https: / / www.genome.jp / ) performed multiple sequence alignment of similar genes and screened genes; the InterPro Scan tool of EMBL-EBI (https: / / www.ebi.ac.uk) was used to predict transmembrane domains; IBS software (download address http: / / ibs.biocuckoo.org / download.php) was used to draw protein structure diagrams, and the Multiple Sequence Alignment by CLUSTALW analysis tool of Genome Net was used to construct the evolutionary tree of the screened genes.

[0043] Example 4: Synthesis of Ceratoides arborescens iron-zinc transporter gene

[0044] The target gene was synthesized by Nanjing GenScript Company, and the subsequent construction of the expression vector was completed using the gene synthesized by the company. The cloning vector used for the synthetic gene was pUC57 and the resistance was Amp.

[0045] Example 5: Construction of low zinc and low iron yeast fusion vector

[0046] The yeast expression vector pFL61 and the cloning vector containing the target gene were digested with restriction endonuclease NotⅠ.

[0047] The linear pFL61 and the target gene fragment were recovered by gel recovery kit, and the yeast expression vector pFL61 was dephosphorylated by CIAP phosphorylase.

[0048] Element volume Linear DNA vector fragment 4 0μL CIAP 10×Reaction Buffer 5μL Diluted CIAP (0.01 U / μl) 5μL 50μL

[0049] After mixing the components of the above system, incubate at 37°C for 30 min.

[0050] Add 5 μL of Diluted CIAP (0.01 U / μl) and continue incubation at 37°C for 30 min.

[0051] Add 100 μL of phenol:chloroform:isoamyl alcohol (25:24:1), mix thoroughly, and centrifuge at 20,000 g for 10 min.

[0052] Transfer the supernatant to a new centrifuge tube, add 10 μL 3M CH3COONa and 300 μL 100% C2H5OH, and centrifuge at 20,000 g for 10 min.

[0053] The supernatant was removed, 500 μL of 100% C2H5OH was added, and the mixture was centrifuged at 20,000 g for 5 min.

[0054] The supernatant was removed, dried in a 50°C metal bath, and then redissolved in 20 μL dd H2O.

[0055] The target gene was connected to the dephosphorylated yeast expression vector pFL61 at a molar ratio of 3:1 using T4 DNA Ligase; after the connection was completed, the competent Escherichia coli DH5α was transformed and spread on LB screening medium to screen positive bacteria.

[0056] After the positive bacteria grow out, use Easy Taq PCR Super Mix to detect the target gene. Select the positive bacteria containing the target gene for amplification, extract the plasmid and use the restriction endonuclease NotⅠ for single enzyme digestion, and select the positive bacteria with the correct target band for preservation.

[0057] Because the use of NotⅠ single restriction enzyme digestion to connect the target gene into the expression vector pFL61 may lead to the reverse insertion of the gene, an additional restriction enzyme site is introduced after the 3' end of the NotⅠ restriction site to determine the correct direction of gene insertion. During cloning, two restriction enzyme sites are designed at the 5' end of the gene, which are NotⅠ and kpnⅠ restriction sites respectively. After the target gene is connected into the expression vector pFL61 using NotⅠ single restriction enzyme digestion, kpnⅠ is used for single restriction enzyme digestion again.

[0058] Example 6: Construction of high zinc yeast fusion vector

[0059] Using KOD FX Neo and Ceratoides arborescens zinc ion transporter-related primer sequences, PCR amplification was performed using the plasmid containing the target gene as a template according to the following reaction system. The relevant primer sequences are as follows:

[0060] F:5'- CGCG GATC CATG TTT ACA GCC CGA CAT GAT CCC ACA C -3';

[0061] R:5'-CCC A AGCT T TTA AGC CCA ACG TCC AAC GAC AGA CAT -3'.

[0062] The amplification system is as follows:

[0063]

[0064] The target gene was amplified by PCR, and the target gene fragment was recovered by electrophoresis detection and connected into the cloning vector PESI®-Blunt Simple Cloning Vector for sequencing.

[0065] After the target gene was sequenced correctly, the yeast expression vector pYUL2 was ligated with the target gene using T4 DNA Ligase at a molar ratio of 3: 1. After the ligation was completed, the competent E. coli DH5α was transformed, and the bacterial solution was spread on LB screening medium to screen positive bacteria.

[0066] After the positive bacteria grow out, use Easy Taq PCR Super Mix to detect the target gene.

[0067] Positive bacteria containing the target gene are selected for expansion culture, the plasmid is extracted and double-digested with restriction endonucleases, and positive bacteria with the correct target band are selected for preservation.

