Application of ZmEIN3b gene in regulating plant root hair trait

Abstract

The application discloses application of a ZmEIN3b gene in regulating a root hair character of a plant. The application obtains an overexpression strain containing the ZmEIN3b gene in corn through a transgenic technology, and 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 ZmEIN3b gene and corn material with excellent root hair phenotype in the future.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant genetic engineering, in particular, to application of ZmEIN3b 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 ZmEIN3b 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 ZmEIN3b gene or biological material containing the gene in regulating root hair traits of plants.

[0005] The ZmEIN3b 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: 4; 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: 4 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 ZmEIN3b 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 ZmEIN3b gene; or

[0013] 2) causing the plant to overexpress a ZmEIN3b 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 ZmEIN3b 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 ZmEIN3b gene in corn through transgenic technology, and the transgenic material exhibits 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 ZmEIN3b gene and the obtaining of corn materials with excellent root hair phenotype. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1The expression of maize ZmEIN3a and ZmEIN3b genes in different tissues and parts of maize in a preferred embodiment of the present invention is shown.

[0027] Figure 2 The relative expression levels of ZmEIN3a and ZmEIN3b genes in maize root hairs in a preferred embodiment of the present invention.

[0028] Figure 3 Figure 1 is a diagram of the root hair phenotype of zmein3a and zmein3b mutants in a preferred embodiment of the present invention, wherein WT: B73 wild type, the background material of the mutant, serves as a control, and zmein3a and zmein3b are mutants.

[0029] Figure 4 Figure 2 shows the root hair phenotype of ZmEIN3a-overexpressing transgenic maize in a preferred embodiment of the present invention. A: Analysis of ZmEIN3a gene expression in transgenic lines; B: Root hair phenotype of transgenic lines; C: Root hair length measurement results of transgenic lines; D: Root hair density measurement results of transgenic lines. In each figure, WT is the KN5585 wild-type control, the background material of the transgenic lines; #1 and #2 are the ZmEIN3a-overexpressing transgenic lines. ** indicates significant difference, P < 0.01.

[0030] Figure 5 Figure 2 shows the root hair phenotype of ZmEIN3b-overexpressing transgenic maize in a preferred embodiment of the present invention. A: ZmEIN3b gene expression analysis in transgenic lines; B: Root hair phenotype of transgenic lines; C: Root hair length measurement results of transgenic lines; D: Root hair density measurement results of transgenic lines. In each figure, WT represents the wild-type KN5585 transgenic background material, and #1 and #2 represent the ZmEIN3b-overexpressing transgenic lines. ** indicates significant difference, P < 0.01. DETAILED DESCRIPTION

[0031] The present invention provides two maize root trait regulatory genes ZmEIN3a and ZmEIN3b and applications thereof in maize breeding.

[0032] The present invention provides two maize EIN3 genes (designated ZmEIN3a and ZmEIN3b) that can effectively increase maize root hair length and number. Functional loss and overexpression analysis of these two genes further clarified their role in regulating maize root hair traits. This has important guiding significance for cultivating high-yielding maize varieties with excellent root traits and is also beneficial for cultivating high-yielding maize varieties under conditions such as poor water and nutrient availability. This invention provides a valuable genetic resource for improving maize root traits.

[0033] The nucleotide sequences of the ZmEIN3a and ZmEIN3b genes are shown in SEQ ID NO:1 and SEQ ID NO:3, and the amino acid sequences of the proteins they encode are shown in SEQ ID NO:2 and SEQ ID NO:4 in the sequence listing. The mRNA sequence of ZmEIN3a consists of 1929 bases and encodes a protein with 642 amino acids; the mRNA sequence of ZmEIN3b consists of 1944 bases and encodes a protein with 647 amino acids. The present invention also includes functionally equivalent amino acid sequences formed by replacing, deleting, or adding one or more amino acids to the amino acid sequences shown in SEQ ID NO:2 and SEQ ID NO:4, as well as nucleotide sequences encoding these amino acid sequences.

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

[0035] The present invention also provides the use of the genes ZmEIN3a and ZmEIN3b, or biomaterials containing the genes ZmEIN3a or ZmEIN3b, in regulating plant growth and development. Overexpressing the genes ZmEIN3a or ZmEIN3b in corn increases the number and length of root hairs in transgenic plants, thereby promoting plant growth.

[0036] The present invention also provides the use of the genes ZmEIN3a and ZmEIN3b, or biological materials containing the genes ZmEIN3a or ZmEIN3b, in regulating plants in adverse environments such as drought or nutrient deficiency.

