CN111718914B - Application of protein ZmTIP1 in regulation and control of plant drought resistance - Google Patents

Abstract

The invention discloses application of protein ZmTIP1 in regulation and control of plant drought resistance. The invention provides an application of ZmTIP1 protein or related biological materials thereof in regulating and controlling plant drought resistance; the ZmTIP1 protein is protein shown in SEQ ID No.1 or SEQ ID No.2 or protein which is substituted and/or deleted and/or added by one or more amino acid residues, or protein which has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% of homology in sequence and has the same function, or fusion protein which is obtained by connecting labels at the N end and/or the C end. The ZmTIP1 is transferred into Arabidopsis or corn to obtain transgenic plant with drought resistance higher than that of un-transgenic acceptor plant, and this shows that ZmTIP1 and its coded protein have drought resistance function and important significance in raising plant drought resistance.

Description

Application of protein ZmTIP1 in regulation and control of plant drought resistance

Technical Field

The invention relates to the technical field of biology, in particular to application of protein ZmTIP1 in regulation and control of plant drought resistance.

Background

During the whole growth period of the plants, factors influencing the normal growth and development of the plants are many, wherein drought is a main stress factor influencing and limiting the growth and development of the plants, and the plants die in a large area and even die in failure in severe cases. At present, the global cultivated land area is gradually reduced and the drought disasters are more and more frequent, and the improvement or the maintenance of the grain yield by improving the drought resistance of crops has great significance. The cultivation of new varieties of drought-resistant crops is one of the main targets of research in the technical field of agricultural science.

The traditional breeding technology has the defects of long period, high blindness, large workload and the like, and the improvement of the grain yield by the traditional breeding has developed to a certain bottleneck. In recent years, with the development of the subjects such as plant molecular biology and genetics and the intensive research on the molecular mechanism of plant stress resistance, the improvement of the stress resistance of crops by introducing stress resistance-related genes into plants by genetic engineering methods has become increasingly mature.

Corn (Zea mays L.) is used as an important grain crop widely planted in China, and has important significance in cloning corn drought resistance genes, improving corn drought resistance and improving corn yield.

Disclosure of Invention

The invention aims to provide application of protein ZmTIP1 in regulation and control of plant drought resistance.

In a first aspect, the invention claims the use of a zmtpip 1 protein or its related biomaterials in modulating drought resistance in plants.

The relevant biological material may be a nucleic acid molecule capable of expressing the ZmTIP1 protein or an expression cassette, recombinant vector, recombinant bacterium, or transgenic cell line containing the nucleic acid molecule.

The ZmTIP1 protein is any one of the following proteins:

(A1) protein with an amino acid sequence of SEQ ID No.1 or SEQ ID No. 2;

(A2) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID No.1 or SEQ ID No.2 and has the same function;

(A3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the amino acid sequence defined in any one of (A1) to (A2) and having the same function;

(A4) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).

In a second aspect, the invention claims the use of the ZmTIP1 protein or its related biomaterials for modulating plant root hair development.

The related biological material is a nucleic acid molecule capable of expressing the ZmTIP1 protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule. The ZmTIP1 protein is any one of the proteins shown in the above (A1) - (A4).

In the foregoing first and second aspects, the activity and/or expression level of the zmtpip 1 protein or its encoding gene in the plant is increased, and drought resistance of the plant is increased; the activity and/or expression level of the ZmTIP1 protein or the coding gene thereof in the plant is reduced, and the drought resistance of the plant is reduced. The activity and/or expression level of the ZmTIP1 protein or the coding gene thereof in the plant is increased, and the length of the root hair of the plant is increased; the activity and/or expression of the ZmTIP1 protein or the coding gene thereof in the plant is reduced, and the length of the root hair of the plant is shortened.

In a third aspect, the invention claims the use of a ZmTIP1 protein or related biological material for the preparation of a product having palmitoyl transferase activity.

The related biological material is a nucleic acid molecule capable of expressing the ZmTIP1 protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule. The ZmTIP1 protein is any one of the proteins shown in the above (A1) - (A4).

In a fourth aspect, the invention claims a method of breeding a plant variety, method a or method B or method C or method D:

the method A comprises the following steps: a method of breeding a plant variety with increased drought resistance may comprise the step of increasing the expression level and/or activity of zmtpip 1 protein in a recipient plant. The ZmTIP1 protein is any one of the proteins shown in the above (A1) - (A4).

The method B comprises the following steps: a method of breeding a plant variety with reduced drought resistance may comprise the step of reducing the expression level and/or activity of zmtpip 1 protein in a recipient plant. The ZmTIP1 protein is any one of the proteins shown in the above (A1) - (A4).

The method C comprises the following steps: a method of growing a plant variety with increased root hair length comprising the step of increasing the expression level and/or activity of zmtpip 1 protein in a recipient plant. The ZmTIP1 protein is any one of the proteins shown in the above (A1) - (A4).

The method D comprises the following steps: a method of growing a plant variety with reduced root hair length comprising the step of reducing the expression level and/or activity of zmtpip 1 protein in a recipient plant. The ZmTIP1 protein is any one of the proteins shown in the above (A1) - (A4).

Further, the present invention claims a method for breeding transgenic plants, method E or method F or method G or method H:

the method E comprises the following steps: a method of breeding transgenic plants with increased drought resistance comprising the steps of: introducing a nucleic acid molecule capable of expressing ZmTIP1 protein into a receptor plant to obtain a transgenic plant; the transgenic plant has increased drought resistance compared to the recipient plant. The ZmTIP1 protein is any one of the proteins shown in the above (A1) - (A4).

Method F: a method of breeding a transgenic plant with reduced drought resistance, comprising the steps of: inhibiting and expressing the encoding gene of ZmTIP1 protein in a receptor plant to obtain a transgenic plant; the transgenic plant has reduced drought resistance compared to the recipient plant. The ZmTIP1 protein is any one of the proteins shown in the above (A1) - (A4).

Method G: a method of growing a transgenic plant with increased root hair length, comprising the steps of: introducing a nucleic acid molecule capable of expressing ZmTIP1 protein into a receptor plant to obtain a transgenic plant; the transgenic plant has an increased root hair length compared to the recipient plant. The ZmTIP1 protein is any one of the proteins shown in the above (A1) - (A4).

Method H: a method of growing a transgenic plant with reduced root hair length, comprising the steps of: inhibiting and expressing the encoding gene of ZmTIP1 protein in a receptor plant to obtain a transgenic plant; the transgenic plant has a reduced root hair length compared to the recipient plant. The ZmTIP1 protein is any one of the proteins shown in the above (A1) - (A4).

In a fifth aspect, the invention claims a method for phenotypic reversion of a deletion mutant of palmitoyl transferase.

The method for phenotype reversion of the palm acyltransferase deletion mutant provided by the invention can comprise the step of introducing a nucleic acid molecule capable of expressing ZmTIP1 protein into the palm acyltransferase deletion mutant. The ZmTIP1 protein is any one of the proteins shown in the above (A1) - (A4).

Wherein, the palm acyltransferase deletion mutant can be a palm acyltransferase deletion yeast mutant or a palm acyltransferase deletion plant mutant (such as an Arabidopsis thaliana mutant). In one embodiment of the invention, the yeast mutant deficient in palmitoyl transferase is specifically yeast palmitoyl transferase gene-deficient mutant strain akr 1; the plant mutant with the palm acyltransferase deletion is specifically an Arabidopsis thaliana palm acyltransferase gene-deficient mutant tip 1-3.

In each of the methods, introduction of a nucleic acid molecule capable of expressing the ZmTIP1 protein into a recipient plant can be achieved by introducing a recombinant expression vector containing a gene encoding the ZmTIP1 protein into the recipient plant.

The recombinant expression vector can be constructed by using the existing plant expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector for plant microprojectile bombardment and the like. Such as pROKII, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Co., Ltd.), etc. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The polyadenylation signal can direct polyadenylation to the 3 'end of the mRNA precursor, and untranslated regions transcribed from the 3' end of Agrobacterium crown gall inducible (Ti) plasmid genes (e.g., nopalin synthase Nos), plant genes (e.g., soybean storage protein genes) all have similar functions. When the gene is used to construct a recombinant plant expression vector, any enhanced promoter (such as cauliflower mosaic virus (CAMV)35S promoter, maize Ubiquitin promoter (Ubiquitin), constitutive promoter or tissue-specific expression promoter (such as seed-specific expression promoter) can be added before the transcription initiation nucleotide, and can be used alone or in combination with other plant promoters Plant cells or plants are identified and selected, and the plant expression vector used is processed, for example, by adding a gene encoding an enzyme or a luminescent compound which can produce a color change (GUS gene, luciferase gene, etc.), a marker gene for antibiotics (e.g., nptII gene conferring resistance to kanamycin and related antibiotics, bar gene conferring resistance to phosphinothricin herbicide, hph gene conferring resistance to hygromycin antibiotic, and frdh gene conferring resistance to methatrexate, EPSPS gene conferring resistance to glyphosate) which can be expressed in plants, or a marker gene for chemical resistance (e.g., herbicide resistance), mannose-6-phosphate isomerase gene providing the ability to metabolize mannose.

