Application of ZmEIN3a gene in regulating plant root hair trait

Abstract

The application discloses application of a ZmEIN3a gene in regulating a plant root hair character, and obtains overexpression strains containing the ZmEIN3a gene in corn through transgenic technology, wherein the transgenic material presents an excellent root hair phenotype character, the root hair length is significantly increased, and the root hair density is also obviously increased. Through application of the gene, the molecular mechanism of the gene in regulating the corn root hair phenotype is determined, which has important guiding significance for cultivating corn multi-root hair and long root hair varieties, high-yield and stress-resistant varieties, and lays a foundation for further research on the function of the ZmEIN3a gene and corn materials with excellent root hair phenotype.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant genetic engineering, in particular, to application of ZmEIN3a gene in regulating root hair traits of plants. BACKGROUND

[0002] Maize is a nutrient-rich food crop, and is also an important agricultural feed and industrial processing raw material. Root hair is an extension structure of root epidermal cells, and plays an important role in the acquisition of nutrients and water of crops, anchoring to soil, and response to biological and non-biological stress. Root hair traits have important influence on the growth status of maize plants, yield, and drought stress adaptation and resistance to lodging of plants. Obtaining excellent maize germplasm resources with ideal root hair phenotype is an important goal of breeders. Roots are located underground and cannot be directly observed. It is difficult to operate and the data error is large by using the method of digging soil roots and auxiliary trait measurement. It is difficult to obtain breeding materials with root hair traits by traditional breeding methods. By genetic manipulation or transgenic technology to change the function of target genes, the root hair traits of maize can be effectively improved. Therefore, obtaining the regulation gene resources of maize root hair traits has important theoretical and application value for maize breeding. SUMMARY

[0003] The purpose of the present application is to provide application of ZmEIN3a gene in regulating root hair traits of plants.

[0004] In order to achieve the purpose of the present application, in the first aspect, the present application provides application of ZmEIN3a gene or biological material containing the gene in regulating root hair traits of plants.

[0005] The ZmEIN3a gene is a gene encoding the following protein (A) or (B):

[0006] (A) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2; or

[0007] (B) a protein derived from (A) by substitution, deletion or addition of one or more amino acids to the sequence shown in SEQ ID NO: 2 and having equivalent function.

[0008] The biological material includes but is not limited to recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, engineered bacteria or non-reproducible plant part.

[0009] Further, overexpression of the ZmEIN3a gene in plants increases the number and length of root hairs of transgenic plants.

[0010] In the present application, the plants include monocotyledonous plants and dicotyledonous plants. Preferably, the plants are Gramineae Zea mays plants, and more preferably, the plants are maize.

[0011] In a second aspect, the present application provides a method for promoting root hair length and increasing root hair density, promoting plant growth, and improving plant stress resistance, the method comprising:

[0012] 1) causing the plant to comprise a ZmEIN3a gene; or

[0013] 2) causing the plant to overexpress a ZmEIN3a gene;

[0014] Further, the overexpression can be achieved by any of the following 1) to 5), or a combination thereof:

[0015] 1) by introducing a plasmid comprising the gene;

[0016] 2) by increasing the copy number of the gene on the chromosome of the plant;

[0017] 3) by changing the promoter sequence of the gene on the chromosome of the plant;

[0018] 4) by operably linking a strong promoter to the gene;

[0019] 5) by introducing an enhancer.

[0020] The stress resistance refers to the plant's ability to survive in adverse environments such as drought or nutrient deficiency.

[0021] In a third aspect, the present application provides the use of a transgenic plant obtained by the method in plant breeding.

[0022] The breeding methods include, but are not limited to, transgenesis, hybridization, backcrossing, selfing, or asexual reproduction.

[0023] By the above technical solution, the present application has at least the following advantages and beneficial effects:

[0024] The present application provides a gene ZmEIN3a that can effectively improve the root hair traits of corn, and the application of the gene can help to clarify the molecular mechanism of the gene in regulating the root hair phenotype of corn, which is of great significance for breeding corn varieties with more root hairs, long root hairs, high yield, and stress resistance.

[0025] The present application obtains overexpression lines containing the ZmEIN3a gene in corn through transgenic technology, and the transgenic materials exhibit excellent root hair phenotype traits, with significantly increased root hair length and density. This lays a foundation for further research on the function of the ZmEIN3a gene and the obtaining of corn materials with excellent root hair phenotype. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1The expression of the ZmEIN3a and ZmEIN3b genes in different tissues and parts of corn in the preferred embodiments of the present application.

