CN111269919A

CN111269919A - Corn gene ZmLIP2 and application thereof

Info

Publication number: CN111269919A
Application number: CN202010195036.5A
Authority: CN
Inventors: 沈亚欧; 梁天虎; 张严玲; 陈珉
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2020-03-19
Filing date: 2020-03-19
Publication date: 2020-06-12
Anticipated expiration: 2040-03-19
Also published as: CN111269919B

Abstract

The invention discloses a corn gene ZmLIP2 and application thereof, and the invention utilizes agrobacterium-mediated transgenic technology to transform the corn gene ZmLIP2 and obtain RNAi interference transformation event of the corn gene ZmLIP 2. The expression level analysis of the plants interfered by RNAi shows that the expression level is obviously reduced compared with that of receptor materials. The gene can negatively regulate and control the absorption and the transportation of the plant to the lead, the relation between the corn ZmLIP2 gene and the lead stress resistance of the plant is determined for the first time, and the importance of the gene in the lead resistance application of the corn is verified.

Description

Corn gene ZmLIP2 and application thereof

Technical Field

The invention relates to a corn gene ZmLIP2 and application thereof, belonging to the field of plant genetic engineering.

Background

Corn is one of the important food crops worldwide. However, with the continuous deterioration of the ecological environment, the content of heavy metals in the soil is increased, the growth and development and the yield quality of the soil are seriously influenced by abiotic stress including the heavy metals, and the crop yield is severely limited. The method is the most effective way for coping with the adversity stress and improving the corn yield by excavating the adversity resistance related genes and researching the adversity response mechanism of the genes. At present, the stress-resistant germplasm resources of corn in China are very deficient, and the stress-resistant germplasm resources obtained by conventional breeding not only have a very difficult process, but also have a long breeding period, so that an ideal resistant variety is difficult to breed. With the rapid development of modern molecular biology, a good opportunity is provided for solving the problem.

According to the accumulation characteristics and the genetic rule of the corn on lead, the mining of the related genes for lead absorption and transport by a molecular marker technology is a fundamental research and important component of strategy breeding aiming at the lead pollution of soil. At present, although research reports on lead adsorption characteristics and possible mechanisms of different corn materials and different tissues exist, no research on positioning of genes related to control of heavy metal lead in corn is reported

Disclosure of Invention

The invention overcomes the defects of the prior art and provides the corn gene ZmLIP2 and application thereof. The invention provides a corn gene ZmLIP2, which defines the relation between the corn ZmLIP2 gene and plant lead stress resistance and verifies the importance of the gene in the application of lead resistance of corn.

The nucleotide sequence of the maize gene ZmLIP2 is shown in SEQ ID No. 1.

Furthermore, the amino acid sequence of the maize gene ZmLIP2 is shown as SEQ ID NO. 2.

Furthermore, the maize gene ZmLIP2 is positioned between the maize chromosome 6 chr06.156,292,425-chr 06.156,294,916.

The application of the maize gene ZmLIP2 in improving the lead-resistant capability of plants.

A method of increasing lead tolerance in a plant, the method comprising: downregulating expression of maize gene ZmLIP2 in plant lines.

Further, the method comprises the following steps: constructing a recombinant vector RNAi-ZmLIP2, transferring RNAi-ZmLIP2 into maize immature embryos through agrobacterium mediation, reducing the expression quantity of maize gene ZmLIP2 in plant strains, and improving the lead-resistant capability of plants.

The application of the maize gene ZmLIP2 in cultivating plants with enhanced lead resistance.

A method of growing plants with increased lead tolerance comprising the steps of: constructing a recombinant vector RNAi-ZmLIP2, transferring RNAi-ZmLIP2 into maize immature embryos or arabidopsis thaliana through agrobacterium mediation to obtain transgenic plants with enhanced lead resistance, and obtaining T₀Plant generation and selfing to obtain T₁And (5) replacing plants to obtain plants with enhanced lead resistance.

The application also protects a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing the maize gene ZmLIP 2.

Has the advantages that:

(1) the invention researches the function of the corn gene ZmLIP2, defines the relation between the corn gene ZmLIP2 and the lead resistance of plants for the first time, and lays a theoretical basis for the application of the gene in improving the lead resistance of the plants.