[0037] The present invention also provides the use of the genes ZmEIN3a and ZmEIN3b, or biological materials containing the genes ZmEIN3a or ZmEIN3b, in preparing transgenic plants.

[0038] The present invention also provides the use of the genes ZmEIN3a and ZmEIN3b, or biological materials containing the genes ZmEIN3a or ZmEIN3b, in plant breeding (including yield trait breeding).

[0039] In the present invention, the plants include monocotyledonous plants and dicotyledonous plants, preferably different inbred varieties of corn of the genus Zea of ​​the Poaceae family.

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

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

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

[0043] Preferably, the plants are different inbred varieties of corn of the genus Zea of ​​the family Poaceae.

[0044] Such methods include, but are not limited to, transgenic, hybridization, backcrossing, selfing, or asexual reproduction.

[0045] Furthermore, the method for overexpressing the gene ZmEIN3a or ZmEIN3b is selected from the following 1) to 5), or an optional combination:

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

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

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

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

[0050] 5) By introducing enhancers.

[0051] Specifically, the present invention provides methods for cloning the maize ZmEIN3a and ZmEIN3b genes and constructing overexpression vectors. Through genetic transformation, transgenic maize lines overexpressing ZmEIN3a or ZmEIN3b, respectively, with superior root hair traits, are obtained. The functions of the maize ZmEIN3a and ZmEIN3b genes can be further investigated. Application of either gene can improve the root hair phenotype of maize.

[0052] Total RNA was extracted from maize B73 and reverse transcribed into cDNA. Gene-specific primers (with homologous recombination arms at both ends) were designed and PCR amplified to obtain the full-length 1929-bp ZmEIN3a and 1944-bp ZmEIN3b genes. The two PCR products were run on agarose gels and excised. Homologous recombination reactions were performed with the excised products of the pCAMBIA3301 overexpression vector (driven by the CaMV35S promoter) digested with NcoI and BstEII. The products were transformed into Escherichia coli and identified by PCR. Plasmids were extracted from positive clones and sequenced to confirm successful vector construction. The recombinant plasmid was transformed into Agrobacterium, and the maize immature embryo material was treated with Agrobacterium culture containing the ZmEIN3a and ZmEIN3b overexpression recombinant plasmids, respectively. Through resistance screening culture and PCR identification of the Bar gene, transgenic maize lines overexpressing ZmEIN3a and ZmEIN3b were obtained, respectively, laying the foundation for obtaining transgenic plants with excellent root hair phenotypes.

[0053] The specific method is as follows:

[0054] 1. Total RNA was extracted from maize B73 leaves and reverse transcribed into cDNA. Primers were designed for PCR amplification to obtain PCR products containing the CDS of the ZmEIN3a and ZmEIN3b genes (with homologous recombination arms at both ends). The upstream and downstream primers used to amplify 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' ZmEIN3a-R: 5'-CGATCGGGGAAATTCGAGCTGGTCACCTCAGTAGAACCAATTGGTCCCG-3'

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

[0057] 2. The plant expression vector pCAMBIA3301 was double-digested with NcoI and BstEII, and the digested products were recovered by agarose gel electrophoresis and excision. The linearized vector and the PCR product recovered from the gel were homologously recombined using a homologous recombinase. The products were transformed into Escherichia coli, and the bacterial suspension was plated on a resistant plate (kanamycin) and incubated inverted overnight at 37°C. Single colonies were selected and shaken, and the suspension was identified by PCR. Plasmids from positive clones were extracted and sequenced to confirm that the sequences were correct and that the insertion orientation in the vector was correct. This confirmed that the maize ZmEIN3a and ZmEIN3b genes had been successfully cloned and constructed into the maize expression vectors.

[0058] Roots and leaves of different parts of the maize inbred line B73 were collected during the seedling and tasting stages. Total RNA was extracted and reverse-transcribed into cDNA for quantitative PCR analysis of the ZmEIN3a and ZmEIN3b genes. The results showed that both ZmEIN3a and ZmEIN3b genes were expressed in primary roots, seminal roots, crown roots, and leaves during the seedling stage, as well as in total roots, stem nodes, interstems, leaves, and tassels during the tasting stage. The expression trends of the two genes were similar across all parts. Figure 1 This indicates that ZmEIN3a and ZmEIN3b genes play an important regulatory role in the growth and development of maize.

[0059] Root hair materials of maize seedlings were further scraped to detect the relative expression levels of ZmEIN3a and ZmEIN3b genes in root hairs. The experimental results showed that both genes were expressed in root hairs, with the expression level of ZmEIN3b slightly higher than that of ZmEIN3a ( Figure 2 ).