[0027] Figure 2 The relative expression amount of the ZmEIN3a and ZmEIN3b genes in the root hairs in the preferred embodiments of the present application.

[0028] Figure 3 The root hair phenotype of the zmein3a and zmein3b mutants in the preferred embodiments of the present application. WT: B73 wild type, the background material of the mutants, as a control, zmein3a and zmein3b are mutants.

[0029] Figure 4 The root hair phenotype of the ZmEIN3a overexpression transgenic corn in the preferred embodiments of the present application. A: the expression amount analysis of the ZmEIN3a gene in the transgenic lines; B: the root hair phenotype diagram of the transgenic lines; C: the root hair length determination results of the transgenic lines; D: the root hair density determination results of the transgenic lines. In each diagram, WT is the KN5585 wild type control, i.e. the background material of the transgenic lines; #1 and #2 represent the ZmEIN3a overexpression transgenic line numbers respectively. ** represents a significant difference, P<0.01.

[0030] Figure 5 The root hair phenotype of the ZmEIN3b overexpression transgenic corn in the preferred embodiments of the present application. A: the expression amount analysis of the ZmEIN3b gene in the transgenic lines; B: the root hair phenotype diagram of the transgenic lines; C: the root hair length determination results of the transgenic lines; D: the root hair density determination results of the transgenic lines. In each diagram, WT is the transgenic background material KN5585 wild type, #1 and #2 represent the ZmEIN3b overexpression transgenic line numbers respectively. ** represents a significant difference, P<0.01. DETAILED DESCRIPTION

[0031] The present application provides two corn root system trait regulating genes ZmEIN3a and ZmEIN3b and their application in corn breeding.

[0032] The present application provides two corn EIN3 genes (named as ZmEIN3a and ZmEIN3b respectively) which can effectively increase the root hair length and the root hair quantity of corn. Through the functional loss and overexpression analysis of the two genes, the role of the two genes in regulating the root hair traits of corn is further clarified, which has important guiding significance for cultivating high-yield varieties with excellent root system traits of corn, and is also beneficial to cultivating high-yield corn varieties under poor water and nutrient conditions. The present application provides valuable gene resources for improving the root system traits of corn.

[0033] The nucleotide sequences of the ZmEIN3a and ZmEIN3b genes are shown as SEQ ID NO: 1 and SEQ ID NO: 3, and the amino acid sequences of the encoded proteins are shown as SEQ ID NO: 2 and SEQ ID NO: 4. The mRNA sequence of ZmEIN3a has 1929 bases, and ZmEIN3a can encode a protein having 642 amino acids; the mRNA sequence of ZmEIN3b has 1944 bases, and ZmEIN3b can encode a protein having 647 amino acids. The present application also includes functionally equivalent amino acid sequences formed by substitution, deletion, or addition of one or more amino acids of the amino acid sequences shown as SEQ ID NO: 2 and SEQ ID NO: 4, and nucleotide sequences encoding these amino acid sequences.

[0034] The present application also provides biological materials containing the gene ZmEIN3a or ZmEIN3b, including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, or non-regenerative plant parts.

[0035] The present application also provides the use of the genes ZmEIN3a and ZmEIN3b, or the biological materials containing the gene ZmEIN3a or ZmEIN3b, in regulating the growth and development of plants. Overexpression of the gene ZmEIN3a or ZmEIN3b in maize increases the number and length of root hairs of transgenic plants, and promotes plant growth.

[0036] The present application also provides the use of the genes ZmEIN3a and ZmEIN3b, or the biological materials containing the gene ZmEIN3a or ZmEIN3b, in regulating the growth and development of plants. Overexpression of the gene ZmEIN3a or ZmEIN3b in maize increases the number and length of root hairs of transgenic plants, and promotes plant growth.

[0037] The present application also provides the use of the genes ZmEIN3a and ZmEIN3b, or the biological materials containing the gene ZmEIN3a or ZmEIN3b, in regulating the growth and development of plants. Overexpression of the gene ZmEIN3a or ZmEIN3b in maize increases the number and length of root hairs of transgenic plants, and promotes plant growth.

[0038] The present application also provides the use of the genes ZmEIN3a and ZmEIN3b, or the biological materials containing the gene ZmEIN3a or ZmEIN3b, in regulating the growth and development of plants. Overexpression of the gene ZmEIN3a or ZmEIN3b in maize increases the number and length of root hairs of transgenic plants, and promotes plant growth.