(2) The invention utilizes agrobacterium-mediated transgenic technology to transform ZmLIP2 gene of corn and obtains RNAi interference transformation event of ZmLIP2 gene of corn. The expression level analysis of the plants interfered by RNAi shows that the expression level is obviously reduced compared with that of receptor materials. Lead stress is carried out on the ZmLIP2 gene interference strain, compared with a wild type, the growth condition of corn under the lead stress is enhanced at the root system level and the leaf blade level, and the lead resistance of the strain is enhanced.

(3) The research of the invention shows that the corn ZmLIP2 gene can negatively regulate and control the absorption and the transportation of the plant to lead, the relation between the corn ZmLIP2 gene and the lead stress resistance of the plant is firstly determined, and the importance of the gene in the lead resistance application of the corn is verified. Provides theoretical basis and practical value for the application of the gene in corn and other plants and the improvement of stress resistance. The corn ZmLIP2 gene has wide application prospect in the field of heavy metal pollution resistance of plants, particularly in the field of soil lead toxicity resistance of corn, and has great economic benefit potential.

(4) The invention provides a method for enhancing lead resistance of plants by using transgenic ZmLIP2, cultivating lead-resistant plants, reducing accumulation of heavy metal lead in plants and reducing toxic action on plants.

Drawings

FIG. 1 is a map of expression elements used in RNAi intervention lines.

FIG. 2 shows the relative gene expression levels of ZmLIP2 gene RNAi strains using qRT-PCR.

FIG. 3 is a photograph showing the phenotype of the RNAi interference strain and the non-transgenic negative control after one week of non-treatment control and lead treatment (3 mmol/L).

FIG. 4 shows the plant phenotype data of RNAi intervention lines and wild type controls after lead treatment (3mmol/L) and non-treatment.

FIG. 5 shows the lead content of the plants after lead treatment (3mmol/L) for the RNAi intervention lines and the wild type control.

Detailed Description

In order to make the technical solutions in the present application better understood, the present invention is further described below with reference to examples, which are only a part of examples of the present application, but not all examples, and the present invention is not limited by the following examples.

The present invention is further defined in the following examples, from which one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions; wherein the present invention introduces the above-mentioned expression vector into plant cells, methods of introduction are well known to those skilled in the art and include, but are not limited to: agrobacterium-mediated transformation, particle gun, electrical stimulation, ovary injection, etc. The selection marker gene used in the present invention is a bialaphos resistance gene (bar), and may further include other selection marker genes and reporter genes. The screening antibiotic selected by the invention is kanamycin, and besides, the screening antibiotic adopted by the invention is the prior art in the field.

Example 1 transcriptome analysis of maize inbred line 178 roots

1-1, lead stress treatment of maize inbred line material

The method comprises the steps of selecting full corn inbred line 178 seeds, disinfecting the seeds with 75% ethanol for 1min, soaking the seeds in 10% H2O2 solution for 15min, continuously shaking the seeds during disinfection, rinsing the seeds with deionized water for 3-5 times until residual H2O2 is rinsed, soaking the seeds in the deionized water for 4 hours, and finally germinating the seeds with filter paper at 28 ℃ in the dark. After about 2-3 days, the germinated corns are transferred to a floating plate, cultured by Hoagland nutrient solution under the conditions of light (16 h)/darkness (8h) at the temperature of 28 ℃ for water culture, the culture solution is changed twice a week, and 6 seedlings with consistent growth are selected in the three-leaf period for stress treatment. The 6 seedlings were transferred to a plastic container containing nutrient solution after endosperm removal. The experiment set up 2 treatments: normal water supply (i.e. complete nutrient, CK) and lead stress treatment (i.e. complete nutrient +3mmol/L Pb (NO3)2, T). The nutrient solution is prepared at present and the pH value is adjusted to about 6.0. And respectively taking root systems stressed for 0h, 24h, 48h and 72h to extract RNA, and carrying out transcriptome sequencing.