[0068] The yeast expression vector pYUL2 correctly linked to the target gene was extracted using an endotoxin-free plasmid extraction kit to transform the high zinc-sensitive yeast mutant △zrc1 / △cot1 competent state. The bacterial solution was spread on SD-Leu screening medium and inverted in a 30°C constant temperature incubator for positive clone screening.

[0069] The positive colonies grown were inoculated into SD-Leu liquid screening medium and cultured at 30°C and 250 rpm.

[0070] Yeast plasmids were extracted and PCR detection was performed using Easy Taq PCR Super Mix to screen positive bacteria containing the target gene fragment for bacterial preservation.

[0071] Example 7. Phenotypic analysis of low zinc and low iron sensitive yeast mutants

[0072] The low zinc-sensitive yeast mutant △zrt1 / △zrt2, the negative control △zrt1 / △zrt2-pFL61 (i.e., △zrt1 / △zrt2 was transformed with the pFL61 empty vector), the positive control △zrt1 / △zrt2-pFL61-At ZIP7 (i.e., At ZIP7 was transformed with △zrt1 / △zrt2) and △zrt1 / △zrt2-pFL61-CaMTP1 (i.e., the low zinc-sensitive yeast mutant △zrt1 / △zrt2 containing the target gene to be phenotypically analyzed) were activated simultaneously on YPD solid medium.

[0073] The low iron sensitive yeast mutant △fet3 / △fet4, the negative control △fet3 / △fet4-pFL61 (i.e., pFL61 empty vector transformed into △fet3 / △fet4), the positive control △fet3 / △fet4-pFL61-At ZIP7 (i.e., At ZIP7 transformed into △fet3 / △fet4), and △fet3 / △fet4-pFL61-CaMTP1 (i.e., the low zinc sensitive yeast mutant △fet3 / △fet4 containing the target gene to be analyzed for phenotype) were activated simultaneously in YPD solid medium. Incubate inverted in a 30℃ constant temperature incubator, select single colonies with the same growth status and inoculate into 5 ml of liquid 1×YPD medium, and culture overnight at 30℃ and 250 rpm. Centrifuge at 700 g for 5 min at room temperature, discard the supernatant, and wash twice with sterile water. Finally, the absorbance value OD600 of the bacterial solution was adjusted to 1.0 with sterile water. Taking OD600=1.0 as the starting concentration, a 10-fold gradient dilution was performed, namely OD600=0.1, OD600=0.01, OD600=0.001, and OD600=0.0001.

[0074] Pipette 10 μl of the diluted gradient bacterial solution and apply it to YPD complete nutrient solid medium and yeast low zinc phenotype medium respectively, and invert and culture in a 30°C constant temperature incubator to observe the growth status of the colonies.

[0075] Example 8: Phenotypic analysis of high zinc sensitive yeast mutants

[0076] The high zinc-sensitive yeast mutant △zrc1 / △cot1, wild type BY4741, negative control △zrc1 / △cot1-pYUL2 (i.e., △zrc1 / △cot1 transformed with pFL61 empty vector), positive control △zrc1 / △cot1-pYUL2-MtMTP3 (i.e., △zrc1 / △cot1 transformed with Mt MTP3) and △zrc1 / △cot1-pYUL2-CaMTP1 (i.e., the high zinc-sensitive yeast mutant △zrc1 / △cot1 containing the target gene to be analyzed by phenotype) were activated simultaneously on YPD solid medium.

[0077] Incubate the cells upside down in a 30℃ constant temperature incubator, select single colonies with the same growth status and inoculate them into 5 ml of liquid 1×YPD medium, and culture them overnight at 30℃ and 250 rpm. Centrifuge at 700 g for 5 min at room temperature, discard the supernatant, and wash twice with sterile water.

[0078] Finally, the absorbance value OD600 of the bacterial solution was adjusted to 1.0 with sterile water. Taking OD600=1.0 as the starting concentration, a 10-fold gradient dilution was performed, namely OD600=0.1, OD600=0.01, OD600=0.001, and OD600=0.0001.

[0079] Pipette 10 μl of the diluted gradient bacterial solution and apply it to YPD complete nutrient solid medium and yeast high zinc phenotype medium respectively, and invert and culture in a 30°C constant temperature incubator to observe the growth status of the colonies.

[0080] Example 9: Transient expression in tobacco epidermal cells

[0081] Using KOD FX Neo DNA Polymerase and primer sequences related to the zinc ion transporter of Ceratoides arborescens, PCR amplification was performed using the plasmid containing the target gene as a template according to the following reaction system.

[0082]

[0083] The target gene fragment was obtained by electrophoresis detection and connected into the cloning vector PESI®-Blunt Simple Cloning Vector for sequencing.