[0039] In the present application, the plants include monocotyledonous plants and dicotyledonous plants, preferably different inbred varieties of the grass family Zea mays.

[0040] The present application also provides a method for promoting the length and density of root hairs of plants, the method comprising:

[0041] 1) causing the plant to contain the gene ZmEIN3a or ZmEIN3b; or

[0042] 2) overexpressing the gene ZmEIN3a or ZmEIN3b in the plant.

[0043] Preferably, the plant is a different inbred variety of Zea mays.

[0044] The method includes, but is not limited to, transgenesis, hybridization, backcrossing, selfing or vegetative propagation.

[0045] Further, the method of overexpressing the gene ZmEIN3a or ZmEIN3b is selected from the following 1) to 5), or optional combinations:

[0046] 1) by introducing a plasmid with the gene ZmEIN3a or ZmEIN3b into the plant;

[0047] 2) by increasing the copy number of the gene ZmEIN3a or ZmEIN3b on the chromosome of the plant;

[0048] 3) by changing the promoter sequence of the gene ZmEIN3a or ZmEIN3b on the chromosome of the plant;

[0049] 4) by operably linking a strong promoter to the gene ZmEIN3a or ZmEIN3b;

[0050] 5) by introducing an enhancer.

[0051] In particular, the present application provides a cloning and overexpression vector construction method of the maize ZmEIN3a and ZmEIN3b genes, and through genetic transformation, a maize transgenic line with excellent root hair traits overexpressing ZmEIN3a or ZmEIN3b is obtained, and the function of the maize ZmEIN3a and ZmEIN3b genes is further studied. Through the application of any one of the two genes, the root hair phenotype of maize is improved.

[0052] The total RNA of corn B73 was extracted and reverse-transcribed into cDNA. The gene-specific primers (with homologous recombination arms at both ends) were designed, and the ZmEIN3a gene with a full length of 1929 bp and the ZmEIN3b gene with a full length of 1944 bp were obtained by PCR amplification. After the two PCR products were run on an agarose gel and the gel was cut and recovered, the recovered products were subjected to a homologous recombination reaction with the recovered product of the NcoI and BstEII double-digested pCAMBIA3301 overexpression vector (driven by the CaMV35S promoter). The product was transformed into E. coli, and then subjected to bacterial liquid PCR identification. The plasmid of the positive clone was extracted and subjected to a sequencing reaction to confirm the successful construction of the vector. The recombinant plasmid was transformed into Agrobacterium, and the corn embryo material was treated with the Agrobacterium liquid containing the ZmEIN3a and ZmEIN3b overexpression recombinant plasmids, respectively. Through resistance screening culture and PCR identification of the Bar gene, the transgenic corn lines overexpressing ZmEIN3a and ZmEIN3b were obtained, respectively, thereby laying a foundation for obtaining transgenic plants with excellent root hair phenotype.

[0053] The specific method is as follows:

[0054] 1. The total RNA was extracted from the corn B73 leaves, and reverse-transcribed into cDNA. The primers were designed to perform PCR to obtain the PCR products containing the CDS of ZmEIN3a and ZmEIN3b genes (with homologous recombination arms at both ends). The upstream and downstream primers for amplifying the ZmEIN3a and ZmEIN3b genes were named ZmEIN3a-F and ZmEIN3a-R, and ZmEIN3b-F and ZmEIN3b-R, respectively.

[0055] ZmEIN3a-F: 5'-GAGAGAACACGGGGGACTCTTGACCATGATGGGAGGCGGGCTGTTGGTGGATCA-3'

[0056] ZmEIN3b-F: 5'-AGAGAACACGGGGGACTCTTGACCATGATGGGAGGCGGGCTGATGATGG-3'

[0057] 2. The plant expression vector pCAMBIA3301 is double enzyme cut by NcoI and BstEII, and then agarose gel electrophoresis is performed and the enzyme cut product is recovered by cutting the gel, the linearized vector is subjected to homologous recombination with the PCR product recovered by cutting the gel, the product is transformed into E. coli, the bacterial liquid is coated on a resistant plate (kanamycin) and cultured at 37 DEG C for inversion overnight, a single colony is picked and shaken, the bacterial liquid is subjected to PCR identification, the positive clone is subjected to plasmid extraction and sequencing, and it is proved that the sequence is correct and the insertion direction on the vector is correct. It is proved that the maize ZmEIN3a and ZmEIN3b genes are successfully cloned and constructed into the maize expression vector.