1-2 extraction of corn total RNA

The method in the RNA extraction reference Trizol kit comprises the following specific steps: (1) cooling the grinder with liquid nitrogen: quickly putting the weighed materials into a mortar and quickly grinding until the materials are ground into superfine powder; (2) adding Trizol into a mortar according to the dosage of 1ml per 0.1 g of material; (3) after 20 minutes, the mortar is disassembled and continuously ground until Trizol in the mortar is transparent, and the Trizol is subpackaged into 2ml centrifuge tubes; (4) adding 300 μ l of chloroform into the centrifuge tube, reversing for 1 minute, mixing thoroughly, standing for 5 minutes, centrifuging for 15min (4 ℃, 12000rpm), and carefully sucking the supernatant into another 2ml centrifuge tube; (5) repeating the step 4, adding chloroform and the subsequent steps, sucking the supernatant again and transferring the supernatant into another 1.5ml centrifuge tube; (6) after the supernatant was aspirated again, total RNA was extracted using Trizol partner of beijing tianenz gene technology ltd. (7) After the RNA precipitate is dried, adding a proper amount of DEPC treatment water to dissolve.

1-3 obtaining of cDNA (Using Takara Co., Ltd.)

reagent Kit (Perfect RealTime)), wherein the reaction system and procedures are as followsThe following:

(1) removal reaction of genomic DNA (Total RNA amount: maximum 1. mu.L)

(2) Reverse transcription reaction, the reaction system is shown below

1-4, spatiotemporal expression Pattern analysis of ZmLIP2 Gene

The expression of ZmLIP2 gene under lead stress condition was relatively quantitatively analyzed by fluorescent quantitative PCR using cDNA from 1-3 treated samples as template and GAPDH as reference gene. The ZmLIP2 gene has the nucleotide sequence shown in sequence 3 and the amino acid sequence of the coded protein shown in sequence 4.

Wherein the reaction system comprises the following steps:

TABLE 3 qRT-PCR amplification System

The reaction procedure is as follows: 30s at 95 ℃; 95 ℃ for 5s, Tm (optimal annealing temperature of primers) for 30s, 40 cycles; 10s at 95 ℃; melting curves were plotted at 65 ℃ for 5s and 65 ℃ to 95 ℃. The sequences of the gene quantitative PCR primers used are shown in the following table:

TABLE 4 qRT-PCR primers

After transcriptome sequencing is carried out on a corn root system under stress, compared with a control condition, the ZmLIP2 gene is a gene which is expressed by the corn root system in response to lead stress and is most obviously up-regulated.

Example 2 acquisition and analysis of ZmLIP2 intervention lines

In the embodiment, Ubi is used as a promoter, NOS is used as a terminator, bar is used as a selective marker gene, kanamycin is used as a selective marker gene, a target gene fragment primer is designed according to a ZmLIP2 sequence, a CUB vector is used as a framework, and a homologous recombination method is adopted to construct an interference vector of a ZmLIP2 gene by utilizing BamHI and Smal I double enzyme digestion. And transforming the constructed interference vector into agrobacterium-infected competent cells. The agrobacterium is infected with wild corn C01 and cultured to obtain T0 generation ZmLIP2 gene interference corn.

The method comprises the following specific steps:

2-1, extraction and purification of corn RNA

Total RNA of the inbred-line-C01 (hereinafter also referred to as wild-type maize) of maize was extracted by Trizol method, as described in reference to 1-2. First strand cDNA Synthesis references 1-3.

Cloning of 2-2, ZmLIP2 Gene

Using the reverse transcription cDNA as a template, and adopting the following primer pairs to carry out PCR amplification:

5'-ACGGATCCCACTAGTCTGGTCACGCTCCCC-3' as the upstream primer shown in SEQ ID NO. 5;

5'-ATCCCGGGGTATAAATTGTAATGACCATAC-3' as shown in SEQ ID NO. 6.