[0060] The present invention conducted root hair phenotype analysis on maize zmein3a mutant and zmein3b mutant (due to EMS mutation, the target protein coding sequence was terminated prematurely). Compared with the control, the root hair length of the mutant material was shorter and the root hair density was sparser ( Figure 3 ), indicating that ZmEIN3a and ZmEIN3b genes regulate maize root hair traits.

[0061] Furthermore, genetic engineering was used to obtain overexpression lines of the ZmEIN3a or ZmEIN3b gene in maize. The experimental results showed that the transgenic materials exhibited excellent root hair phenotypes, with significantly increased root hair length and root hair density ( Figure 4 and 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 and add pre-cooled isopropanol at a ratio 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, 12,000 rpm (approximately 13,400 g) for 10 min, discard the liquid, add pre-cooled 75% ethanol solution at a ratio of 1 ml 75% ethanol / ml Trizol, centrifuge at 4°C, 10,000 rpm (approximately 5,180 g) for 5 min, discard the liquid, and repeat this operation twice.

[0075] (6) Centrifuge at 4°C, 12,000 rpm (approximately 13,400 g) for 1 min, carefully aspirate excess ethanol, and then open the lid and let it air dry for about 1 min to evaporate the excess ethanol.

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

[0077] RNA samples were frozen at -80°C.

[0078] 1.2.2 cDNA Synthesis

[0079] (1) Removal of genomic DNA:

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

[0081] (2) Reverse transcription of RNA:

[0082] cDNA was synthesized using Primer Script™ RT reagent Kit, and the reaction solution was prepared on ice.

[0083] Take 10 μl of the reaction solution for removing genomic DNA, 1 μl of RTPrimerMix, 4 μl of 5× PrimeScriptBuffer2, 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] Primers were designed for PCR amplification of the cDNA of the ZmEIN3a and ZmEIN3b genes so that both ends of the PCR products contained regions that could undergo homologous recombination with the enzyme-digested vector. The upstream and downstream primers for amplifying the ZmEIN3a and ZmEIN3b genes were 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 reverse-transcribed cDNA as a template. A 50 μl amplification system consisted of 25 μl of 2× reaction buffer, 4 μl of 2.5 mM dNTPs, 1 μl of upstream and downstream primers, 1 μl of cDNA, 1 μl of KOD Fx-1-1 enzyme, and ddH2O to 50 μl. The amplification protocol was as follows: 94°C for 3 min; 35 cycles of 94°C for 30 sec, 55°C for 30 sec, and 72°C for 1.5 min; and 72°C for 10 min. The product was then refrigerated at 4°C until ready for use. The PCR product was subjected to agarose gel electrophoresis and recovered.

[0088] The plant overexpression vector pCAMBIA3301 (purchased from Weimi Biotechnology (Jiangsu) Co., Ltd.) was double-digested with NcoI and BstEII. 50 μl of the digestion system consisted of 5 μl of 10× FastDigest Green Buffer, 1 μl each of NcoI and BstEII (10 U / μl), 1 μg of the vector plasmid, and ddH2O to 50 μl. Gently mix and incubate at 37°C in a water bath for 2 h. The digestion products were subjected to agarose gel electrophoresis, and the target band was recovered by excision.

[0089] The steps for agarose gel recovery of DNA samples are as follows:

[0090] (1) Cut the DNA band of the correct size from the agarose gel (try to remove the excess part) and place it in a new centrifuge tube, and weigh the gel.

[0091] (2) Add an 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 incubate in a 58°C water bath until the gel is completely melted, which takes about 20 minutes.

[0092] (3) The sol product was cooled to room temperature, added to an adsorption column (the adsorption column was placed in a collection tube) and centrifuged at 10,000 rpm (about 5,180 g) for 1 min.

[0093] (4) Discard the waste liquid in the collection tube, add 700 μl Washing Buffer, and centrifuge at 10,000 rpm (about 5,180 g) for 1 min.

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

[0095] (6) Discard the solution and centrifuge the adsorption column at 10,000 rpm (about 5,180 g) for 2 minutes.

[0096] (7) Place the adsorption column in a new centrifuge tube and drop an appropriate amount (e.g., 30 μl) of elution buffer or ddH2O onto the center of the adsorption membrane. Allow to stand at room temperature for 3-5 minutes. Centrifuge at 10,000 rpm (approximately 5180 g) for 2 minutes to collect the DNA solution.

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

[0098] E. coli transformation:

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

[0100] (2) Heat shock in a 42°C water bath for 35 seconds and immediately place on ice for 2 minutes.

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

[0102] (4) Spread 200 μl of bacterial solution on a solid LB plate containing kanamycin and incubate inverted at 37°C overnight.