In the present invention, when the receptor is a plant, the promoter for promoting transcription of the encoded gene in the recombinant expression vector is a 35S promoter or a Zmubi1 promoter. More specifically, the recombinant vector is a recombinant plasmid (named pGZ) obtained by inserting the gene encoding the ZmTIP1 protein into a multiple cloning site (such as Sma I and Sac I, and located at the downstream of a 35S promoter) of a pGKX vector. Or the recombinant vector is a recombinant plasmid (named pSBIII-ZmTIP 1) obtained by inserting the encoding gene of the ZmTIP1 protein into a multiple cloning site (such as Sma I, positioned at the downstream of Zmubi1 promoter) of the pSB11 vector and recombining with the pSB1 vector^MO17). When the receptor is yeast, the recombinant font is specifically a recombinant plasmid obtained by inserting the encoding gene of the ZmTIP1 protein into the multiple cloning site (such as Hind III and BamH I) of a pYES2/NTA vector.

In the above method, the introduction of the recombinant expression vector into the recipient plant may specifically be: plant cells or tissues are transformed by conventional biological methods using Ti plasmids, Ri plasmids, plant viral vectors, direct DNA transformation, microinjection, conductance, agrobacterium mediation, etc., and the transformed plant tissues are grown into plants. Transformed cells, tissues or plants are understood to comprise not only the end product of the transformation process, but also transgenic progeny thereof.

In the above aspects, the "nucleic acid molecule capable of expressing the ZmTIP1 protein" is a gene encoding the ZmTIP1 protein.

Further, the encoding gene of the ZmTIP1 protein can be any one of the following DNA molecules:

(B1) a DNA molecule shown as SEQ ID No.3 or SEQ ID No. 4;

(B2) a DNA molecule that hybridizes under stringent conditions to the DNA molecule defined in (B1) and encodes the ZmTIP1 protein;

(B3) a DNA molecule which has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% homology with the DNA sequence limited by (B1) or (B2) and encodes the ZmTIP1 protein.

In the above genes, the stringent conditions may be as follows: 50 ℃ in 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in2 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing at 50 ℃ in 1 XSSC, 0.1% SDS; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; can also be: in a solution of 6 XSSC, 0.5% SDS at 65 ℃ and then washed once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

In the present invention, the drought resistance may be embodied in any one of:

1) the transgenic plant has a root hair length longer than the recipient plant under drought stress;

2) the seedling survival rate of the transgenic plant under drought stress is higher than that of the receptor plant;

3) the transgenic plant has higher yield than the recipient plant under drought stress.

In each of the above aspects, the plant may be a monocot or a dicot;

further, the monocotyledon may be a gramineae plant; the dicotyledonous plant can be a crucifer;

still further, the gramineous plant may be corn; the cruciferous plant may be arabidopsis thaliana.

Experiments prove that the gene ZmTIP1 from corn (Zea mays L.) is transferred into arabidopsis thaliana or corn to obtain transgenic plants, the drought resistance of the transgenic plants is higher than that of receptor plants which are not transgenic, and the ZmTIP1 and the protein coded by the same have the function of drought resistance and have important significance in breeding and research for improving the drought resistance of plants.

Drawings

FIG. 1 shows palmitoyl transferase activity of ZmTIP1 protein encoded by different haplotypes. A is ZmTIP1 which can complement the phenotype of yeast palmitoyl transferase gene-deficient mutant; b is a phenotype that ZmTIP1 protein coded by two genotypes can complement arabidopsis tip1-3 root hair development defects; c is 35S, ZmTIP1/tip1-3 transgenic Arabidopsis root hair length statistics. 11, 13, 17 and 4, 9, 24 in B and C represent different transformation events or transgenic lines.

FIG. 2 is T₃Transfer the root hair development and drought phenotype of ZmTIP1 Arabidopsis plants. A is the length observation of root hair of ZmTIP1 transgenic Arabidopsis thaliana under simulated drought conditions with PEG8000 of different concentrations for 35S; b is a schematic diagram of a measuring region of the length of the root hair; c is fluorescence quantitative PCR detection 35S, relative expression quantity of ZmTIP1 transgenic Arabidopsis thaliana ZmTIP1 gene; d is the root hair length statistics of ZmTIP1 transgenic Arabidopsis under the simulated drought condition of PEG8000 with different concentrations for 35S; e is 35S, ZmTIP1 transgenic arabidopsis drought phenotype; f is 35S, and the survival rate of the ZmTIP1 transgenic arabidopsis is counted after rehydration.

FIG. 3 is T₃The generation transferred the root hair development phenotype of ZmTIP1 maize and ZmTIP1 mutant. A is Ubi: length observation of ZmTIP1 transgenic maize; b is fluorescence quantitative PCR detection Ubi, relative expression quantity of ZmTIP1 transgenic corn ZmTIP1 gene; c is Ubi, ZmTIP1 transgenic corn root hair length statistics; d is the genotype identification of ZmTIP1 mutant; e is RT-PCR to identify the expression quantity of ZmTIP1 gene in the ZmTIP1 mutant; f is root hair length observation of ZmTIP1 mutant; g is the root hair length statistics for ZmTIP1 mutant.

FIG. 4 is T₃Survival rate statistics results of the ZmTIP 1-transferred corn and the ZmTIP1 mutant after drought treatment and rehydration. A is Ubi, ZmTIP1 drought phenotype analysis of transgenic corn; b is drought phenotype analysis of ZmTIP1 mutant; c is Ubi, the survival rate of the transgenic corn is counted after the transgenic corn is rehydrated by ZmTIP 1;d is the survival rate statistics of the ZmTIP1 mutant after rehydration.

FIG. 5 is T₃Statistics of field drought phenotypes of transgenic ZmTIP1 maize and ZmTIP1 mutants. A is Ubi, ZmTIP1 transgenic corn plant height statistics under different drought treatment conditions in the field; b is Ubi, ZmTIP1 transgenic corn pollen dispersing time statistics under different drought treatment conditions in the field; c is Ubi, statistics of the interval time between the pollen scattering and the silking of ZmTIP1 transgenic corn under different drought treatment conditions in the field; d is Ubi, namely statistics of yield of ZmTIP1 transgenic corn single plants under different drought treatment conditions in the field; e is the statistics of the plant height of the ZmTIP1 mutant under the conditions of normal field watering and drought treatment; f is statistics of the powder scattering time of the ZmTIP1 mutant under the conditions of normal field watering and drought treatment; g is the statistics of the interval time between powder scattering and silk throwing; h is the statistics of the yield of the single ZmTIP1 mutant plant under the conditions of normal field watering and drought treatment.

FIG. 6 is a subcellular localization observation of ZmTIP1 protein. A is 35S, ZmTIP1-GFP transgenic arabidopsis subcellular localization observation; b is ZmTIP1 protein located in Golgi body, trans-Golgi network and vacuole precursor.

FIG. 7 shows the result that ZmTIP1 protein regulates the subcellular localization of ZmCPK28 protein. A is ZmCPK28 protein and its mutant ZmCPK28-C3A-GFP, ZmCPK28-C4A-GFP and ZmCPK28-C3&4A-GFP, and is used for subcellular localization observation in corn protoplast. B is Western Blot for detecting subcellular localization of ZmCPK28-GFP and ZmCPK28-C3&4A-GFP proteins in corn protoplasts (T represents total protein; S represents cytoplasmic protein; P represents membrane protein); c is co-immunoprecipitation of ZmCPK28-Myc in maize protoplasts with ZmTIP 1-GFP.

In each figure, indicates a significant difference at P < 0.05 compared to WT results; indicates that the difference was very significant at P < 0.01 compared to WT results.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The vectors pGKX and pGK-CsGFP: described in the document "Qin F, Sakuma Y, Tran LS, Maruyama K, Kidokoro S, et al (2008) Arabidopsis DREB 2A-interaction proteins functions as RING E3 genes and novel regulatory plant stress-responsive gene expression plant Cell 20: 1693. The public is available from the plant institute of Chinese academy of sciences.

Agrobacterium tumefaciens GV3101+ pSoup strain: described in the literature "Scholthof HB, Alvarado VY, Vega-Arreguin JC, Ciomperlik J, Odokonyero D, et al (2011) Identification of an ARGONAUTE for antiviral RNA screening in Nicotiana benthamiana plant physical 156: 1548-1555". Publicly available from the plant institute of Chinese academy of sciences;

vector pBI 121: described in the document "Qin F, Kakimoto M, Sakuma Y, Maruyama K, Osakabe Y, et al (2007) Regulation and functional analysis of ZmDREB2A in response to gravity and heat strains in Zea Mays L.plant J50: 54-69". Publicly available from the plant institute of Chinese academy of sciences;

agrobacterium tumefaciens GV3101 strain: described in "hanging Y, Zhang D, Wang X, Tang W, Wang W, et al (2013) Arabidopsis thaliana chromamat remodelling factor PICKLE interactions with transformation factor HY5to transformation plant Cell 25: 242-. Publicly available from the plant institute of Chinese academy of sciences;

columbia ecotype Arabidopsis thaliana (Columbia ecotype)): described in the document Yamaguchi-Shinozaki K, Shinozaki K (1994) A novel cis-acting element in an Arabidopsis gene infected in responsive to gravity, low-temperature, or high-salt strain plant Cell 6: 251-264. The public is available from the plant institute of Chinese academy of sciences.

