ZmMYB36 gene and its application

Abstract

The present invention provides a ZmMYB36 gene, wherein the gene has a nucleotide sequence as shown in SEQ ID NO.1. The gene provided by the present invention and the application of the gene can provide a basis and direction for corn breeding and directional breeding. The gene of the present invention is of great significance for breeding new low-phosphorus tolerant varieties and research on corn tolerance to low phosphorus. For example, the gene of the present invention can be overexpressed to improve the tolerance of plants to low phosphorus.

Description

Technical Field

[0001] The present invention relates to the field of molecular biotechnology, in particular to a ZmMYB36 gene and an application thereof. Background Art

[0002] Corn is one of the three major food crops in the world, with the characteristics of high yield, great potential for yield increase, and wide application. As a food, feed and economic crop. As the second largest nutrient element after nitrogen, phosphorus is a macronutrient necessary for the growth and development of corn, and is also an important component of nucleic acids, phospholipids, ATP, etc.

[0003] The abundance of phosphorus in field soil is high, but due to chelation (phosphate ions will form insoluble substances with cations such as calcium, magnesium, iron, and aluminum), the effective phosphorus content in the soil is low, and phosphorus is distributed heterogeneously in the soil, especially its poor mobility in the soil. Corn has a high demand for phosphorus, so corn is prone to symptoms of phosphorus deficiency during the seedling stage and the critical period of female ear development.

[0004] Phosphorus is still an important factor limiting plant growth in natural and farmland ecosystems. When plants are deficient in phosphorus, their growth will be severely restricted. Phosphorus deficiency in corn during the seedling stage will promote root growth, increase root dry weight, reduce aboveground dry weight, increase root-crown ratio, and promote the accumulation of anthocyanins in the aboveground part. However, phosphorus deficiency during the maturity stage will affect corn grain setting and reduce yield.

[0005] During the growth of corn, phosphorus fertilizer can promote the growth and development of corn root system, and can also significantly increase the leaf area index and chlorophyll content of corn. Phosphorus fertilizer can also significantly increase corn grain yield and quality. Fertilization can effectively solve the problem of crop phosphorus deficiency, but in the process of corn production, excessive application of chemical fertilizers will cause agricultural non-point source pollution, causing serious adverse effects on the ecological environment. In addition, phosphate rock is a non-renewable resource, and phosphorus resources will eventually be exhausted.

[0006] Plants respond to low-phosphorus stress through a series of morphological and physiological changes. The root system is an important trait for plants to absorb and obtain nutrients. When plants suffer from low-phosphorus stress, they will promote the growth of the root system, expand the contact area between the root system and the soil, and maximize the interception of phosphorus in the soil. Plants can also absorb phosphorus by promoting mycorrhizal symbiosis. Plants release signal substances such as sugars into the soil to promote mycorrhizal symbiosis and expand the contact area between plant roots and soil. In this symbiosis, fungi absorb Pi through the hyphal network and then release it into the root endodermis through arbuscules. Symbiotic plants obtain most of the phosphates. AM symbiosis also improves plant stress resistance and soil stability. According to Advanced Plant Nutrition, edited by Li Chunjian, low phosphorus will also promote dicotyledonous plants to secrete organic acids and protons into the soil. Inorganic phosphorus in the soil mostly exists in the form of insoluble CaPO4, FePO4 and AlPO4. Releasing protons under low phosphorus conditions can lower the pH value in the soil and dissolve insoluble substances. At the same time, organic acids can chelate with iron and aluminum ions to release phosphorus for plant absorption and utilization. Low phosphorus will also increase the acid phosphatase activity of dicotyledonous plants, and the acid phosphatase secreted into the soil can hydrolyze organic phosphorus in the soil and release phosphorus under acidic conditions. The acid phosphatase in the plant body can catalyze the decomposition of phospholipids in old leaves into phosphorus, realizing the reuse of phosphorus in the plant body.