[0103] Positive monoclonal colonies on the transformation plates were picked and cultured in liquid culture. For the recombinant plasmids linked to the pCAMBIA3301 vector, PCR identification was performed using the bacterial suspension as a template with ZmEIN3a gene-specific primers (5'-CCGTCATGGAGAGGCCAAA-3' and 5'-GGTAGTCGACGCCGCTAGACAT-3'). For the recombinant plasmids linked to the CAMBIA3301 vector, PCR identification was performed using ZmEIN3b gene-specific primers (5'-GTCAATAGCATGATGCAGCAAC-3' and 5'-AATTGGTCCCGTCGTTCTTCT-3'). For clones with clear PCR amplification bands of the correct size, plasmids were extracted and sent for sequencing. The steps for plasmid extraction are as follows:

[0104] (1) Equilibration step of adsorption column: Place adsorption column CP3 in a collection tube, add 500 μl of equilibration solution BL to 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 later use.

[0105] (2) Take 1.5 ml of bacterial solution and place it in a new centrifuge tube. Centrifuge at 12000 rpm (about 13400 g) for 1 min. Remove the supernatant as much as possible (if the bacterial solution is large, the bacteria can be collected in the same centrifuge tube through multiple centrifugation).

[0106] (3) Add 250 μl of solution P1 (RNase A has been added to P1 and mixed thoroughly) to the centrifuge tube containing the bacterial cells, and suspend the bacterial cells thoroughly using a vortex oscillator.

[0107] (4) Add 250 μl of solution P2 to the centrifuge tube and gently invert the tube 6-8 times to fully lyse the bacteria.

[0108] (5) Add 350 μl of solution P3 to the centrifuge tube and immediately and gently invert it upside down 6-8 times to mix thoroughly. A white flocculent precipitate will appear. Centrifuge at 12000 rpm (about 13400 g) for 10 min.

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

[0110] (7) Add 500 μl of deproteinized solution PD to the adsorption column CP3, centrifuge at 12,000 rpm (about 13,400 g) for 30-60 seconds, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0111] (8) Add 600 μl of rinse solution PW to the adsorption column (please make sure that anhydrous ethanol has been added to PW and mixed evenly), centrifuge at 12000 rpm (about 13400 g) for 30-60 seconds, pour out the waste liquid in the collection tube, and place the adsorption column in the collection tube.

[0112] (9) Repeat step 8.

[0113] (10) Place the adsorption column in a collection tube and centrifuge at 12,000 rpm (about 13,400 g) for 2 min to remove the residual rinse solution in the adsorption column.

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

[0115] Among them, solutions P1, P2, P3, deproteinization solution PD, and rinse solution PW were from the plasmid DNA mini-extraction kit of Nanjing Novozymes Biotech Co., Ltd.

[0116] The ZmEIN3a plant expression recombinant plasmid and the ZmEIN3b plant expression recombinant plasmid were sequenced to confirm that the inserted fragments were accurate, 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 this 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 this gene is shown in SEQ ID NO. 4.

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

[0119] The ZmEIN3a plant expression recombinant plasmid and the ZmEIN3b plant expression recombinant plasmid were transformed into Agrobacterium EHA105 competent cells, respectively. Positive single colonies were picked and identified by PCR using specific primers for the ZmEIN3a gene (5'-CCGTCATGGAGAGGCCAAA-3' and 5'-GGTAGTCGACGCCGCTAGACAT-3'). The ZmEIN3b recombinant plasmid was identified by PCR using specific primers for the ZmEIN3b gene (5'-GTCAATAGCATGATGCAGCAAC-3' and 5'-AATTGGTCCCGTCGTTCTTCT-3'), demonstrating that the target recombinant plasmids were successfully transformed into Agrobacterium.

[0120] Agrobacterium EHA105 transformation method:

[0121] 1. Take the competent Agrobacterium cells stored at -80℃ and thaw them on ice.

[0122] 2. Under sterile conditions, add the plasmid to be transformed into the freshly thawed competent cell suspension. Add 1 μg (no more than 10 μl) of plasmid DNA to every 100 μl of competent cells. Mix gently and let stand in an ice water bath for 5 minutes.

[0123] 3. Place the centrifuge tube in liquid nitrogen for 5 minutes.

[0124] 4. Quickly place the centrifuge tube in a 37°C water bath and let it stand for 5 minutes without shaking the water surface. Then quickly transfer it to an ice water bath and let it stand for 5 minutes.