Wherein the forward primer is a BamH I site, the downstream primer is a SmaI site, the PCR amplification system is 2. mu.l of forward primer (5. mu. mol/. mu.L), 2. mu.l of downstream primer (5. mu. mol/. mu.L), 5. mu.l of 5 XPhusonHFbuffer (GC), 5. mu.l of dNTP mixture (2.5mmol/L), 0.25. mu.l of Phusion DNA polymerase, 1. mu.l of cDNA template, and ddH2O to make up the total volume to 11.75. mu.l, the amplification conditions are pre-denaturation at 98 ℃ for 30 seconds, denaturation at 98 ℃ for 10 seconds, annealing at 59 ℃ for 25 seconds, extension at 72 ℃ for 1min and 40 sec, circulation for 30 times, extension at 72 ℃ for 10 min, 4. mu.l of PCR product is ligated with pMD19-TSimple Vector, the procedure is carried out according to the instruction of TaKaRa product pMD19-T Sivector, then the ligation product is transformed into 5 α, the Escherichia coli is cultured in a liquid medium containing ampicillin (100mg of ampicillin), and the plasmid containing 100mg of ampicillin is cultured overnight.

2-3 construction of plant recombinant vector RNAi-ZmLIP2

RNA of maize inbred line C01 is extracted, cDNA is obtained by reverse transcription as a template, PCR amplification is carried out by using an upstream primer and a downstream primer to obtain a PCR amplification product (ZmLIP2 gene) of 1775bp, the reverse complementary fragment is subjected to double digestion by using restriction enzymes BamHI and Smal I, then the PCR amplification product is connected with a BamHI and Smal I double digestion pCUB expression vector large fragment, the operation steps are carried out according to the instruction of T4 DNA ligase of Fermentas company, then the connection product is transformed into Escherichia coli DH5 α competent cells, the Escherichia coli DH5 α competent cells are cultured overnight on a solid culture medium containing spectinomycin (50mg/L), white colonies are picked up, the LBA is cultured in LB liquid culture medium containing spectinomycin (50mg/L), the alkali method is used for extracting BamHI and carrying out digestion identification to obtain a gene fragment with the size of 1775bp as a positive plasmid, the LBA of the positive plasmid is named as a ZRNAi-mLIP 2, a partial structure is shown in a schematic diagram, a map, a ZmRNAi-464 transgenic agrobacterium is inserted into a Target gene region for expression of ZmLIP-464, and the ZmLIP-464 transgenic Agrobacterium strain can be obtained.

2-4 Agrobacterium-mediated transformation of maize immature embryos and acquisition of resistant plants

One day before the infection, activated Agrobacterium colony LBA4404/RNAi-ZmLIP2 (single Agrobacterium colony carrying recombinant plasmid) was picked and inoculated into liquid YEP medium containing 50mg/L rifampicin and 50mg/L spectinomycin, and shake-cultured at 28 ℃ and 220rpm overnight; and (3) putting the agrobacterium tumefaciens solution into an 80ml centrifuge tube, centrifuging at 6000rpm for 5 minutes, removing a supernatant, collecting a precipitate, resuspending the precipitate by using an AB induction culture solution, and culturing on a shaking table at 28 ℃ for at least 5 hours to perform precipitation and dip dyeing. Picking young wild corn (corn inbred line C01) embryos 10-12 days after pollination, putting the young wild corn embryos into a presoaking culture solution, centrifuging at 2700rpm for 5 seconds, and removing a culture medium; adding 2ml of presoaking culture medium for resuspension, and centrifuging at 2700rpm for 5 seconds; water bath at 46 ℃ for 3 minutes, ice bath for 1 minute, centrifuging at 2700rpm for 5 seconds, discarding the culture medium, adding 2ml of preimpregnation culture solution, centrifuging at 4 ℃ and 14000rpm for 10 minutes, and discarding the culture solution; transferring the bacterial liquid cultured by the AB induction culture medium into an 80ml centrifuge tube, centrifuging at 6000rpm for 5 minutes, collecting thalli, and resuspending the thalli by using a staining culture solution; adding 1ml of the bacterial liquid into a centrifugal tube filled with the immature embryos, gently and uniformly mixing for several times, standing for 5 minutes at room temperature, and removing the bacterial liquid; transferring the young embryo to a co-culture medium, performing dark culture at 28 ℃ for one week, transferring to a screening medium, and performing subculture once every two weeks; screening twice, transferring to a differentiation culture medium, transferring the differentiated seedling to a rooting culture medium, and hardening the seedling after the seedling grows to have more than 3 robust roots; hardening seedlings for 7 days, and then moving to a greenhouse for planting to obtain T0 generation interference ZmLIP2 corns. The preinfection medium in this example was MS medium supplemented with 1.5mg/L of 2,4-D (Murashige and Skoog,1962), and the screening medium was N6 supplemented with 2.0mg/L of 2,4-D, 2,4-D being 2, 4-dichlorophenoxyacetic acid.