Agrobacterium tumefaciens LBA4404 strain: described in the literature "Ishida, Y., heii, Y. & Komari, T (2007) Agrobacterium-mediated transformation of mail. 1614-. Publicly available from the plant institute of Chinese academy of sciences;

maize inbred line A188: nature protocol, described in the literature "Ishida, y., heii, Y. & Komari, T (2007) Agrobacterium-mediated transformation of mail.nature protocol.2: 1614-. The public is available from the plant institute of Chinese academy of sciences.

Vectors PSB1 and PSB 11: described in the literature "Komari et al (1996) Vectors carrying two separate T-DNAs for transformation of high Plant medium by Agrobacterium tumefaciens and transformation of transformations from selection markers. the Plant journal.10(1), 165-174". The public is available from the plant institute of Chinese academy of sciences.

Maize inbred line B73: the term "Schnable, P.S. et al (2009) The B73 mail gene: complex, diversity, and dynamics. science 326: 1112-. The public is available from the plant institute of Chinese academy of sciences.

Maize inbred line MO 17: described in the literature "Song et al, (2017) Transcriptomics and Alternative partitioning analysis derived Lines B73and Mo17in Response to additive Rhopalosiphum padi infection. front Plant Sci.10; 8-1738'. The public is available from the plant institute of Chinese academy of sciences.

Arabidopsis thaliana palmitoyl transferase gene-deficient variant tip1-3 and yeast palmitoyl transferase gene-deficient mutant strain akr 1: described in The literature "Hemsley et al, (2005) The TIP GROWTH DEFECTIVE1S-Acyl transfer enzymes regulation Plant Cell GROWTH in Arabidopsis, The Plant Cell 17; 2554, 2563 ". The public is available from the plant institute of Chinese academy of sciences.

Yeast expression vector pYES 2/NTA: invitrogen, catalog number V8252-20.

Yeast AH 109: product of Clontech, cat.No. 630489.

Two mutants of the maize ZmTIP1 gene (MT1 and MT 2): freely provided by Maize Genetics collaboration storage Center, the number of MT1 on the website is UFmu-06452, and the number of MT2 on the website is UFmu-03169.

Maize inbred line W22 is described in The documents "Springer et al, (2018) The mail W22genome genes a fundamental for functional genetics and transplason biology. Nature genetics 50(9): 1282. sup. 1288", publicly available from The plant research institute of The Chinese academy of sciences.

Maize inbred line CIMBL55 is described in the literature "Wang et al, (2016) Genetic variation in ZmVPP1 distributions to gravity complete in maize seeds. Nature genetics 48(10): 1233-41", publicly available from the plant research institute of the Chinese academy of sciences.

Example 1 obtaining of protein ZmTIP1 and Gene encoding it

1. Protein ZmTIP1 and its coding gene clone

Taking drought-tolerant corn inbred line CIMBL55 and sensitive corn inbred line MO17 seeds, accelerating germination for three days at 28 ℃, transferring the germinated seeds into nutrient soil or nutrient solution for culturing for 1 week, taking whole seedlings, quickly freezing and grinding the seedlings in liquid nitrogen, extracting total RNA, carrying out reverse transcription to obtain cDNA, and carrying out PCR amplification by taking the cDNA as a template and primers F1 and R1 as primers. And carrying out 1% agarose gel electrophoresis on the amplification product to obtain a PCR amplification product of 1920 bp.

After sequencing, the PCR product derived from the sensitive maize inbred line MO17 has the nucleotide indicated by 164-2083 of SEQ ID No.3, and the PCR product derived from the drought-tolerant maize inbred line CIMBL55 has the nucleotide indicated by 164-2083 of SEQ ID No. 4. The genes shown in SEQ ID No. 3and SEQ ID No.4 are named as ZmTIP1, and the protein coded by the gene is named as ZmTIP 1. SEQ ID No. 3and SEQ ID No.4 are different genotypes, the protein shown in SEQ ID No.1 is coded by SEQ ID No.3, and the protein shown in SEQ ID No.2 is coded by SEQ ID No. 4. In SEQ ID No. 3and SEQ ID No.4, the 5 'non-coding region is located at positions 1-163, the coding region is located at positions 164 and 2083, and the 3' non-coding region is located at positions 2084 and 2800.

The primer sequences are as follows:

F1：5’-ATGGCGTCGGAGATCGAG-3’；

R1：5’-TCACAATGGGATAAGAGACC-3’。

2. construction of recombinant vector pGZ

ZmTIP1^MO17The coding sequence of the gene (position 164-2083 of SEQ ID No. 3) and ZmTIP1^CIMBL55The two ends of the coding sequence of the gene (position 164-2083 of SEQ ID No. 4) are respectively added with the restriction enzyme sites SacI and SmaIRecognizing the sequence, carrying out double enzyme digestion on SacI and SmaI, and connecting the enzyme digestion product with a pGKX vector skeleton large fragment subjected to the same double enzyme digestion to obtain two recombinant vectors pGZ corresponding to two ZmTIP1 genotypes. The promoter for promoting the ZmTIP1 gene in the recombinant vector pGZ is a 35S promoter.

3. Obtaining of recombinant Agrobacterium tumefaciens

The recombinant vector pGZ is transformed into agrobacterium tumefaciens GV3101+ pSoup strain to obtain recombinant agrobacterium X containing recombinant vector pGZ (plasmid is extracted, and if the plasmid is pGZ by sequencing verification, the recombinant agrobacterium is a positive clone). A control was also set for the transfer of pGKX empty vector into the Agrobacterium tumefaciens GV3101+ pSoup strain.

4. Obtaining ZmTIP1 transgenic Arabidopsis thaliana

Transforming wild Columbia ecological arabidopsis thaliana and palmitoyl transferase gene defect variant tip1-3 by using a flower bud soaking method for the recombinant agrobacterium X, and harvesting seeds of T1 generation; screening T1 generation seeds by using an MS culture medium containing 30mg/L kanamycin, and planting and harvesting resistant seedlings to obtain T2 generation seeds; seeds of T2 generation are screened by MS culture medium containing 30mg/L kanamycin, and kanamycin-resistant seedlings with kanamycin resistance separation ratio of 3:1 are selected and planted, so that ZmTIP1 arabidopsis thaliana is transformed from T2 generation.

Extracting T2 generation ZmTIP1 Arabidopsis plant RNA, reverse transcribing to obtain cDNA as template, taking gene Actin2 in Arabidopsis as internal reference, and primer FC1 and RC 1. The expression level of ZmTIP1 gene was determined by using specific primers F2 and R2, the sequences of which are as follows:

F2：5’-AATGGGAATGATACAGATGACAAGT-3’；

R2：5’-TTGGCTATGAGGTTTTGAGTTTGCT-3’；

FC1：5’-GGTAACATTGTGCTCAGTGGTGG-3’；

RC1：5’-AGACACACCATCACCAGAATCC-3’。

selecting positive T2 generation with high ZmTIP1 gene expression quantity to transfer ZmTIP1 arabidopsis thaliana for seed collection to obtain T3 generation transfer seeds, and screening the T3 generation seeds by using MS culture medium containing 30mg/L kanamycin to obtain homozygous T3 generation ZmTIP1 transgenic strain which can not generate kanamycin resistance separation any more.

T1 represents the seeds of the current generation and the plants grown by the seeds; t2 represents the seeds produced by selfing of T1 generation and the plants grown from it; t3 represents the seeds produced by selfing of T2 and the plants grown from it.

The experiment was also carried out with the empty control of recombinant Agrobacterium X replaced by Agrobacterium tumefaciens GV3101+ pSoup strain transformed into pGKX empty vector.

The flower bud soaking method comprises the following specific steps:

inoculating the recombinant agrobacterium X into LB liquid culture medium containing 50mg/L kanamycin and 5mg/L tetracycline, carrying out shaking culture at 28 ℃ until OD600 is 0.8, centrifuging at 25 ℃ for 1 minute at 4000 rpm, removing supernatant, carrying out heavy suspension on the thalli by using a heavy suspension solution (the solvent is water, the concentrations of solutes of sucrose and silwet77 are 50g/L and 0.02 percent (volume percentage content), respectively, obtaining an infection solution, dotting the infection solution on a flower bud and a growth point by using a pipette, covering the bud and the growth point by using a film, keeping moisture for 2 days, placing the flower bud and the growth point under normal conditions, and harvesting seeds.

5. ZmTIP1 can complement phenotype of yeast palmitoyl transferase gene-deficient mutant

Taking cDNA of drought-tolerant corn inbred line CIMBL55 and sensitive corn inbred line MO17 as templates, carrying out PCR amplification by specific primers F1 and R1 (the sequence is the same as above), and carrying out PCR amplification on a target gene (ZmTIP 1)^MO17The coding sequence of the gene: position 164-2083 of SEQ ID No. 3; ZmTIP1^CIMBL55The coding sequence of the gene: 164 th and 2083 th sites of SEQ ID No. 4) and respectively transformed into a yeast palmitoyl transferase gene-deficient mutant strain akr1 (containing a reporter gene URA) by linking HindIII and BamHI enzyme cutting sites into a yeast expression vector pYES2/NTA, taking a transformed empty vector pYES2/NTA as a negative control and an untransformed wild-type yeast AH109 as a positive control, inducing the expression of a target gene by galactose, shaking the strain overnight at 30 ℃ and 37 ℃ by using an SC-URA liquid medium until the OD600 of the strain is 2.2-2.4, and taking a small amount of the strain to observe the morphology of yeast cells under a microscope.