[0007] The Chinese patent with publication number CN106068858A discloses a method for studying the effect of phosphorus application on spring corn yield and soil available phosphorus content. The phosphorus application rate is 130.2 kg·hm -2 , spring corn yield is 1197.1kg·hm -2 ; Olsen-P in soil is 18.7 mg kg -1 , phosphorus leaching is 26.2 mg kg -1 However, reliance on exogenous phosphate fertilizer application is still constrained by soil properties.

[0008] Therefore, it is urgent to provide new corn materials or varieties that are tolerant to low phosphorus stress and can increase grain yield. In addition, on the one hand, due to differences in understanding among those skilled in the art; on the other hand, because the applicant studied a large number of documents and patents when making the present invention, but due to space limitations, not all details and contents are listed in detail, but this is by no means that the present invention does not have the characteristics of these prior arts, on the contrary, the present invention already has all the characteristics of the prior art, and the applicant reserves the right to add relevant prior art in the background art. Summary of the invention

[0009] In view of the shortcomings of existing corn genetic materials and technologies, the present invention provides a ZmMYB36 gene having a nucleotide sequence as shown in SEQ ID NO.1. Figure 1The ZmMYB36 gene structure shown is located on chromosome 2 of maize (Chr. 2: 187758433-187760985 bp), which includes 3 exons and 2 introns (the broken lines between exons represent introns).

[0010] The present invention provides a ZmMYB36 protein having an amino acid sequence as shown in SEQ ID NO.2. Figure 2 The schematic diagram of the protein domain information of ZmMYB36 is shown. ZmMYB36 encodes 346 amino acids. Its MYB DNA-binding domain is predicted, and it is found that it has two MYB DNA binding functional domains between amino acids 14-62 and 68-113, respectively, and belongs to the R2R3 MYB family.

[0011] The present invention provides an application of a ZmMYB36 gene in improving the tolerance of corn to low-phosphorus stress. The nucleotide sequence of the ZmMYB36 gene is shown in SEQ ID NO.1.

[0012] According to a preferred embodiment, the use comprises overexpression of the ZmMYB36 gene in corn.

[0013] The present invention provides an application of a ZmMYB36 gene in regulating the growth of a corn female ear. The nucleotide sequence of the ZmMYB36 gene is shown in SEQ ID NO.1.

[0014] The present invention provides an application of a ZmMYB36 gene in regulating corn grain number. The nucleotide sequence of the ZmMYB36 gene is shown in SEQ ID NO.1.

[0015] In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as adding genes that express enzymes or luminescent compounds that can produce color changes in plants (GUS gene, GFP gene, luciferase gene, etc.), antibiotic markers with resistance (gentamicin marker, kanamycin marker, etc.) or chemical agent resistance marker genes (such as herbicide resistance genes), etc. Considering the safety of transgenic plants, no selective marker genes can be added, and transformed plants can be directly screened by adversity.

[0016] According to a preferred embodiment, the primer pair for amplifying the full length of the ZmMYB36 gene or any fragment thereof also falls within the protection scope of the present invention.

[0017] According to a preferred embodiment, the expression cassette, transgenic cell line and recombinant bacteria containing the coding gene of the plant low-phosphorus stress tolerance related protein also fall within the protection scope of the present invention.

[0018] According to a preferred embodiment, a recombinant vector containing a gene encoding the above-mentioned protein related to plant tolerance to low-phosphorus stress also falls within the protection scope of the present invention.

[0019] The present invention provides application of a ZmMYB36 gene in cultivating corn resistant to low-phosphorus stress.

[0020] According to a preferred embodiment, the maize lines overexpressing the ZmMYB36 gene increase the ear length and grain number under low phosphorus stress.

[0021] The present invention provides a biological material containing a ZmMYB36 gene, wherein the nucleotide sequence of the ZmMYB36 gene is shown as SEQ ID NO.1.

[0022] According to a preferred embodiment, the biological material is selected from recombinant DNA, expression cassette, transposon, plasmid vector, phage vector, virus vector, engineered bacteria or non-renewable plant parts.