2-5, molecular identification of Zea mays interfering with ZmLIP2 gene

Extracting T0 generation interference ZmLIP2 corn plant genome DNA by adopting a CTAB method (Sambrook and Russell, molecular cloning experimental guidance, 2001), and respectively designing primer detection marker genes bar and Ubi-LIP2 fusion fragments, wherein the sequences of the primer pairs are as follows:

the marker gene bar gene primer pair is as follows:

an upstream primer: 5'-GTGGAGAGGCTATTCGGCTATGACTG-3', as shown in SEQ ID NO. 7;

a downstream primer: 5'-AGCTCTTCAGCAATATCACGGGTAGC-3', as shown in SEQ ID NO. 8.

The length of a marker gene bar amplification sequence is 650bp, and the amplification conditions are as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30 seconds, annealing at 59 ℃ for 30 seconds, extension at 72 ℃ for 40 seconds, and circulation for 35 times; extension at 72 ℃ for 10 min. The primer pair of the Ubi-LIP2 fusion fragment (the upstream primer is a promoter region sequence in an expression vector, and the downstream primer is a ZmLIP2 gene sequence) is as follows:

an upstream primer: 5'-GCCTTCATACGCTATTTATTTGCT-3', as shown in SEQ ID NO. 9;

a downstream primer: 5'-GGCTTTACACTTTATGCTTCCG-3', as shown in SEQ ID NO. 10.

The length of the target fragment amplification sequence is 550bp, and the amplification conditions are as follows: the amplification conditions were: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30 seconds, annealing at 61 ℃ for 30 seconds, extension at 72 ℃ for 40 seconds, and circulation for 35 times; extension at 72 ℃ for 10 min.

The empty vector pCUB:: UBI was transferred to wild type maize in the same manner to obtain T0 generation of transgenic pCUB:: UBI maize. The result of the detection by the same method is not obviously different from that of the wild corn. And (4) sowing the T0 generation plants, and selfing to obtain T1 generation plants.

2-6, carrying out lead stress on the positive transgenic interference plant and analyzing the phenotype of the positive transgenic interference plant

The method comprises the steps of selecting plump seeds of ZmLIP2 interference lines of T1 generations, treating the seeds by a method of 1-1, sampling each material 14 days after lead treatment, and performing RNA extraction, phenotype identification and lead content determination. Wherein, the RNA extraction method refers to 1-2, MAS6 is used for digestion of samples, and an inductively coupled plasma MASs spectrometer is used for determination of lead content.

The results of the expression analysis of the positive transgenic plants of the wild type and the intervention strain after lead treatment are shown in FIG. 2, wherein WT represents wild type control, RNAi-1 and RNAi-2 represent two transformation events of the intervention strain, and the significant reduction of the expression of the intervention strain can be seen. Phenotype pictures of wild type control and the intervention strain before and after lead treatment are shown in fig. 3, and non-transgenic negative control and the intervention strain of the non-lead treatment group and non-transgenic negative control and the intervention strain of the lead treatment group are respectively shown from left to right in fig. 3. As can be seen from the figure, under normal growth conditions, the growth vigor of the interference strain is not obviously different from that of the non-transgenic negative control, and the root length, the leaf size and the plant height of the interference strain are not obviously different. Under lead treatment, the leaves of the overground part of the interference strain plants are longer than the wild type, the root branches are more than the wild type, and the plant height and the root length are longer than the wild type. The interference strain plant can reduce the lead stress toxicity of the corn plant and ensure the normal growth of the plant. From this, it can be seen that the ZmLIP2 gene has a negative regulatory effect. The interference plant can obviously enhance the resistance of the corn seedling stage to lead stress and is induced by the lead stress. As shown in FIG. 4, A, B, C in FIG. 4 shows the growth conditions of plant height, fresh weight of leaves, and fresh weight of roots, respectively. It can also be seen from fig. 4 that there was no significant difference in plant height, fresh leaf weight, and fresh root weight of wild type, the intervention lines, when treated with no lead. And when the lead is treated, the damage of the interference strain caused by the lead stress is obviously lower than that of the wild type. This also illustrates the negative regulatory effect of the gene. The lead contents of aerial parts and roots at the seedling stage were measured, and the results are shown in FIG. 5, from which it can be seen that the lead contents of RNAi intervention lines were generally lower than those of wild type plants (WT) regardless of aerial parts or roots. The results of the expression quantity, the phenotype picture and the lead content are combined to obtain that the ZmLIP2 gene interference strain can reduce the accumulation of heavy metal lead in the plant body, thereby reducing the toxicity on the plant. This further illustrates the important role of the ZmLIP2 gene in negative regulation of lead stress in maize.