As shown in FIG. 1A, the yeast akr1 mutant showed a sticky and elongated phenotype at 30 ℃ and 37 ℃ while the ZmTIP1 was transformed^MO17And ZmTIP1^CIMBL55Of (2) a yeastThe cell morphology is normal and similar to that of wild-type AH109 yeast cells, indicating that the ZmTIP1 protein encoded by the two genotypes can restore the phenotype of the akr1 mutant yeast.

Further converting ZmTIP1^MO17And ZmTIP1^CIMBL55The coding region (CDS) segment is constructed on an arabidopsis thaliana overexpression vector pGKX, and an arabidopsis thaliana palmitoyl transferase gene defect mutant tip1-3 is transformed through agrobacterium mediation to obtain T₃Seeds of homozygous generations were grown on MS + 3% Sucrose solid plates for 6 days to observe Arabidopsis root hair length. Meanwhile, wild type Arabidopsis thaliana and Arabidopsis thaliana (no-load control) transformed with pGKX empty vector were set as controls.

As a result, as shown in B and C in FIG. 1, the tip1-3 mutant had significantly shorter root hairs as compared with the Arabidopsis wild type, and two genotypes, 35S, ZmTIP1^MO17、35S:ZmTIP1^CIMBL55The root hair length of the transgenic plants can be restored to the wild type level. The length of the root hair of arabidopsis thaliana (no-load control) transferred into the pGKX empty vector is basically consistent with that of the wild type of arabidopsis thaliana, and no statistical difference exists.

The above results show that: the ZmTIP1 protein encoded by the two genotypes has palmitoyl transferase activity and plays an important role in root hair development.

Example 2 functional study of protein ZmTIP1 and the Gene encoding it

One, overexpression of ZmTIP1 gene to improve drought resistance of arabidopsis thaliana

Taking T obtained according to step 4 of example 1₃Transfer ZmTIP1^MO17Arabidopsis lines (TL1 and TL14, transformation receptor is Arabidopsis Columbia ecotype), ZmTIP1^CIMBL55Arabidopsis lines (TL8 and TL9, transformation recipients are Arabidopsis Columbia ecotype), wild type Arabidopsis Col and Arabidopsis transformed with pGKX empty vector (no-load control). 0g/L, 100g/L and 150g/L of PEG8000 were added to 1/2MS + 0.5% Sucrose solid medium in this order, photographed by a Nikon style microscope (A in FIG. 2) after 6 days of light exposure, and the root hair length was measured by Image J software, and at least 500-600 root hairs were measured for each strain, and the region indicated by the arrow in B in FIG. 2 was the main root region for measuring the root hair length. Compared with wild type, in the absence of treatmentAnd over-expression of ZmTIP1 under different concentrations of PEG treatment^MO17And ZmTIP1^CIMBL55The length of the root hair of the plant is obviously longer than that of the wild type (the length of the root hair of the non-loaded control arabidopsis thaliana is basically consistent compared with that of the wild type, and no statistical difference exists), and the fact that the length of the root hair of the arabidopsis thaliana can be obviously increased by increasing the expression quantity of the two genotypes of the zmtelpi 1 on the transcription level is shown (C and D in figure 2). Further, after growing in a pot containing 100g of nutrient soil under normal conditions for 25 days, drought treatment (i.e., stopping watering) is performed, and after about 13 days of water cut, rosette leaves of Col are seriously dried down and the rosette leaves of TL1 and TL14 and TL8 and TL9 are rehydrated when the rosette leaves are seriously wilted. The survival rate of each line of plants was counted 3 days after rehydration (plants showing normal growth were defined as surviving plants, plants showing severe drought and abnormal growth were defined as dead plants; the survival rate was the percentage of the number of surviving plants in each line to the total number of plants). The experiment is repeated for 3 times, the number of plants of each strain is not less than 30 plants, and the average value is taken for statistical analysis.

The results are shown in Table 1, E and F in FIG. 2, and it can be seen that after drought treatment, two genotypes T were observed₃The survival rate of the transgenic ZmTIP1 Arabidopsis line is obviously higher than that of wild Arabidopsis (the survival rate of the unloaded control Arabidopsis is basically consistent with that of the wild type, and no statistical difference exists).

TABLE 1 results of survival (%) of ZmTIP1 transgenic Arabidopsis plants after drought treatment

Line of plants	Repetition of 1	Repetition 2	Repetition of 3	Mean. + -. standardDifference (D)
					Col	26.25	15.625	21.4	21.09±5.3
TL1	93.75	97.92	92.71	94.79±2.75
					TL14	100	83.93	85.42	89.78±8.88
TL8	100	88.39	93.75	94.04±5.80
					TL9	100	95.54	100	98.51±2.58

Note: indicates a significant difference at P < 0.05 compared to CK results, and indicates a very significant difference at P < 0.01 compared to CK results.

Second, evaluation of drought resistance of ZmTIP 1-transgenic maize and ZmTIP1 mutant

1. Construction of recombinant vector pSBIII

ZmTIP1^MO17The coding sequence of the gene (164-2083 th site of SEQ ID No. 3) is connected into the pSB11 vector (located at the downstream of Zmbi 1 promoter) through Sma I enzyme cutting site, the rest sequences on the vector are kept unchanged, and the pSB11 vector connected with the target fragment is recombined with the pSB1 vector to obtain the recombinant vector pSBIII-ZmTIP1^MO17. Meanwhile, a recombinant vector pSBIII is obtained after the pSB11 vector which is not connected with the target fragment and the pSB1 vector are recombined.

2. Obtaining of recombinant Agrobacterium tumefaciens

Mixing the recombinant vector pSBIII-ZmTIP1^MO17Transforming Agrobacterium tumefaciens LBA4404 strain to obtain recombinant Agrobacterium Y containing recombinant vector pSBIII (extracting plasmid, sequencing verifying that the plasmid is pSBIII-ZmTIP 1)^MO17And then the recombinant agrobacterium is a positive clone).

Meanwhile, a control for transferring a pSBIII empty vector into the Agrobacterium tumefaciens LBA4404 strain is set.

3. Obtaining transgenic maize

Transforming the recombinant agrobacterium Y into a maize inbred line A188 by an agrobacterium-mediated maize genetic stable transformation method to obtain T₀Plants were grown and planted in the greenhouse (16 h-light/8 h-dark).

Extraction of T₀Transferring ZmTIP1 corn leaf RNA, reverse transcribing to obtain cDNA, and PCR identifying with specific primer F3 and R3 to obtain T0 generation ZmTIP1 corn.

The sequences of the primers are as follows:

F3：5’-TATGCCATGACTACGACAG-3’；

R3：5’-CCGATCTAGTAACATAGATGACTACC-3’。

positive T₀Inbreeding of the plant generations to obtain T₁Seed generation; t is₁The generation plants are identified by PCR with the same method to obtain positive plants, and T is obtained after selfing₂And (5) seed generation. Positive individuals are identified by the same PCR identification method and selfed to obtain T3 generation homozygous lines, the segregation ratio of T2 generation plants is 3:1, and at least 24 positive individuals are identified by T3 generation PCR to be used as homozygous lines. Breeding homozygous linesSeeds from T3 and T4 generations were obtained for subsequent drought phenotype testing.

Extraction of T₃Transferring ZmTIP1 corn RNA, reverse transcribing to obtain cDNA, qPCR quantifying the cDNA of ZmTIP1 gene with specific primers F2 and R2, using corn gene ZmUbi2 as reference, primers FC2 and RC2, and A188 as reference.

The sequences of the primers are as follows:

F2：5’-AATGGGAATGATACAGATGACAAGT-3’；

R2：5’-TTGGCTATGAGGTTTTGAGTTTGCT-3’。

FC2：5’-TGGTTGTGGCTTCGTTGGTT-3’；

RC2：5’-GCTGCAGAAGAGTTTTGGGTACA-3’。

the results are shown in B in FIG. 3, the expression level of ZmTIP1 of ZmTIP1 maize ML1, ML3, ML5 and ML9 of T3 generation is obviously higher than that of wild maize, which indicates that the ZmTIP1 maize ML1, ML3, ML5 and ML9 of T3 generation are positive transgenic maize.

T above₀Represents a plant grown in the current generation of transformation; t is₁Generation represents T₀Seeds produced by generation selfing and plants grown from the seeds; t is₂Generation represents T₁Seeds produced by generation selfing and plants grown from the seeds, and so on.

The experiment was also set with an empty control in which recombinant Agrobacterium Y was replaced with Agrobacterium tumefaciens LBA4404 strain transformed into pSBIII empty vector.