[0058] The different types of roots and leaf materials of the maize inbred line B73 at the seedling stage and the tasseling stage are collected, total RNA is extracted and reverse transcribed into cDNA, and the fluorescent quantitative PCR analysis of the ZmEIN3a and ZmEIN3b genes is performed. The experimental results show that the ZmEIN3a and ZmEIN3b genes are expressed in the primary root, seed root, crown root and leaf at the seedling stage, and the total root, stem node, interstem, leaf and tassel at the tasseling stage, and the expression amount trends of the two genes in each part are similar. Figure 1 It is proved that the ZmEIN3a and ZmEIN3b genes have important regulation effects on the growth and development of maize.

[0059] The root hair material of the maize seedling root is further scraped, and the relative expression amount of the ZmEIN3a and ZmEIN3b genes in the root hair is detected. The experimental results show that the two genes are expressed in the root hair, and the expression amount of ZmEIN3b is slightly higher than that of ZmEIN3a. Figure 2

[0060] The root hair phenotype analysis of the maize zmein3a mutant and zmein3b mutant (the target protein coding sequence is terminated in advance due to EMS mutation) is performed, compared with the control, the root hair length of the mutant material is shorter, and the root hair density is also sparser, Figure 3 It is proved that the ZmEIN3a and ZmEIN3b genes regulate the root hair traits of maize.

[0061] Further, the overexpression strain containing the ZmEIN3a or ZmEIN3b gene in maize is obtained through the transgenic technology, and the experimental results show that the transgenic material presents excellent root hair phenotype traits, the root hair length is significantly increased, and the root hair density is also obviously increased. Figure 4 Figure 5

[0062] ​​​Under the same growth conditions, the root hair length and density of ZmEIN3a overexpression transgenic maize and ZmEIN3b overexpression transgenic maize are obviously better than that of wild type control KN5585, while the root hair phenotype of zmein3a and zmein3b mutants are obviously weaker than that of its wild type background material B73. It is illustrated that no matter in which maize background material, ZmEIN3a and ZmEIN3b are involved in the regulation of root hair phenotype, and overexpression of ZmEIN3a or ZmEIN3b can obviously improve the root hair length and density.

[0063] The present application effectively improves the maize root hair phenotype by overexpression of ZmEIN3a or ZmEIN3b through transgenic technology, and provides a train of thought for obtaining excellent maize root hair phenotype material.

[0064] The following examples are used to illustrate the present application, but not to limit the scope of the present application. If not specifically indicated, the technical means used in the examples are the conventional means familiar to those skilled in the art, and the raw materials used are commercially available goods.

[0065] Example 1 Cloning of maize ZmEIN3a and ZmEIN3b genes and construction of plant expression vector

[0066] 1.1 Experimental materials: maize B73 seedlings, E. coli competent strain DH5α, Agrobacterium strain EHA105, plant overexpression vector pCAMBIA3301, plant total RNA extraction reagent Trizol, reverse transcription kit, restriction endonuclease NcoI and BstEll, homologous recombinase, plasmid extraction kit, gel recovery kit, etc.

[0067] 1.2 Experimental method:

[0068] 1.2.1 Extraction of total RNA from maize (Trizol method)

[0069] The operation steps are as follows:

[0070] (1) Put the maize material into a centrifuge tube (with a small steel ball of 5 mm in diameter), add a small amount of liquid nitrogen, and put it into a grinder at 50 Hz for 30-60 sec.

[0071] (2) According to the amount of 50-100 mg sample / ml Trizol, add Trizol to the ground material, mix well until the material is completely dissolved in the liquid, and stand on ice for 5 min.

[0072] (3) According to the amount of 200 μl chloroform / ml Trizol, add chloroform and mix well by hand (note: do not use vortex shaker), stand on ice for 10 min, centrifuge at 4℃, 12000 rpm (about 13400 g) for 15 min.

[0073] (4) Transfer the supernatant to a new 1.5 ml centrifuge tube, add pre-cooled isopropanol according to the amount of 0.5 ml isopropanol / ml Trizol, mix well, and precipitate at -20°C for more than 30 min.