While the invention has been described in detail in the foregoing by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that certain modifications and improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

SEQUENCE LISTING

<110> Sichuan university of agriculture

<120> corn gene ZmLIP2 and application thereof

<130>2020

<160>10

<170>PatentIn version 3.3

<210>1

<211>2492

<212>DNA

<213> sequence

<400>1

ccatgtataa attgtaatga ccatacgaaa aacaaattaa agtgcaaaat gtgtaaccca 60

tcagtatgtc atagctcaca tttttacatg cttggcgtca tcacaaaaaa aaagattact 120

cacttgtgaa caccccaatt taactgccac aaccaccaag tccaagtggc tccaaggtcc 180

cacatattga gctccgtaca cgccaagtgc acgaagccac ttcatctgcg tggcccttcc 240

acgtttgaag ccttcgaagc tgaacacacc cgcccttatc attgctcctc ggtttcctcc 300

tcctcgttat cttcgtcgtc gtctgaagaa gaagatgacg aggagtctga atggtccgat 360

tctttgtctt ccttcggctc cggggcggcg gcaggcggtg gagcggcagg ggccggtggg 420

tcaggcggca tcgccgtgag gaagcctagc gtcatgacga cgtcggccat cagcggtcgc 480

acggagtcct cttcctgcaa gcacatcgca gccatggcga ccacctggtt caacgctttg 540

gcagggtatt ccttctttat gagtggatca accagctcat ggtatctctt ctggtccttg 600

aacatcggca tggcctgaaa atgtgtcgtg cagatgcatg ccatgccatg ccatgccaat 660

ataatgcagc tcattagcaa aagcaacgtt gaagaagcag caaaaacgcg tagcttataa 720

tacttaccca ggtgacaacg ttctgctcgt ccaccggctt gctggtatcg acggccctcc 780

tcccggagat gagctgcacc agcaccaccc cgaagctgta cgcgtcggac ttcatggtgg 840

cctgccctgt gcgatcgtac tccggcgcgc agcagccgaa ggagcccatc atgggcgacg 900

ccaccaccgg catgctgccc ccggcctggc cgagctgcgc gagcccgaag tcggagagct 960

tgggcgtgag cttctcgtcg agaagtatgt gggaggcctt gaattccccg tacaccaccg 1020

gcgggttcgc tgtctcgtgc aggtactcca gcccctgcgc ggcgccgtgc gccaccttca 1080

tcctcgtgca ccagtccatc ggcttcttgt ccgccggcag atctggcgcg cgccggcagc 1140

ttgttcagca gagacataga cgacgaaact gacaaattct gcaggagctt gccagttgcc 1200

agataccggc gtgcacgtac gtacgtaccg aacaggtggt cttctaaggt gccggcgggc 1260

acggactcgt acaccagcag ccgctggtct ccgtcagcgc agtagccaat gatgtcgacg 1320

aggttctcgt ggtggagctg gctgagcttg gcgaccccgg tcaagaacgc cttgttgtcc 1380

tggaacccat gcttgtccag ctgcttgatg gccacggtct ggagcaaaag tagcagaggc 1440

acgccatgca tcacacattt cattcagaga gacgcatgga cgacgacacg tgaggcgtgg 1500

cagttcacgt acgtacctgc ccgctcttct ctaaccggcc cttgtagacc cggaagaagc 1560

cgccttcacc gacgaggttg tacggcgtga agtggtctgt ggcggcggcg agctcgcgga 1620

acgcgaacgc ctgccccgtg atggcgagcc gcagcgccga gtcatcggcg gaagcctcgt 1680

gcgcctggtt ccctgcatcg cgagatcaaa tcaaattcgt tgggcgttgc aggcgcgacg 1740

agatttggac gtgctaagcg agatgcatga agaaaccgat cgaagcagag agggccatct 1800