[0023] The plasmid vector, phage vector, and viral vector proposed in the present invention can be a genetic vector, which contains a specific nucleic acid sequence and can transfer the target nucleic acid sequence into a host cell, so that the target nucleic acid sequence is integrated into the genome of the host cell to obtain a recombinant cell.

[0024] The present invention provides a method for cultivating a plant resistant to low phosphorus stress, wherein the expression of the ZmMYB36 gene is increased by genetic engineering to improve the tolerance of corn to a low phosphorus environment. Preferably, the plant is a grass plant. Particularly preferably, the plant is corn.

[0025] According to a preferred embodiment, the plant expression vector carrying the ZmMYB36 gene of the present invention can be transformed into plant cells or tissues through conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated transformation, etc.

[0026] As a transcription factor, ZmMYB36 is specifically induced to express in the root by low phosphorus stress, and its expression level increases 5 times as the phosphorus deficiency time increases to 25 days. Analysis of the phenotypes of transgenic strains throughout the growth period found that overexpression of ZmMYB36 significantly increased root and aboveground biomass and improved the root-crown ratio under low phosphorus conditions, which was opposite to the phenotype of the ZmMYB36 knockout strain; and under low phosphorus conditions, the ZmMYB36 overexpression strain increased corn yield indicators such as grain number and ear length, indicating that the ZmMYB36 gene has an effect on corn female ear development. ZmPHR1s, an important transcription factor that responds to low phosphorus stress, regulates and promotes the expression of ZmMYB36. Based on the above findings, the present invention provides more effective gene resources and new breeding goals for cultivating high-yield crops and improving phosphorus use efficiency (PUE) under low phosphorus conditions in agriculture.

[0027] According to a preferred embodiment, corn tolerant to low phosphorus stress is cultivated by gene editing. Specifically, the present invention provides a method for cultivating corn tolerant to low phosphorus stress, comprising the following steps:

[0028] Integrating the ZmMYB36 gene into a plasmid vector to construct an overexpression vector;

[0029] Constructing genetically engineered Agrobacterium containing the overexpression vector;

[0030] The genetically engineered Agrobacterium is transformed into corn callus to obtain a homozygous mutant strain with stable inheritance.

[0031] The present invention is based on the comparison of knockout (CRISPR) and overexpression (OE) of the ZmMYB36 gene in corn, and finds that the ZmMYB36 gene is involved in the pathway induced by low phosphorus in corn, and based on the phenotypic comparison of transgenic strains and wild-type strains, finds that the ZmMYB36 gene has the effect of promoting phosphorus utilization and increasing corn ear length in corn. The method of the present invention can be used to cultivate new corn varieties resistant to low phosphorus. The gene of the present invention is of great significance for cultivating new varieties resistant to low phosphorus, and the research on corn tolerance to low phosphorus. For example, the gene of the present invention can be overexpressed to improve the tolerance of plants to low phosphorus. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of the ZmMYB36 gene structure;

[0033] Figure 2 This is a schematic diagram of the protein domain information of ZmMYB36;

[0034] Figure 3 is the expression level of maize ZmMYB36 gene under nutrient stress;

[0035] Figure 4This is a subcellular localization analysis diagram of ZmMYB36 protein;

[0036] Figure 5 To screen the expression level of ZmMYB36 gene in the obtained ZmMYB36 CRISPR strain and OE strain;

[0037] Figure 6 Statistical graphs of ear weight, grain weight, number of grains per ear, and ear length of ZmMYB36 transgenic lines;

[0038] Figure 7 This is a plant height phenotype analysis diagram of the ZmMYB36 CRISPR strain and the OE strain;

[0039] Figure 8 This is the observation diagram of the aboveground and root phenotypes of the ZmMYB36 transgenic line during maturity;

[0040] Fig. 9 The biomass statistics of ZmMYB36 transgenic lines at maturity;

[0041] Fig.10 This is the phenotype analysis diagram of the female ear of the ZmMYB36 transgenic strain in sand culture at maturity;

[0042] Fig.11 The phosphorus concentration and utilization efficiency of different parts of the transgenic strain ZmMYB36 during the reproductive growth period of sand culture.