[0074] (5) Centrifuge at 4°C, 12000 rpm (about 13400 g) for 10 min, discard the liquid, add pre-cooled 75% ethanol solution according to 1 ml 75% ethanol / ml Trizol, centrifuge at 4°C, 10000 rpm (about 5180 g) for 5 min, discard the liquid, and repeat the two operations twice.

[0075] (6) Centrifuge at 4°C, 12000 rpm (about 13400 g) for 1 min, carefully suck out the excess ethanol, and then open the cover and air for about 1 min to volatilize the excess ethanol.

[0076] (7) Add 20-50 μl of RNase-free water, incubate at 55-60°C for 5-10 min to obtain the RNA solution.

[0077] The RNA sample is stored at -80°C.

[0078] 1.2.2 Synthesis of cDNA

[0079] (1) Removal of genomic DNA:

[0080] Take <1 μg of total RNA, add 5×gDNA Eraser Buffer 2 μl, gDNA Eraser 1 μl, and supplement with RNase-free water to a total volume of 10 μl, mix gently, incubate at 42°C for 2 min (or treat at room temperature for 5 min), and then place on ice.

[0081] (2) Reverse transcription of RNA reaction:

[0082] The synthesis of cDNA uses Primer Script™ RT reagent Kit, and the reaction solution is prepared on ice.

[0083] Take 10 μl of the genomic DNA removal reaction solution, 1 μl of RT Primer Mix, 4 μl of 5×PrimeScript Buffer 2, 1 μl of PrimeScript RT Enzyme Mix I, and 4 μl of RNase-free water to 20 μl, mix gently and centrifuge briefly, incubate at 37°C for 15 min, incubate at 85°C for 5 sec, place the centrifuge tube on ice to terminate the reaction, and store at -20°C.

[0084] 1.2.3 Cloning of ZmEIN3a and ZmEIN3b genes

[0085] The primers were designed to amplify the cDNA of ZmEIN3a and ZmEIN3b genes by PCR, so that the PCR products contain regions that can be homologously recombined with the digested vector. The upstream and downstream primers for ZmEIN3a and ZmEIN3b genes are ZmEIN3a-F and ZmEIN3a-R, and ZmEIN3b-F and ZmEIN3b-R, respectively.

[0086] ZmEIN3a-F: 5'-GAGAGAACACGGGGGACTCTTGACCATGATGGGAGGCGGGCTGTTGGTGGATCA-3' ZmEIN3a-R: 5'-CGATCGGGGAAATTCGAGCTGGTCACCTCAGTAGAACCAATTGGTCCCG-3' ZmEIN3b-F: 5'-AGAGAACACGGGGGACTCTTGACCATGATGGGAGGCGGGCTGATGATGG-3' ZmEIN3b-R: 5'-CGATCGGGGAAATTCGAGCTGGTCACCTCAGTAGAACCAATTGGTCCCGTCGT-3'

[0087] PCR amplification was performed using the synthesized cDNA as a template, and the 50 μl amplification system contained 25 μl of 2x reaction buffer, 4 μl of dNTPs (2.5 mM each), 1 μl of each of the upstream and downstream primers, 1 μl of cDNA, 1 μl of KOD Fx-1-1 enzyme, and ddH2O to a total volume of 50 μl. The amplification program was as follows: 94°C for 3 min; 94°C for 30 sec, 55°C for 30 sec, 72°C for 1.5 min, 35 cycles; 72°C for 10 min, and then storage in a refrigerator at 4°C. The PCR products were subjected to agarose gel electrophoresis, and the gel was cut and recovered.

[0088] The plant overexpression vector pCAMBIA3301 (purchased from Uni Biotech (Jiangsu) Co., Ltd.) was digested with NcoI and BstEII, and the 50 μl digestion system contained 5 μl of 10x FastDigest Green Buffer, 1 μl of each of the enzymes NcoI and BstEII (10 U / μl), 1 μg of the vector plasmid, and ddH2O to a total volume of 50 μl. The mixture was gently mixed and incubated at 37°C for 2 h. The digestion products were subjected to agarose gel electrophoresis, and the target band was cut and recovered.

[0089] The agarose gel recovery of the DNA sample was performed according to the following steps:

[0090] (1) The correct size of the DNA band was cut from the agarose gel (as much as possible) and placed in a new centrifuge tube, and the weight of the gel was measured.

[0091] (2) Add equal volume of binding buffer to the gel (if the gel weighs 0.1 g, its volume can be considered as 100 μl, then add 100 μl of binding buffer), and put it in 58°C water bath until the gel is completely melted, which takes about 20 min.