caccgccacc gggcttgggc ggcgccgcgg ccgttggagc ggcggcggga gctgggaagt 1860

acgcggcggg cgcctgcacc atcgcgggtg gcgtcatgtt agacgccggg gccgcgtcgc 1920

ccgggctctc cgcgtcggcg tcgtcgttgt tgccctttcc gccgccgaag cacggaaagc 1980

agctcatctt ccttttccct ctgcgcctcc ccgtggcggc gaccctgtgg acgcggggcg 2040

agcggcgtgg gcgcgagcga gcgacgagcc gatgactcga caggggcaag aatgctgggc 2100

acgcacgcac gcacggacgg accgagccgc cggccgccgt cactgcgcgc cgaccatgcc 2160

ccggcgtctc agcatcgacg cggcgtctcc acttcgtgga acgccatgcc cgagctcgcc 2220

gcacgcgcac caaatgaaac ccgcccgtcc ggtgcggagc aggggagcgt gaccagacta 2280

gtggtggctt gctcaggaga ccggagaggg gcacaagacc accacgagac gagaacgacg 2340

gtcgatggct atggcaatgg aaggcaggcc ttggagatcg agagatccaa gggccttccc 2400

agagggaggg gagaccggag gaggacaccg ataccggtgg aggggaggcg ggaggagagg 2460

gatgctaagt ttggcgcgtg gagtgaatcg ag 2492

<210>2

<211>426

<212>PRT

<213> sequence

<400>2

Met Ser Cys Phe Pro Cys Phe Gly Gly Gly Lys Gly Asn Asn Asp Asp

1 5 10 15

Ala Asp Ala Glu Ser Pro Gly Asp Ala Ala Pro Ala Ser Asn Met Thr

20 25 30

Pro Pro Ala Met Val Gln Ala Pro Ala Ala Tyr Phe Pro Ala Pro Ala

35 40 45

Ala Ala Pro Thr Ala Ala Ala Pro Pro Lys Pro Gly Gly Gly Asn Gln

50 55 60

Ala His Glu Ala Ser Ala Asp Asp Ser Ala Leu Arg Leu Ala Ile Thr

65 70 75 80

Gly Gln Ala Phe Ala Phe Arg Glu Leu Ala Ala Ala Thr Asp His Phe

85 90 95

Thr Pro Tyr Asn Leu Val Gly Glu Gly Gly Phe Phe Arg Val Tyr Lys

100 105 110

Gly Arg Leu Glu Lys Ser Gly Gln ThrVal Ala Ile Lys Gln Leu Asp

115 120 125

Lys His Gly Phe Gln Asp Asn Lys Ala Phe Leu Thr Gly Val Ala Lys

130 135 140

Leu Ser Gln Leu His His Glu Asn Leu Val Asp Ile Ile Gly Tyr Cys

145 150 155 160

Ala Asp Gly Asp Gln Arg Leu Leu Val Tyr Glu Ser Val Pro Ala Gly

165 170 175

Thr Leu Glu Asp His Leu Phe Asp Leu Pro Ala Asp Lys Lys Pro Met

180 185 190

Asp Trp Cys Thr Arg Met Lys Val Ala His Gly Ala Ala Gln Gly Leu

195 200 205

Glu Tyr Leu His Glu Thr Ala Asn Pro Pro Val Val Tyr Gly Glu Phe

210 215 220

Lys Ala Ser His Ile Leu Leu Asp Glu Lys Leu Thr Pro Lys Leu Ser

225 230 235 240

Asp Phe Gly Leu Ala Gln Leu Gly Gln Ala Gly Gly Ser Met Pro Val

245 250 255

Val Ala Ser Pro Met Met Gly Ser Phe Gly Cys Cys Ala Pro Glu Tyr

260 265 270

Asp Arg Thr Gly Gln Ala Thr Met Lys Ser AspAla Tyr Ser Phe Gly

275 280 285

Val Val Leu Val Gln Leu Ile Ser Gly Arg Arg Ala Val Asp Thr Ser

290 295 300

Lys Pro Val Asp Glu Gln Asn Val Val Thr Trp Ala Met Pro Met Phe

305 310 315 320

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

325 330 335

Glu Tyr Pro Ala Lys Ala Leu Asn Gln Val Val Ala Met Ala Ala Met

340 345 350