The agrobacterium-mediated gene transformation method comprises the following specific steps:

the recombinant Agrobacterium Y was inoculated in YEB broth containing 50mg/L spectinomycin and shake-cultured at 28 ℃ to OD₆₀₀Is 0.5. Placing the young maize embryo into a 2mL centrifuge tube filled with preservation solution, carrying out heat treatment at 46 ℃ for 3min, and centrifuging at4 ℃ at 2000 rpm for 10 min. Adding the prepared recombinant agrobacterium into the treated immature embryo, culturing for 3 days in the dark at 22 ℃, and transferring to a new culture medium for 7-10 days in the dark at 28 ℃. Screening by phosphinothricin with different concentrations, transferring to a differentiation culture medium, transferring to a rooting culture medium for culture after differentiation, and transferring to nutrient soil after a certain size.

4. Obtaining of maize ZmTIP1 gene mutant

Two mutants of the Maize ZmTIP1 gene (MT1 and MT2) as well as wild type W22 seed were provided free of charge Genetics Cooperation Stock Center, the mutant is formed by inserting Mu transposon into a ZmTIP1 gene promoter region and a first intron region respectively, the mutant seeds are sowed in a greenhouse, leaf DNA is extracted, PCR amplification identification is carried out on the mutant MT1 by using specific primers F4 and R4, amplification identification is carried out on the mutant MT2 by using specific primers F1 and R4, amplification identification is carried out on the wild type W22 by using specific primers F6 and R6, the identified mutant is hybridized with W22 to obtain F1 generation seeds, the heterozygote mutant is identified by using the same PCR identification method, then backcross is carried out twice with W22 to obtain BC2F1 generation seeds, the heterozygote single plant is identified by PCR to obtain BC2F2 generation seeds, the BC2F2 generation seeds are sowed in the field, the leaf DNA is extracted, and the homozygous mutant single plant identified by PCR is selfed to obtain BC2F3 generation homozygous mutant seeds for subsequent phenotype test (D in figure 3). Root RNA of a BC2F3 homozygous mutant strain is extracted and reverse transcribed to obtain cDNA, the cDNA of gene ZmTIP1 is semi-quantified by using specific primers F7 and R7, the gene ZmUbi2 of corn is used as an internal reference, the primers are FC2 and RC2, and the wild type W22 is used as a control, and as a result, as shown in E in figure 3, the ZmTIP1 genes of mutants MT1 and MT2 are hardly expressed.

The sequences of the primers are as follows:

F4：5’-GGTGCTCAACAGACCCTACTC-3’；

R4：5’-AGAGAAGCCAACGCCAWCGCCTCYATTTCGTC-3’。

F6：5’-AAAATCCTAGTTACGCACCG-3’；

R6：5’-CATAAACCAGCTCCGAAA-3’。

F7：5’-CCACGCGCTTCAGTGGGCCGCA-3’；

R7：5’-CACCGTTCTGCTGCGAAACT-3’。

in the above primers, W represents A or T; y represents T or C.

5. ZmTIP1 involved in phenotypic analysis of corn root hair development

Selecting corn seeds A188, ML1, ML3, ML5 and ML9 which are identical and plump in size and are transferred into pSBIII empty vector, and 10% H₂O₂And (3) disinfecting for 20min, washing the mixture by using distilled water, soaking the mixture for 6 to 8 hours by using saturated calcium sulfate, and putting the soaked mixture on wet filter paper to accelerate germination for 2 to 3 days at the temperature of 28 ℃. Seeds with consistent shoots were picked and transferred to 25cm × 25cm Agar plates at pH5.7, after 4 days of vertical culture, maize root hairs were photographed using an Olympus fluoroscope SEX16 microscope and root hair length was determined using Image J software. The test is repeated three times, each strain is repeated for at least 600- & lt700 & gt root hairs of 20 plants are observed, and the average value is taken for statistical analysis.

The results are shown in fig. 3, a and C, and the root hair length of 4 transgenic lines was significantly longer than wild type 7 days after germination (no-load control maize root hair length was essentially identical compared to wild type, with no statistical difference).

The root hairs of wild type W22, mutant MT1 and MT2 were photographed and the length of the root hairs was measured by the above method, and the average value was taken for statistical analysis.

Results as shown in fig. 3F and G, the length of root hairs of ZmTIP1 mutant was significantly shorter than the wild type 7 days after germination.

6. ZmTIP1 involved in phenotypic analysis of drought resistance of corn

T to be sown for 7 days₃Transferring ZmTIP1 corn strains (ML1, ML3, ML5 and ML9) and wild type corn A188 plants and corn A188 transferred into pSBIII empty carrier into a pot filled with 2Kg of nutrient soil, carrying out drought treatment (namely stopping watering) for about 20 days after the corn strains grow for 10 days under normal conditions, carrying out rehydration when the leaf wilting degree of the transgenic plants is obviously different from the wild type, and carrying out statistics on the survival rate of each strain after 7 days of rehydration (the plants with normal leaf color and normal growth are defined as survival plants, the plants with scorched leaves and normal growth are defined as death plants, and the survival rate is the percentage of the number of the survival plants in each strain to the total number of the plants). The experiment is repeated for 3 times, the number of plants of each strain is not less than 30 plants, and the average value is taken for statistical analysis.

The results are shown in Table 2, A and C in FIG. 4, and it can be seen that the drought treatment was carried out for 20 days, T₃The transgenic ZmTIP1 has less leaf withering degree than wild corn and high survival rateIn wild-type maize (no-load control maize survival rate was essentially identical compared to wild-type, no statistical difference).

TABLE 2 survival (%) results of transgenic maize plants after drought treatment

Line of plants	Repetition of 1	Repetition 2	Repetition of 3	Mean. + -. standard deviation of
					WT	30.56	32.78	27.78	30.37±2.50**
ML1	70.00	69.44	50.00	63.15±11.39**
					ML3	71.67	50.00	60.00	60.56±10.84**
ML5	80.00	66.67	70.56	72.41±6.86**
					ML9	94.44	90.00	81.67	88.70±6.49**

Note: indicates that the difference was very significant at P < 0.01 compared to WT results.

Statistics of survival rates for W22, MT1 and MT2 in the same manner as described above are shown in table 3, B and D in fig. 4, and it can be seen that after 20 days of drought treatment, leaf withering degree of ZmTIP1 mutant MT1 and MT2 is less than that of wild-type maize, and survival rate is significantly lower than that of wild-type maize.

TABLE 3 survival (%) results of mutant plants after drought treatment

Line of plants	Repetition of 1	Repetition 2	Repetition of 3	Mean. + -. standard deviation of
					WT	63.88	51.38	50.00	55.09±7.65**
MT1	16.67	22.22	31.48	23.46±7.48**
					MT2	25.93	25.00	33.33	28.09±4.57**

Note: indicates that the difference was very significant at P < 0.01 compared to WT results.

Therefore, the expression quantity of the ZmTIP1 protein coding gene is obviously related to the drought resistance of the corn in the seedling stage, the survival rate of the corn in the seedling stage can be obviously reduced by reducing the expression quantity of the ZmTIP1 gene, the survival rate of the corn in the seedling stage can be obviously improved by improving the expression quantity of the ZmTIP1 gene, and the drought resistance of the corn is improved.

7. Field drought phenotype analysis of ZmTIP1 maize and ZmTIP1 mutants

WT, ML1, ML3, ML5 and ML9 were tested for tolerance under drought treatment conditions in field experimental conditions. In this experiment 3 treatments were set, named WW (normal watering), MD (mild drought) and SD (severe drought). WW treatment irrigates sufficient water throughout the growing season to ensure normal corn growth, MD treatment stops water supply 21 days after seeding, but waters once after the corn is tasseled; the water supply was stopped 21 days after the SD treatment sowing until the corn was harvested. The plant height, the powder scattering time (DTA), the powder scattering and spinning interval (ASI) and the yield of each plant are respectively counted. The experiment is repeated for 4 times, the number of plants of each strain is not less than 10 plants, and the average value is taken for statistical analysis.

The results are shown in A, B, C and D in FIG. 5, and under normal watering conditions, the agronomic traits were not significantly different from WT except that ML1 and ML5 showed earlier pollen dispersal than wild type. Under the mild drought condition, the plant height of the transgenic line ML1 is obviously higher than that of the wild type, the pollen scattering time is earlier than that of the wild type, the ASI of 4 transgenic lines are obviously smaller than that of the wild type, and the single-plant yield is obviously higher than that of the wild type. Under severe drought conditions, the mean value of the ASI of the transgenic line is smaller than that of the wild type, and the mean value of the yield of a single plant is higher than that of the wild type but does not reach a significant level.

In the above method, two treatments, WW and MD, were set for wild-type WT, mutants ML1 and ML2, and statistics were made for plant height, pollen Dispersal Time (DTA), pollen dispersal and silking interval (ASI), and individual plant yield, respectively.

Results as shown by E, F, G and H in fig. 5, the traits were not significantly different from WT except that the yield of ML1 was lower than that of wild type under normal watering conditions. However, under mild drought conditions, the plant height of ML2 is significantly lower than that of the wild type, and the yield per plant of 2 mutants is significantly lower than that of the wild type.

The results show that the expression of the coding gene of the protein ZmTIP1 in plants can increase the yield of a single plant and improve the drought resistance of the plants, and the reduction of the expression level of the coding gene of the protein ZmTIP1 can obviously reduce the yield of the single plant.