[0092] (3) Cool the sol product to room temperature, add it to the adsorption column (put the adsorption column in a collection tube) and centrifuge at 10,000 rpm (about 5180 g) for 1 min.

[0093] (4) Discard the waste liquid in the collection tube, add 700 μl of washing buffer, and centrifuge at 10,000 rpm (about 5180 g) for 1 min.

[0094] (5) Repeat step (4).

[0095] (6) Discard the solution, and centrifuge the adsorption column at 10,000 rpm (about 5180 g) for 2 min.

[0096] (7) Put the adsorption column in a new centrifuge tube, and add an appropriate amount (e.g. 30 μl) of elution buffer or ddH2O to the middle of the adsorption membrane, and let it stand at room temperature for 3-5 min. Centrifuge at 10,000 rpm (about 5180 g) for 2 min, and collect the DNA solution.

[0097] The reaction system for the ligation of the target gene and the vector is as follows: 1 μl of homologous recombinase, 3 μl of PCR recovery product, 30-50 ng of linearized vector, and ddH2O to 10 μl. Mix gently, and ligate at 50°C for 15 min. After the reaction, put the centrifuge tube on ice.

[0098] Transformation of E. coli:

[0099] (1) Add 5 μl of the above reaction solution to 50 μl of E. coli DH5α competent cells, mix gently, and let stand on ice for 30 min (do not shake).

[0100] (2) Heat shock at 42°C for 35 sec, and immediately put on ice for 2 min.

[0101] (3) Add 500 μl of LB liquid medium, and culture at 37°C on a shaker at 200 rpm for 1 h.

[0102] (4) Take 200 μl of the bacterial solution, and spread on an LB solid plate containing kanamycin, and culture at 37°C overnight.

[0103] The positive monoclonal colonies on the selection plate were picked and cultured in liquid medium. For the recombinant plasmid of ZmEIN3a and pCAMBIA3301 vector, the gene specific primers (5'-CCGTCATGGAGAGGCCAAA-3' and 5'-GGTAGTCGACGCCGCTAGACAT-3') were used for PCR identification with the bacterial solution as the template. For the recombinant plasmid of ZmEIN3b and CAMBIA3301, the gene specific primers (5'-GTCAATAGCATGATGCAGCAAC-3' and 5'-AATTGGTCCCGTCGTTCTTCT-3') were used for PCR identification. For the clones with clear PCR amplification bands of correct size, the plasmid was extracted and sent for sequencing. The operation steps of plasmid extraction are as follows:

[0104] (1) Equilibrium step of the adsorption column: place the adsorption column CP3 into the collection tube, add 500 μl of the equilibrium solution BL into the adsorption column, centrifuge at 12000 rpm (about 13400 g) for 1 min, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube for standby.

[0105] (2) Take 1.5 ml of the bacterial solution and place it into a new centrifuge tube, centrifuge at 12000 rpm (about 13400 g) for 1 min, and try to aspirate the supernatant (when the bacterial solution is more, the bacterial bodies can be collected into the same centrifuge tube by multiple centrifugation).

[0106] (3) Add 250 μl of solution P1 (RNase A has been added into P1 and mixed) into the centrifuge tube with bacterial bodies, and suspend the bacterial bodies thoroughly with a vortex shaker.

[0107] (4) Add 250 μl of solution P2 into the centrifuge tube, and gently turn the centrifuge tube up and down for 6-8 times, so that the bacterial bodies are fully lysed.

[0108] (5) Add 350 μl of solution P3 into the centrifuge tube, immediately gently turn up and down for 6-8 times, mix thoroughly, at this time, white flocculent precipitate appears, centrifuge at 12000 rpm (about 13400 g) for 10 min.

[0109] (6) Transfer the supernatant (note that try not to aspirate the precipitate) into the adsorption column CP3 (the adsorption column is placed in the collection tube), centrifuge at 12000 rpm (about 13400 g) for 30-60 sec, discard the waste liquid in the collection tube, and place the adsorption column CP3 back into the collection tube.

[0110] (7) Add 500 μl of the deproteinization solution PD into the adsorption column CP3, centrifuge at 12000 rpm (about 13400 g) for 30-60 sec, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.

[0111] (8) Add 600 μl of rinse solution PW (make sure the ethanol has been added to PW and mixed well) to the column, centrifuge at 12000 rpm (about 13400 g) for 30-60 sec, discard the waste in the collection tube, and place the column in the collection tube.