Cys Leu Gln Glu Glu Asp Ser Val Arg Pro Leu Met Ala Asp Val Val

355 360 365

Met Thr Leu Gly Phe Leu Thr Ala Met Pro Pro Asp Pro Pro Ala Pro

370 375 380

Ala Ala Pro Pro Pro Ala Ala Ala Pro Glu Pro Lys Glu Asp Lys Glu

385 390 395 400

Ser Asp His Ser Asp Ser Ser Ser Ser Ser Ser Ser Asp Asp Asp Glu

405 410 415

Asp Asn Glu Glu Glu Glu Thr Glu Glu Gln

420 425

<210>3

<211>20

<212>DNA

<213> Artificial sequence

<400>3

gacaagaagc cgatggactg 20

<210>4

<211>20

<212>DNA

<213> Artificial sequence

<400>4

gccttgaatt ccccgtacac 20

<210>5

<211>30

<212>DNA

<213> Artificial sequence

<400>5

acggatccca ctagtctggt cacgctcccc 30

<210>6

<211>30

<212>DNA

<213> Artificial sequence

<400>6

atcccggggt ataaattgta atgaccatac 30

<210>7

<211>26

<212>DNA

<213> Artificial sequence

<400>7

gtggagaggc tattcggcta tgactg 26

<210>8

<211>26

<212>DNA

<213> Artificial sequence

<400>8

agctcttcag caatatcacg ggtagc 26

<210>9

<211>24

<212>DNA

<213> Artificial sequence

<400>9

gccttcatac gctatttatt tgct 24

<210>10

<211>22

<212>DNA

<213> Artificial sequence

<400>10

ggctttacac tttatgcttc cg 22

Claims

1. The maize gene ZmLIP2 is characterized in that the nucleotide sequence is shown as SEQ ID No. 1.

2. The maize gene ZmLIP2 of claim 1, located between chr06.156,292,425 and chr06.156,294,916 of maize chromosome 6.

3. Use of the maize gene ZmLIP2 as defined in any one of claims 1-2 for increasing lead tolerance in plants.

4. A method of increasing lead tolerance in a plant, the method comprising: downregulating expression of maize gene ZmLIP2 in plant lines.

5. The method of claim 4, comprising the steps of: constructing a recombinant vector RNAi-ZmLIP2, transferring RNAi-ZmLIP2 into maize immature embryos through agrobacterium mediation, reducing the expression quantity of maize gene ZmLIP2 in plant strains, and improving the lead-resistant capability of plants.

6. Use of the maize gene ZmLIP2 as defined in any one of claims 1-2 for breeding plants with increased lead tolerance.

7. A method of growing plants with increased lead tolerance comprising the steps of: constructing a recombinant vector RNAi-ZmLIP2, transferring RNAi-ZmLIP2 into maize immature embryos or arabidopsis thaliana through agrobacterium mediation to obtain transgenic plants with enhanced lead resistance, and obtaining T₀Plant generation and selfing to obtain T₁And (5) replacing plants to obtain plants with enhanced lead resistance.

8. A recombinant vector, expression cassette, transgenic cell line or recombinant bacterium comprising the maize gene ZmLIP2 of any of claims 1-2.