Example 3 investigation of subcellular localization of ZmTIP1 protein

1. Construction of recombinant vectors

ZmTIP1 shown in 164 th-2082 th site of SEQ ID No.3 was inserted between SmaI and SacI of the vector pGK-CsGFP (located downstream of the 35S promoter), and the remaining sequences on the vector were kept unchanged, and the resulting recombinant vector was named pGK-CsGFP-ZmTIP 1.

2. Obtaining of recombinant Agrobacterium tumefaciens

pGK-CsGFP-ZmTIP1 vector linked with ZmTIP1 sequence shown in 164 th and 2082 th of SEQ ID No.3 is transformed into Agrobacterium tumefaciens GV3101+ pSoup strain to obtain recombinant Agrobacterium tumefaciens Y containing the recombinant vector (plasmid is extracted, and sequencing verifies that the plasmid is recombinant pGK-CsGFP-ZmTIP1 plasmid containing ZmTIP1, and the recombinant Agrobacterium tumefaciens is positive clone).

3. Transfer 35S acquisition of ZmTIP1-GFP Arabidopsis thaliana

Transforming wild Columbia ecological arabidopsis thaliana by using the recombinant agrobacterium tumefaciens Y through a flower bud soaking method to obtain T1 generation seeds; screening T1 generation seeds by using an MS culture medium containing 30mg/L kanamycin, and planting and harvesting resistant seedlings to obtain T2 generation seeds; seeds of T2 generation are screened by MS culture medium containing 30mg/L kanamycin, and kanamycin-resistant seedlings with kanamycin resistance separation ratio of 3:1 are selected and planted to obtain T2 generation ZmTIP1-GFP arabidopsis thaliana.

Extracting T2-generation ZmTIP1-GFP arabidopsis thaliana plant RNA, carrying out reverse transcription to obtain cDNA as a template, taking gene Actin2 in arabidopsis thaliana as an internal reference, and taking primers of FC1 and RC 1. The expression level of ZmTIP1 gene was determined by using specific primers F2 and R2, the sequences of which are as follows:

F2：5’-AATGGGAATGATACAGATGACAAGT-3’；

R2：5’-TTGGCTATGAGGTTTTGAGTTTGCT-3’；

FC1：5’-GGTAACATTGTGCTCAGTGGTGG-3’；

RC1：5’-AGACACACCATCACCAGAATCC-3’。

selecting positive T2 generation with high ZmTIP1 gene expression quantity to transfer ZmTIP1-GFP arabidopsis thaliana for seed collection, obtaining T3 generation transfer seeds, screening the T3 generation seeds by using MS culture medium containing 30mg/L kanamycin to obtain homozygous T3 generation ZmTIP1-GFP transgenic strain which can not generate kanamycin resistance separation any more.

T1 represents the seeds of the current generation and the plants grown by the seeds; t2 represents the seeds produced by selfing of T1 generation and the plants grown from it; t3 represents the seeds produced by selfing of T2 and the plants grown from it.

The experiment is also provided with the no-load control of replacing the recombinant agrobacterium Y with the agrobacterium tumefaciens GV3101+ pSoup strain which is transferred into the pGK-CsGFP empty vector

4. Subcellular localization observation of ZmTIP1 protein

T3 generation homozygous ZmTIP1-GFP transgenic line was seeded on MS plate, and after 5 days of light exposure, subcellular localization of the transgenic line ZmTIP1 protein was observed with Leica TCS SP5 fluorescence microscope, and as a result, as shown in A in FIG. 6, ZmTIP1 protein was localized not only to the cytoplasmic membrane but also in a punctate distribution in the cytoplasm. To further determine the specific localization of the ZmTIP1 protein in the cytoplasm, ZmTIP1-GFP homozygous strain was hybridized with RFP-tagged Arabidopsis transgenic strain WAVE 2R (vacuolar precursor/PVC), WAVE 3R (trans-Golgi network tagged), Wave 9R (Tonoplast), and WAVE 22R (Golgi) to obtain F1 seeds, F1 seeds were sown on MS plates and co-localized with confocal microscopipe (olympus 1000MPE,488nm for GFP and 543nm for RFP) 5 days after exposure to light, resulting in co-localization of the mTIP1 protein with PVC, Golgi and TGN/EE, but not with Tonoplast, as shown in B in FIG. 6. In summary, the zmtpip 1 protein is localized to the cytoplasmic membrane, golgi apparatus, trans-golgi network and vacuolar precursors.

5. ZmTIP1 protein regulating plasma membrane localization of ZmCPK28 protein

3 independent ZmTIP1-GFP transgenic lines of T3 generation and a control pGK-CsGFP transgenic line are sown on an MS plate, visible light culture is carried out for 10 days at 22 ℃, 6-8 g of plant material is taken and fully ground and crushed by a mortar, 7ml of 2 × extraction Buffer (50mM Na-Phosphate, pH 7.4; 150mM NaCl; 1mM DTT, 1 × protease inhibitor cocktail tables; 0.1% Nonidet P-40) is added for continuous grinding (operation on ice) for 5min, and the plant material is transferred into a 15ml centrifuge tube to be fully shaken and uniformly mixed for 1min, 13,000g and centrifuged for 20min at4 ℃. The supernatant was transferred into 1.5ml centrifuge tubes (about 6-8), 15. mu.L of Anti-GFP mAb-Magnetic Beads (MBL) pre-washed 3 times with 2 × extraction Buffer was added to each tube, and the mixture was mixed at4 ℃ for 2 h. The Beads were collected by a magnetic stand, washed 3-4 times with 2 × extraction Buffer to remove non-specifically bound proteins, and then washed 3-4 times with 2 × extraction Buffer without NP-40. Finally, after eluting with 100 μ L of Elution buffer containing 0.1M Gly-HCl (pH3.0), rapidly adding 1/10 volumes of 1M Tris-HCl (pH6.7) to neutralize protein so as to avoid protein denaturation, transferring the eluate to a mass spectrum platform of Chinese agriculture university for detection of the interacting protein, and finally determining the protein detected by 3 transgenic strains simultaneously as possible interacting protein. Wherein AtCPK21(Calcium-Dependent Protein Kinase 21, At4g04720) is repeatedly detected for many times, and the two conserved cysteine residues are contained in the N-terminal amino acid sequence MGSCCS of the homologous gene ZmCPK28(GRMZM2G 168706) in corn, and the two conserved cysteine residues can be modified by palmitoyl transferase to influence the subcellular localization of the target Protein.

ZmCPK28-GFP and ZmCPK28-C3A-GFP, ZmCPK28-C4A-GFP and ZmCPK28-C3&4A-GFP which mutate the two conserved cysteine residues are constructed on a pSB11 vector, the 4 recombinant vectors are respectively transformed into protoplasts of corn W22 and ZmTIP1 mutants MT1 and MT2, and the protoplasts are subjected to subcellular localization observation by a Leica TCS SP5 fluorescence microscope.

Results as shown in fig. 7 a, ZmCPK28 protein was localized mainly to the plasma membrane, while localization in the cytoplasm was very weak. After the N-terminal single cysteine mutant is used, the localization of partial ZmCPK28 protein is transferred from the cell membrane to the cytoplasm, and if the two cysteines are all mutated, the ZmCPK28 protein is mainly localized to the cytoplasm, but is very weakly localized to the plasma membrane. In line with this, the ZmCPK28 protein was localized predominantly in the cytoplasm in zmtpip 1 mutants MT1 and MT 2. To further verify the localization of ZmCPK28 protein, total protoplast protein was further extracted, cell membrane (P) and cytoplasmic component (S) proteins were isolated, and the localization pattern of ZmCPK28 was examined using Western Blot, and as a result, as shown in fig. 7B, ZmCPK28 was mainly localized to the plasma membrane in wild-type W22, and after two conserved cysteine residues were mutated, the localization of ZmCPK28 protein was transferred to the cytoplasm, and also in zmtpk 1 mutant MT2, ZmCPK28 protein was mainly localized to the cytoplasm, and only a small amount of protein was localized to the cell membrane.

To further confirm whether ZmTIP1 interacts with ZmCPK28, ZmCPK28 protein fused with Myc tag, and ZmTIP1 protein fused with GFP tag was constructed into vector pGKX, and 35S promoter on pGKX vector was replaced with Zmubi1 promoter to transform maize B73 protoplast.

The results are shown in fig. 7, C, where zmtpip 1 interacts with ZmCPK28 protein.

In summary, the zmcip 1 protein may modify ZmCPK28 by palmitoylation, affecting its subcellular localization.

<110> university of agriculture in China

Application of <120> protein ZmTIP1 in regulation and control of plant drought resistance

<130> GNCLN190306

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<170> PatentIn version 3.5

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<213> Zea mays L.