[0125] 5. Add 800 μl of 2×YT or LB liquid culture medium without antibiotics and culture at 28-30℃ with shaking for 2-3 hours to allow the bacteria to recover and express resistance.

[0126] 6. Collect the bacteria by centrifugation at 5000 rpm for 1 min, retain about 100 μl of supernatant, gently pipette to resuspend the bacteria, and evenly spread the bacterial liquid onto LB solid culture medium plates containing the corresponding antibiotics. After the liquid in the plates is completely absorbed, invert the plates and incubate at 28-30°C for 48-72 hours.

[0127] The ZmEIN3a and ZmEIN3b genes were genetically transformed into immature embryos of maize KN5585 using the Agrobacterium transformation method. The steps are as follows:

[0128] Agrobacterium containing the plant expression recombinant plasmid was cultured overnight and centrifuged at 4000 rpm for 1 minute to enrich the cells. Freshly peeled maize embryos (approximately 1 mm) were quickly placed in a 2 ml plastic centrifuge tube containing an appropriate amount of suspension. The suspension was discarded, leaving the maize embryos. 1.0 ml of the Agrobacterium suspension was added to the centrifuge tube and allowed to stand for 5 minutes. The maize embryos adsorbed with Agrobacterium in the centrifuge tube were suspended and poured onto the co-culture medium. In a laminar flow hood, excess Agrobacterium suspension on the embryo surface was removed as much as possible. The embryos were co-cultured at 23°C in the dark for 3 days. The maize embryos were then transferred to a resting medium and cultured in the dark at 28°C for 6 days. After 6 days of incubation, the best-performing embryos were selected and placed on a selection medium containing bialaphos. After two weeks of selection, they were transferred to a fresh selection medium and cultured for 2 weeks. The resistant callus was transferred to a differentiation medium for differentiation culture. After approximately three weeks of incubation at 25°C under a 16-hour light / 8-hour dark photoperiod, the differentiated corn seedlings were transferred to a rooting medium and cultured until roots developed. The seedlings were then transferred to small pots filled with nutrient soil for growth. After the plants developed 8-10 leaves, they were transplanted to a greenhouse for growth until the offspring seeds were harvested. Leaves from the corn plants were extracted, and the Bar gene fragment was amplified by PCR (primers 5'-CCATCGTCAACCACTACATCGAGACA-3' and 5'-CTTCAGCAGGTGGGTGTAGAGCGT-3') to identify the transgenic lines.

[0129] Quantitative PCR was used to assess the expression levels of the target genes in ZmEIN3a and ZmEIN3b overexpressing strains. The upstream primer for analyzing ZmEIN3a expression was qRT-ZmEIN3a-F, and the downstream primer was qRT-ZmEIN3a-R. The upstream primer for analyzing ZmEIN3b expression was qRT-ZmEIN3b-F, and the downstream primer was qRT-ZmEIN3b-R. Actin was used as an internal reference gene, with the upstream primer ZmActin-F and the downstream primer 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'-AATTGGTCCCGTCGTCTTCT-3'

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

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

[0136] The results are as follows Figure 4 As shown in A, in the ZmEIN3a overexpression line, the expression level of the ZmEIN3a gene was significantly higher than that of the wild type, about 5 and 3 times that of the control material, respectively. Figure 5 As shown in A, in the ZmEIN3b overexpression lines, the expression levels of ZmEIN3b were approximately 2-fold and 6-fold that of the wild type, respectively.

[0137] Example 3 Analysis of root hair phenotypes of ZmEIN3a and ZmEIN3b transgenic plants

[0138] According to Example 2, ZmEIN3a overexpressing transgenic lines and ZmEIN3b overexpressing lines were obtained in corn. Compared with the WT control KN5585, the root hair length of ZmEIN3aOE lines #1 and #2, and the root hair density of ZmEIN3bOE lines #1 and #2 were significantly increased. The results are shown in FIG. Figure 4 (BD) and Figure 5 (BD).

[0139] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. Application of the ZmEIN3b gene or biological materials containing the gene in regulating maize root hair traits; The amino acid sequence of the protein encoded by the ZmEIN3b gene is shown in SEQ ID NO: 4; The biological material is an expression cassette, a transposon, a plasmid vector, a viral vector or an engineered bacterium; Overexpression of the ZmEIN3b gene in corn increases the number and length of root hairs of transgenic plants.

2. A method for promoting the length and density of corn root hairs, characterized in that: The method comprises: Overexpression of the ZmEIN3b gene in maize; The amino acid sequence of the protein encoded by the ZmEIN3b gene is shown in SEQ ID NO:

4.

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 plant obtained according to the method of claim 2 or 3 in plant breeding; The breeding goal is 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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