[0084] The correctly sequenced gene containing zinc-iron transporter of Ceratonia arborescens was connected into the entry vector pENTR3C and transformed into competent Escherichia coli DH5α. Easy Taq PCR Super Mix was used for bacterial PCR and restriction enzyme digestion verification to screen positive bacteria containing the target gene and maintain the bacteria.

[0085] Take the verified pENTR3C-target gene fusion vector and the binary expression vector pGWB605 plasmid. Evenly mix the pENTR3C-target gene fusion vector and the pGWB605 expression vector plasmid, and connect the target gene into pGWB605 using Gateway cloning. The reaction system is as follows.

[0086] Gateway LR Clonase Enzyme Mix reaction system:

[0087] Reagent volume

[0088] Entry clone (100-300 ng) 1-10 μl

[0089] Destination vector (150 ng / μl) 2 μl

[0090] 2×LR Clonase Reaction Buffer 4 μl

[0091] TE buffer, PH8.0 to 16 μl

[0092] Gateway cloning reaction conditions:

[0093] The pENTR 3C-target gene entry vector and the pGWB605 expression vector were mixed according to the above reaction system and incubated at 25 °C for 1 h.

[0094] Add 4 μl of proteinase K and incubate at 37°C for 10 min.

[0095] The reaction product was transformed into competent E. coli DH5α, spread on LB screening medium, and inverted in a 37°C constant temperature incubator to screen positive bacteria.

[0096] After the positive bacteria grow out, use Easy Taq PCR Super Mix to detect the target gene.

[0097] Positive bacteria containing the target gene were selected for shake culture, plasmids were extracted and verified using restriction endonucleases, and positive bacteria with the correct target band were selected for preservation.

[0098] The Agrobacterium containing pGWB605-target gene and the Agrobacterium containing vesicle transport-related marker protein were activated on LB solid medium and cultured upside down in a constant temperature incubator at 28 °C.

[0099] Single clones of Agrobacterium were picked and inoculated into LB liquid medium containing 20 μM acetosyringone (AS) and 10 mM MES, and cultured in a shaking incubator at 28°C and 250 rpm until the absorbance OD600 value reached 0.8.

[0100] The cells were collected by centrifugation at 3200 g for 10 min at room temperature, resuspended in Mg-MES buffer, and the OD600 value of the bacterial solution was adjusted to 0.5.

[0101] The adjusted bacterial solution was placed in a constant temperature incubator at 28 °C for 3 h.

[0102] Agrobacterium containing pGWB605-target gene and Agrobacterium containing intracellular vesicle transport-related marker protein were mixed in a volume ratio of 1:1 and injected into the lower epidermis of tobacco, placed in a growth chamber for light culture, and the luminescence position was recorded under laser confocal microscope.

[0103] The expression of fluorescent protein was observed starting 48 hours after injection and was observed every 24 hours until the fluorescent protein appeared.