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Lys Thr Gly Leu Thr Pro Ala Gln Leu Ala Ala Asp Lys Asn His Arg

245 250 255

Gln Val Ala Phe Phe Leu Asp Asn Ala Arg Arg Val His Gly Arg Gly

260 265 270

Cys Gly Ala Asn Thr Arg Phe Gly Lys Leu Ser Lys Leu Gly Leu Ala

275 280 285

Pro Leu Leu Trp Cys Thr Ile Ile Gly Met Leu Ile Thr Tyr Thr His

290 295 300

Ser Val Ile Ser Gly Gln Tyr Ala Met Thr Thr Thr Ala Pro Phe Gly

305 310 315 320

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

325 330 335

Phe Tyr Lys Cys Ser Arg Lys Asp Pro Gly Tyr Ile Asn Ile Asn Ala

340 345 350

Arg Gly Ser Gln Asn Gln Arg Asp Asp Glu Pro Leu Leu Lys Met Glu

355 360 365

Leu Glu Asn Pro Ala Leu Leu Ser Gly Asn Trp Ser Gln Leu Cys Ile

370 375 380

Thr Cys Lys Ile Val Arg Pro Val Arg Ser Lys His Cys Ser Thr Cys

385 390 395 400

Asp Arg Cys Val Glu Gln Phe Asp His His Cys Pro Trp Val Ser Asn

405 410 415

Cys Ile Gly Lys Lys Asn Lys Trp Glu Phe Phe Met Phe Leu Thr Leu

420 425 430

Glu Val Phe Ala Met Ile Ile Thr Gly Ser Ala Ala Ile Ile Arg Ile

435 440 445

Val Arg Asp Pro Asn Ser Pro Ser Ser Phe Gly Ala Trp Ile His Tyr

450 455 460

Ser Ala Phe Gln His Pro Gly Val Val Ser Phe Leu Ala Leu Asp Cys

465 470 475 480

Phe Leu Phe Phe Gly Val Ala Val Leu Thr Val Val Gln Ala Ser Gln

485 490 495

Ile Ala Arg Asn Ile Thr Thr Asn Glu Met Ala Asn Ser Met Arg Tyr

500 505 510

Ala Tyr Leu Arg Gly Pro Gly Gly Arg Phe Arg Asn Pro Tyr Asp His

515 520 525

Gly Ile Arg Lys Asn Cys Ser Asp Phe Leu Leu Asn Gly Tyr Asn Glu

530 535 540

Asp Thr Glu Arg Leu Glu Gln Thr Leu Pro Thr Asp Glu Glu Met Gly

545 550 555 560

Met Ile Gln Met Thr Ser Ala Val Ser Gln Gln Asn Gly Asp Asn His

565 570 575

Leu His His Gly Asn Gly Thr Asp His Ser Cys Ala Val Ser Gln Ala

580 585 590

Asn Ser Lys Pro His Ser Gln Val Gly Ser Ser Gln Cys Cys Asp His

595 600 605

Ser Lys Arg Thr Asp Arg Thr Pro Leu Gly Leu Gly Leu Gly Leu Gly

610 615 620

Arg Asn Ser Ala Ser Arg Gln Tyr Val Arg Ser Leu Ile Pro Leu

625 630 635

<210> 3

<211> 2800

<212> DNA

<213> Zea mays L.

<400> 3

cttccctctc gttctccgct tccgcctccg cctccggcca cgagtcacca gggaagggac 60

aaacgcacaa aatcaacgaa cgcccagcgc ccagtcaaac actacccctt cctccctccc 120

ccctcgccgc ctccccggac cccgccccga ccccaccgaa cccatggcgt cggagatcga 180

ggtgctcgag gacaccacca cctcctcgac ttccctcgtc gcggccgcgt ccacagtccc 240

ttctctcgcg gagggcgcgg aggcgccggc ggaggacgac tcgctgaaga acgacgtgta 300

caccgcggcg gcgtacggcg atctggagaa gctgcagcga ctggtggagg gggagggccg 360

cccagtcacc gagcccgacg gcgggggcta ccacgcgctt cagtgggccg cactcaacaa 420

ccgcgttgcc gccgcgcagt acatccttga gcatggagca gacataaatg ctgtggatca 480

cactggacaa acagcacttc actggagtgc tgtacgtggt catattcaag ttgctgaact 540

acttctgaaa gaaggagcta aggtggatgc tactgattta tacgggtatc aggccacgca 600

tgttgcagca cagtatggtc agactgcatt catttaccac attgttgcaa aatggaatgc 660

tgatccagat atccctgata atgatggaag gagcccttta cactgggctg cttataaggg 720

atttgcagac tccatacggc ttcttttgtt tttggatgct tataggggac ggcaagacaa 780

agaaggttgt actccattac attgggctgc tattcggggg aatcttgagg catgcactgt 840

cttagttcag gttggcaaaa aggatgattt gatggtgaaa gacaaaactg gcttaactcc 900

agcacagctt gctgccgata agaatcatcg gcaagttgca tttttcctcg acaatgctag 960

aagggtacac ggcaaaggat gtggtgcgaa caccagattt gggaaattgt caaaattagg 1020

gctcgctcct cttctttggt gcaccattat tggcatgctt attacataca cacactctgt 1080

tatatcagga caatatgcca tgactacgac agcaccattt gggatattcg catggtcagg 1140

agtttttctt gcaactgctg gcttggtcat gttctataaa tgtagcagga aagatccagg 1200

ttacatcaac ataaatgcaa ggggctcgca aaatcaaagg gatgatgaac cgttgctgaa 1260

gatggagtta gaaaatcctg cacttctttc tggcaactgg tcacaacttt gtataacctg 1320

caaaatagtc agacctgttc gttcaaaaca ttgttctaca tgtgatcgct gcgtggagca 1380

gtttgaccac cactgccctt gggtatctaa ttgcatagga aagaagaaca aatgggaatt 1440

cttcatgttc ctcactctag aagtttttgc aatgatcatt actggctctg ctgccattat 1500

aagaattgta agagatccaa attctccatc atcctttggt gcttggattc attattctgc 1560

gtttcagcat cctggggtgg tttcatttct cgcattggat tgttttcttt tctttggtgt 1620

tgcagttctt acagttgttc aagcatcaca gatagcaagg aacattacaa caaatgagat 1680

ggcaaactcc atgagatatg catacctcag aggcccaggt ggcagattca ggaatccgta 1740

tgatcatggg attcgcaaga actgctctga cttcttgtta aatggataca atgaggacac 1800

tgaacggcta gagcagacat tgcccactga tgaggaaatg ggaatgatac agatgacaag 1860

tgcagtttcg cagcagaacg gtgacaatca tttacatcat ggtaatggca ctgaccatag 1920

ttgcgctgtt tcacaagcaa actcaaaacc tcatagccaa gtgggttcgt ctcagtgttg 1980

tgaccacagt aagaggactg ataggacacc gttgggccta ggattgggcc ttggacgaaa 2040

cagtgcgtcc cggcagtatg ttcggtctct tatcccattg tgatccatca tttggcgatc 2100

atgtgtttgt ttgactgtag attggatttg ttcacttatt ctgttatacg tgcccttgac 2160

atggttcacc atggctgggc agggttaacg ttattgtgcc atgagctccg gaatactaag 2220

atatatctgg tgttgtagtt tagcgttgaa ctcagagaag ttgaaggtaa cgacctggtt 2280

tgcgagtatc agctgatcag cagtctggaa tcagagagtt tgtgcagaaa atgtaagagt 2340

tatggactca tggcagatgc agtagcatga gatgtcttca gaattgttgt gtacatcagg 2400

ttcggtggta cccccattgt ttagcgtgat acgagtttgg aaaaaaaaaa agtagtcgta 2460

aattgagaaa attgtaaatt gattagtaag ctcactgcaa cactagctgt tttcctagtt 2520

ttcattcatt tttgggtctg gttattgtta tgaaccacgg aaagaatttt cggttttcgg 2580

gtatgggtac ctcagttata tttggtctgg ttatggtact tactggtggt gcatgtttgt 2640

gtgagtgctt agagtttagg ctatggccat cagatcatgt atatgactat atgactgtgt 2700

aaaacactgt tttttatctg attgtgtaaa acagtgtttt tgtatcgtac actattgttt 2760

tatagattga agtttaaaat aagagatgtg aatgagtaag 2800

<210> 4

<211> 2800

<212> DNA

<213> Zea mays L.