[0112] (9) Repeat step 8.

[0113] (10) Place the column in the collection tube, centrifuge at 12000 rpm (about 13400 g) for 2 min to remove any residual rinse solution from the column.

[0114] (11) Move the column to a new collection tube, add 50-100 μl of elution buffer EB to the middle of the membrane, let stand at room temperature for 2 min, centrifuge at 12000 rpm (about 13400 g) for 2 min, and collect the plasmid solution in the collection tube.

[0115] Wherein, the solutions P1, P2, P3, the deproteinized solution PD, and the rinse solution PW are from the Plasmid DNA Extraction Kit from Nanjing Norgen Biotek Corp.

[0116] The ZmEIN3a plant expression recombinant plasmid and the ZmEIN3b plant expression recombinant plasmid were sequenced to confirm that the inserted fragments were accurate and correct, indicating that the correct ZmEIN3a and ZmEIN3b plant expression recombinant plasmids were obtained, respectively.

[0117] The full length of the ZmEIN3a gene is 1929 bp, the nucleotide sequence is shown in SEQ ID NO: 1, and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 2. The full length of the ZmEIN3b gene is 1944 bp, the nucleotide sequence is shown in SEQ ID NO: 3, and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 4.

[0118] Example 2 Genetic transformation of ZmEIN3a and ZmEIN3b genes and identification of corn overexpression materials

[0119] The ZmEIN3a plant expression recombinant plasmid and the ZmEIN3b plant expression recombinant plasmid are respectively transformed into Agrobacterium EHA105 competent cells, positive single colonies are picked, and the ZmEIN3a plant expression recombinant plasmid is subjected to PCR identification with specific primers (5'-CCGTCATGGAGAGGCCAAA-3' and 5'-GGTAGTCGACGCCGCTAGACAT-3') of the ZmEIN3a gene, and the ZmEIN3b recombinant plasmid is subjected to PCR identification with specific primers (5'-GTCAATAGCATGATGCAGCAAC-3' and 5'-AATTGGTCCCGTCGTTCTTCT-3') of the ZmEIN3b gene, so as to prove that the target recombinant plasmid is successfully transformed into the Agrobacterium.

[0120] Agrobacterium EHA105 transformation method:

[0121] 1. The Agrobacterium competent cells stored at -80°C are thawed on ice.

[0122] 2. Under sterile conditions, 1 μg (volume not more than 10 μl) of plasmid DNA is added to the just-thawed competent cell suspension, 100 μl of competent cells are added, gently mixed, and placed in an ice water bath for 5 min.

[0123] 3. The centrifuge tube is placed in liquid nitrogen for rapid freezing for 5 min.

[0124] 4. The centrifuge tube is quickly placed in a 37°C water bath for 5 min, without shaking the water surface, and then quickly transferred to an ice water bath for 5 min.

[0125] 5. 800 μl of 2xYT or LB liquid medium without antibiotics is added, and the bacteria are cultured at 28-30°C for 2-3 h, so that the bacteria recover and express resistance.

[0126] 6. The bacteria are collected by centrifugation at 5000 rpm for 1 min, about 100 μl of supernatant is reserved, the bacteria are resuspended by gently blowing, and the bacterial solution is uniformly coated on LB solid medium plates containing the corresponding antibiotics. After the liquid in the plates is completely absorbed, the plates are inverted and cultured at 28-30°C for 48-72 h.

[0127] The Agrobacterium transformation method is used to genetically transform the ZmEIN3a and ZmEIN3b genes of the maize KN5585 embryonic material, and the operation steps are as follows:

[0128] The Agrobacterium liquid containing the plant expression recombinant plasmid was cultured overnight, and the bacterial cells were enriched by centrifugation at 4000 rpm for 1 min. Fresh corn young embryos (about 1 mm) were quickly put into 2 ml plastic centrifuge tubes containing an appropriate amount of suspension, the suspension was discarded, and the corn embryos were left. 1.0 ml of Agrobacterium suspension was added to the centrifuge tube, and the mixture was placed for 5 min. The corn young embryos with Agrobacterium adsorbed were suspended and poured into the co-culture medium. The excess Agrobacterium liquid on the surface of the young embryos was removed as much as possible in a clean bench, and the co-culture was carried out at 23°C in the dark for 3 days. Then the corn young embryos were transferred to the resting medium, and cultured at 28°C in the dark for 6 days. The young embryos with better growth state were selected and placed on the selection medium containing double proline, and cultured for two weeks. The resistant callus was transferred to the differentiation medium for differentiation culture, and was cultured at 25°C under a light cycle of 16 h light / 8 h dark for about 3 weeks. The differentiated corn seedlings were transferred to the rooting medium for further culture until roots grew. Then the seedlings were transferred to a small pot containing nutrient soil for growth. When the plants grew 8-10 leaves, they were transplanted to a greenhouse for growth until the offspring seeds were harvested. The corn plant leaves were taken, the genome was extracted, the Bar gene fragment was amplified by PCR (the primers used were 5'-CCATCGTCAACCACTACATCGAGACA-3' and 5'-CTTCAGCAGGTGGGTGTAGAGCGT-3'), and the transgenic lines were identified.

[0129] The expression levels of the target genes in the ZmEIN3a overexpression lines and the ZmEIN3b overexpression lines were detected by fluorescent quantitative PCR. The upstream primer for analyzing the expression amount of the ZmEIN3a gene was qRT-ZmEIN3a-F, and the downstream primer was qRT-ZmEIN3a-R. The upstream primer for analyzing the expression amount of the ZmEIN3b gene was qRT-ZmEIN3b-F, and the downstream primer was qRT-ZmEIN3b-R. Actin was used as an internal reference gene, the upstream primer sequence was ZmActin-F, and the downstream primer sequence was ZmActin-R.

[0130] qRT-ZmEIN3a-F: 5'-CCGTCATGGAGAGGCCAAA-3'

[0131] qRT-ZmEIN3a-R: 5'-GGTAGTCGACGCCGCTAGACAT-3'

[0132] qRT-ZmEIN3b-F: 5'-GTCAATAGCATGATGCAGCAAC-3'

[0133] qRT-ZmEIN3b-R: 5'-AATTGGTCCCGTCGTTCTTCT-3'

[0134] ZmActin-F: 5'-GATTCCTGGGATTGCCGAT-3'

[0135] ZmActin-R: 5'-TCTGCTGCTGAAAAGTGCTGAG-3'

[0136] Results are shown in Figure 4 As shown in Fig. 2A, the expression level of ZmEIN3a gene in ZmEIN3a overexpression lines was significantly higher than that of wild type, which was about 5 and 3 times of the control material, respectively. As shown in Fig. 2B, the expression level of ZmEIN3b in ZmEIN3b overexpression lines was about 2 and 6 times of the wild type, respectively. Figure 5

[0137] Example 3 Root hair phenotype analysis of ZmEIN3a and ZmEIN3b transgenic plants

[0138] According to Example 2, ZmEIN3a overexpression transgenic lines and ZmEIN3b overexpression lines were obtained in maize, respectively, wherein the root hair length of ZmEIN3a OE lines #1 and #2 and ZmEIN3b OE lines #1 and #2 were significantly increased, and the root hair density was also significantly increased, compared with the WT control KN5585, and the results are shown in Fig. 3A and Fig. 3B. Figure 4 (B-D) and Figure 5 (B-D).

[0139] Although the present application has been described in detail with general description and specific embodiments above, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of the present application claimed.​

Claims

1. Application of the ZmEIN3a gene or biological materials containing the gene in regulating plant root hair traits; The amino acid sequence of the protein encoded by the ZmEIN3a gene is shown in SEQ ID NO: 2; The biological material is an expression cassette, a transposon, a plasmid vector, a viral vector or an engineered bacterium; The plant root hair traits are the number and length of corn root hairs.

2. A method for promoting the length and density of corn root hairs, characterized in that: The method comprises: causing corn to overexpress the ZmEIN3a gene; The amino acid sequence of the protein encoded by the ZmEIN3a gene is shown in SEQ ID NO:

2.

3. The method according to claim 2, characterized in that The overexpression method is selected from the following 1) to 5), or an optional combination: 1) by introducing a plasmid carrying the gene; 2) by increasing the copy number of the gene on the plant chromosome; 3) by changing the promoter sequence of the gene on the plant chromosome; 4) by operably linking a strong promoter to the gene; 5) By introducing enhancers.

4. Use of the transgenic corn obtained according to the method of claim 2 or 3 in plant breeding to promote the length and density of corn root hairs.

5. The use according to claim 4, characterized in that Breeding methods include transgenics, hybridization, backcrossing, selfing or asexual reproduction.

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