[0104] Having generally described the present invention above, the present invention can be further understood by reference to certain specific examples provided herein, which are intended to illustrate rather than limit the present invention. Sequence Listing <110> Mengcao Ecological Environment (Group) Co., Ltd. <120> A gene regulating zinc transport in Ceratoides arborescens and its application <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1060 <212> DNA <213> Ceratoidesarborescens(L osin-sk. Tsien.etC.G.MaEurotiaarborescens AL os.] <400> 1 gcggccgcgg taccatgttt acagcccgac atgatcccac accggaagac aatgaatgcg 60 taatgacaga tgaacaaatt aatgatacat ataatttaac cgttcgtatt gtatctgttt 120 ttgttctctt gattgtatct tttcttggtg ctgcaatttc tgtggtctcg agtcgagtca 180 aatgtttaca tatcaatccc atcattatca acactggcaa gtttttcggc agtggagtcg 240 tgctagcaac gggtttcatt catatgctac ctggtggcat gaaattctta aatgatcctt 300 gtctaccgga ttcatggaat gtttacagtg cttacggtgg actttttgct atgatagcag 360 cattaatcat gcaatttatc gaatttgttg ctcatcaacg gtatcaatca ttgacgacgt 420 ctaaaactca tccaacaatt gaagaatctg aaaggcaagg ccaggaagag attaaagtat 480 ccgtcatcga tgttgttacg tcaaatgtgg aaataccaga tgcaaatcat cattgtcacg 540 gaattactct ccaagatgac gcacaaaaac ataaaataag tacatattta ctcgaatttg 600 gcatagctct tcattcggtt ctcatcggtt taacattggg tacaacgaca gattcgttcg 660 ttgctctatt tattgcactt agttttcatc agttttttga agccattgca cttggtgcac 720 agattgctcg tttagaacat atctctttga aatctgccag tttcatggtc atattttttg 780 ctttaacaac accgattgga atcgccatcg gtattggtgt tcatgcgaag acatataatc 840 ctaaatcggt tgcttcatta cttgtcaatg gaattctcga ttcaatatca gccggcatcc 900 ttatttatgt ggccttagtt aatttgatta cagcagagat gggcgttggt gctcatgcgt 960 ttcacaaatt gagaaaacgc ttaaaattcc tgtactttgt agcgttatat gcaggtgttg 1020 cagccatgtc tgtcgttgga cgttgggctt aagcggccgc 1060 <210> 2 <211> 345 <212> PRT <213> Ceratoides arborescens (L osin-sk. Tsien.etC.G.MaEurotia arborescens A.L os.) <400> 2 Met Phe Thr Ala Arg His Asp Pro Thr Pro Glu Asp Asn Glu Cys Val 1 5 10 15 Met Thr Asp Glu Gln Ile Asn Asp Thr Tyr Asn Leu Thr Val Arg Ile 20 25 30 Val Ser Val Phe Val Leu Leu Ile Val Ser Phe Leu Gly Ala Ala Ile 35 40 45 Ser Val Val Ser Ser Arg Val Lys Cys Leu His Ile Asn Pro Ile Ile 50 55 60 Ile Asn Thr Gly Lys Phe Phe Gly Ser Gly Val Val Leu Ala Thr Gly 65 70 75 80 Phe Ile His Met Leu Pro Gly Gly Met Lys Phe Leu Asn Asp Pro Cys 85 90 95 Leu Pro Asp Ser Trp Asn Val Tyr Ser Ala Tyr Gly Gly Leu Phe Ala 100 105 110 Met Ile Ala Ala Leu Ile Met Gln Phe Ile Glu Phe Val Ala His Gln 115 120 125 Arg Tyr Gln Ser Leu Thr Thr Ser Lys Thr His Pro Thr Ile Glu Glu 130 135 140 Ser Glu Arg Gln Gly Gln Glu Glu Ile Lys Val Ser Val Ile Asp Val 145 150 155 160 Val Thr Ser Asn Val Glu Ile Pro Asp Ala Asn His His Cys His Gly 165 170 175 Ile Thr Leu Gln Asp Asp Ala Gln Lys His Lys Ile Ser Thr Tyr Leu 180 185 190 Leu Glu Phe Gly Ile Ala Leu His Ser Val Leu Ile Gly Leu Thr Leu 195 200 205 Gly Thr Thr Thr Asp Ser Phe Val Ala Leu Phe Ile Ala Leu Ser Phe 210 215 220 His Gln Phe Phe Glu Ala Ile Ala Leu Gly Ala Gln Ile Ala Arg Leu 225 230 235 240 Glu His Ile Ser Leu Lys Ser Ala Ser Phe Met Val Ile Phe Phe Ala 245 250 255 Leu Thr Thr Pro Ile Gly Ile Ala Ile Gly Ile Gly Val His Ala Lys 260 265 270 Thr Tyr Asn Pro Lys Ser Val Ala Ser Leu Leu Val Asn Gly Ile Leu 275 280 285 Asp Ser Ile Ser Ala Gly Ile Leu Ile Tyr Val Ala Leu Val Asn Leu 290 295 300 Ile Thr Ala Glu Met Gly Val Gly Ala His Ala Phe His Lys Leu Arg 305 310 315 320 Lys Arg Leu Lys Phe Leu Tyr Phe Val Ala Leu Tyr Ala Gly Val Ala 325 330 335 Ala Met Ser Val Val Gly Arg Trp Ala 340 345

Claims

1. A zinc transport gene CaMTP1 isolated from Ceratoides arborescens, characterized in that: Its nucleotide sequence is shown in SEQ NO.

1.

2. The protein encoded by the zinc transport gene CaMTP1 according to claim 1, characterized in that The amino acid sequence of the protein is shown in SEQ NO.

2.

3. A recombinant expression vector containing the zinc transport gene CaMTP1 according to claim 1.

4. The recombinant expression vector according to claim 3, characterized in that: The recombinant expression vector is a recombinant plant expression vector.

5. The use of the zinc transport gene CaMTP1 isolated from Ceratoides arborescens according to claim 1, characterized in that: The specific method is as follows: the zinc transport gene CaMTP1 is connected to an expression regulatory element to obtain a recombinant plant expression vector; the recombinant plant expression vector is transformed into a recipient plant or plant cell to cultivate a transgenic plant.

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