<400> 4

cctcaacctt ccctctcgtt ctccgcctcc gcctccggcc acgagtcacc agggaaggga 60

caaacgcaca aaatcaacga acgcccagcg cccagtcaaa cactacccct tcctccctcc 120

ccctcgccgc ctccccggac cccgccccga ccccaccgaa cccatggcgt cggagatcga 180

ggtgctcgag gacaccacca cctcctcgac tttcctcgtc gcggccgcgt ccacagtccc 240

ttctgccgcg gagggcgcgg aggcgccggc ggaggacgac tcgctgaaga acgacgtgta 300

caccgcggcg gcgtacggcg atctggagaa gctgcagcgg ctggtggagg gggagggccg 360

cccagtcacc gagcccgacg gcgggggcta ccacgcgctc cagtgggccg cactcaacaa 420

ccgcgttgcc gccgcgcagt acatccttga gcatggagca gacataaatg ctgtggatca 480

cactggacaa acagcacttc actggagtgc tgtacgtggt catattcaag ttgctgaact 540

acttctgaaa gaaggagcta aggtggatgc tactgattta tatgggtatc aggccacaca 600

tgttgcagca cagtatggtc agactgcatt catttaccac attattgcaa aatggaatgc 660

tgatccagat atccctgata atgatggaag gagcccttta cactgggctg cttataaggg 720

atttgcagac tccatacggc ttcttttgtt tttggatgct tataggggac ggcaagacaa 780

agaaggttgt actccattac attgggctgc tattcggggg aatcttgagg catgcactgt 840

cttagttcag gttggcaaaa aggatgattt gatggtgaaa gacaaaactg gcttaactcc 900

agcacagctt gctgccgata agaatcatcg gcaagttgca tttttcctcg acaatgctag 960

aagggtacac ggcagaggat gtggtgcgaa caccagattt gggaaattgt caaaattagg 1020

gctcgctcct cttctttggt gcaccattat tggcatgctt attacataca cacactctgt 1080

tatatcagga caatatgcca tgactacgac agcaccattt gggatattcg catggtcagg 1140

agtttttctt gcaactgctg gcttggtcat gttctataaa tgtagcagga aagatccagg 1200

ttacatcaac ataaatgcaa ggggctcgca aaatcaaagg gatgatgaac cgttgctgaa 1260

gatggagttg gaaaatcctg cacttctttc tggcaactgg tcacaacttt gtataacctg 1320

caaaatagtc agacctgttc gttcaaaaca ttgttctaca tgtgatcgct gcgtggagca 1380

gtttgaccac cactgccctt gggtatctaa ttgcatagga aagaagaaca aatgggaatt 1440

cttcatgttc ctcactctag aagtttttgc aatgatcatt actggctctg ctgccattat 1500

aagaattgta agggatccaa attctccatc atcctttggt gcttggattc attattctgc 1560

gtttcagcat cctggggtgg tttcatttct cgcattggat tgttttcttt tctttggtgt 1620

tgcagttctt acagttgttc aagcatcaca gatagcaagg aacattacaa caaatgagat 1680

ggcaaactcc atgagatatg catacctcag aggcccaggt ggcagattca ggaatccgta 1740

tgatcatggg attcgcaaga actgctctga cttcttgtta aatggataca atgaggacac 1800

tgaacggcta gagcagacat tgcccactga tgaggaaatg ggaatgatac agatgacaag 1860

tgcagtttcg cagcagaacg gtgacaatca tttacatcat ggtaatggca ctgaccatag 1920

ttgcgctgtt tcacaagcaa actcaaaacc tcatagccaa gtgggttcgt ctcagtgttg 1980

tgaccacagt aagaggactg ataggacacc gttgggccta ggattgggcc ttggacgaaa 2040

cagtgcgtcc cggcagtatg ttcggtctct tatcccattg tgatccatca tttggcgatc 2100

atgtgtttgt ttgactgtag attggatttg ttcacttatt ctgttatacg tgcccttgac 2160

atggttcacc atggctgggc agggttaaac gttgttgtgc catgagctcc ggaatactaa 2220

gatatatctg gtgttgtagt ttagcgttga actcagagaa gttgaaggta acgacctggt 2280

ttgcgagtat cagctgatca gcagtctgga atcagagagc ttgtgcagaa aatgtaagag 2340

ttatggactc atggcagatg cagtagcatg agatgtcttc agaattgttg tgtacacatc 2400

aggttctgtg gtacccccat tgtttagcgt gatacgagtt tgggaaaaaa aagtagtcgt 2460

aaattgagga aattgtaaat tgattagtaa gctcactgca acactagctg ttttcctagt 2520

tttcattcat ttttgggtct ggttattgtt atgaaccacg gaaagaatat ttggttttcg 2580

ggtatgggta cctcagttat atttggtctg gttatggtac ttactggtgg tgcatgtttg 2640

tgtgagtgct tagagtttag gctatggcca gcagatatgt atatgaccgt gtaaaacact 2700

gtttttgtat tgtagactat tattttatag attggagttt aaaataggag atgtgaatga 2760

gtaatctact agagatgtcc ttaggacact gttgttacaa 2800

Claims (14)

1. The application of ZmTIP1 protein or its related biological material in regulating plant drought resistance;

   the related biological material is a nucleic acid molecule capable of expressing the ZmTIP1 protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule;

   the ZmTIP1 protein is any one of the following proteins:

   (A1) protein with an amino acid sequence of SEQ ID No.1 or SEQ ID No. 2;

   (A2) a corn-derived protein having a homology of 99% or more with the amino acid sequence defined in (a1) and having the same function;

   (A3) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A2).
2. 2. Use according to claim 1, characterized in that: the activity and/or expression level of the ZmTIP1 protein or the coding gene thereof in the plant is increased, and the drought resistance of the plant is improved; the activity and/or expression level of the ZmTIP1 protein or the coding gene thereof in the plant is reduced, and the drought resistance of the plant is reduced.
3. 3. The ZmTIP1 protein or its related biological material is used in regulating and controlling the growth of plant root and hair;

   the related biological material is a nucleic acid molecule capable of expressing the ZmTIP1 protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule;

   the ZmTIP1 protein is any one of the following proteins:

   (A1) protein with an amino acid sequence of SEQ ID No.1 or SEQ ID No. 2;

   (A2) a corn-derived protein having a homology of 99% or more with the amino acid sequence defined in (a1) and having the same function;

   (A3) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A2).
4. 4. Use according to claim 3, characterized in that: the activity and/or expression level of the ZmTIP1 protein or the coding gene thereof in the plant is increased, and the length of the root hair of the plant is increased; the activity and/or expression of the ZmTIP1 protein or the coding gene thereof in the plant is reduced, and the length of the root hair of the plant is shortened.
5. 5. Use according to claim 1 or 3, characterized in that: the nucleic acid molecule capable of expressing the ZmTIP1 protein is a DNA molecule shown in SEQ ID No.3 or SEQ ID No. 4.
6. 6. Use according to claim 1 or 3, characterized in that: the plant is a monocotyledon or a dicotyledon.
7. 7. Use according to claim 6, characterized in that: the monocotyledon is a gramineous plant; the dicotyledonous plant is a cruciferous plant.
8. 8. Use according to claim 7, characterized in that: the gramineous plant is corn; the cruciferous plant is arabidopsis thaliana.
9. 9. A method of breeding a plant variety by method a or method B or method C or method D:

   the method A comprises the following steps: a method for breeding a plant variety with improved drought resistance, comprising the step of increasing the expression level and/or activity of ZmTIP1 protein in a recipient plant;

   the method B comprises the following steps: a method for breeding a plant variety with reduced drought resistance, comprising the step of reducing the expression level and/or activity of ZmTIP1 protein in a recipient plant;

   the method C comprises the following steps: a method for producing a plant variety having an increased root hair length, comprising the step of increasing the expression level and/or activity of ZmTIP1 protein in a recipient plant;

   the method D comprises the following steps: a method for producing a plant variety having a reduced root hair length, comprising the step of reducing the expression level and/or activity of ZmTIP1 protein in a recipient plant;

   the ZmTIP1 protein is any one of the following proteins:

   (A1) protein with an amino acid sequence of SEQ ID No.1 or SEQ ID No. 2;

   (A2) a corn-derived protein having a homology of 99% or more with the amino acid sequence defined in (a1) and having the same function;

   (A3) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A2).
10. 10. A method of growing a transgenic plant of method E or method F or method G or method H:

    the method E comprises the following steps: a method of breeding a transgenic plant with improved drought resistance comprising the steps of: introducing a nucleic acid molecule capable of expressing ZmTIP1 protein into a receptor plant to obtain a transgenic plant; the transgenic plant has increased drought resistance compared to the recipient plant;

    method F: a method of breeding a transgenic plant with reduced drought resistance comprising the steps of: inhibiting and expressing the encoding gene of ZmTIP1 protein in a receptor plant to obtain a transgenic plant; the transgenic plant has reduced drought resistance as compared to the recipient plant;

    method G: a method of breeding transgenic plants with increased root hair length comprising the steps of: introducing a nucleic acid molecule capable of expressing ZmTIP1 protein into a receptor plant to obtain a transgenic plant; an increase in root hair length of said transgenic plant compared to said recipient plant;

    method H: a method of breeding transgenic plants with reduced root hair length comprising the steps of: inhibiting and expressing the encoding gene of ZmTIP1 protein in a receptor plant to obtain a transgenic plant; the transgenic plant has a reduced root hair length compared to the recipient plant;

    the ZmTIP1 protein is any one of the following proteins:

    (A1) protein with an amino acid sequence of SEQ ID No.1 or SEQ ID No. 2;

    (A2) a corn-derived protein having a homology of 99% or more with the amino acid sequence defined in (a1) and having the same function;

    (A3) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A2).
11. 11. The method according to claim 9 or 10, characterized in that: the nucleic acid molecule capable of expressing the ZmTIP1 protein is a coding gene of the ZmTIP1 protein;

    the encoding gene of the ZmTIP1 protein is a DNA molecule shown in SEQ ID No.3 or SEQ ID No. 4.
12. 12. The method according to claim 9 or 10, characterized in that: the plant is a monocotyledon or a dicotyledon.
13. 13. The method of claim 12, wherein: the monocotyledon is a gramineous plant; the dicotyledonous plant is a cruciferous plant.
14. 14. The method of claim 13, wherein: the gramineous plant is corn; the cruciferous plant is arabidopsis thaliana.

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