[0043] Fig.12 The yeast one-hybridization and gel retardation migration results of His-ZmPHR1.1 and His-ZmPHR1.2 with the P1BS cis-acting element in the promoter region of ZmMYB36 gene, respectively;

[0044] Fig.13 The dual luciferase activity results of 62K-ZmPHR1.1 and 62K-ZmPHR1.2 activating ZmMYB36 gene transcription;

[0045] Fig.14 The results of yeast one-hybrid experiment of MRE (MYB transcription factor binding element) of ZmMYB36 and ZmPHT1;1, ZmPHT1;2 and ZmPHT1;6 gene promoters;

[0046] Fig.15 The yeast one-hybrid and dual-luciferase results of ZmMYB36 binding to the MRE element of the ZmGA1 gene promoter;

[0047] Fig.16 Yeast one-hybrid, gel retardation migration and chromatin immunoprecipitation-PCR results of ZmMYB36 binding to ZmYIGE1 gene;

[0048] Fig.17 Gel retardation migration and dual luciferase results for ZmMYB36 binding to ZmNPF5.6. DETAILED DESCRIPTION

[0049] The following is a detailed description with reference to the accompanying drawings.

[0050] The V2 stage in the present invention refers to the stage when corn has two fully expanded leaves. The V4 stage in the present invention refers to the stage when corn has four fully expanded leaves. The V7 stage in the present invention refers to the stage when corn has seven fully expanded leaves.

[0051] Example 1

[0052] This example provides materials and culture methods for ZmMYB36 gene overexpression and knockout corn strains.

[0053] The materials include maize inbred line B73 (wild type), ZmMYB36 overexpression strain and gene knockout strain.

[0054] Maize inbred line B73 (wild type), overexpression strains of ZmMYB36, and gene knockout strains were placed in an artificial climate chamber and cultured with nutrient solution. Maize inbred line B73 (wild type), overexpression strains of ZmMYB36, and gene knockout strains were placed in an artificial climate chamber and cultured with nutrient solution.

[0055] The steps for seed germination are as follows:

[0056] (1) Soaking the seeds in a 10% hydrogen peroxide solution for 30 minutes to sterilize the seeds; (2) After washing with deionized water three times, soaking the seeds in a saturated calcium sulfate solution for 5 hours;

[0057] (3) Germinate the seeds on moist filter paper in the dark. When the seeds have two leaves and one heart, select the seeds that are growing well and

[0058] Uniform seedlings, with the endosperm removed;

[0059] (4) Move the five seedlings into a 2 L porcelain jar for cultivation. During the cultivation period, aeration is allowed. Place

[0060] Normal nutrient solution;

[0061] (5) After three days of cultivation, the roots were treated with low nitrogen, low phosphorus or low potassium and cultivated for one week before sampling. The experiment was repeated six times. When sampling, the roots were washed with deionized water and quickly cooled in liquid nitrogen and stored in a -80°C refrigerator.

[0062] The nutrient solution formula is: 2mM NH4NO3, 2mM CaCl2, 0.2mM Fe-EDTA, 0.75mM K2SO4, 0.65mMMgSO4, 0.1mM KCl, 0.25mM KH2PO4, 1х10 -3 mM H3BO3, 1х10 -3 mM MnSO4, 1х10 -4 mM CuSO4, 1х10 -3 mM ZnSO4, 5х10 -6 mM(NH4)6MoO4.

[0063] Low nitrogen conditions are 1 / 50 of the normal nitrogen supply level. Low phosphorus is 1 / 100 of the normal phosphorus supply level. Low potassium is 1 / 100 of the normal potassium supply level.

[0064] The culture method of montmorillonite medium is:

[0065] (1) Three wild-type maize lines, three ZmMYB36 overexpression lines, and three ZmMYB36 knockout lines were selected, and 12 replicates were set for each line;

[0066] (2) Move the well-growing seedlings into pots with a diameter of 25 cm and a height of 40 cm, transplant 4 seedlings into each pot, and water them with deionized water once a day;

[0067] (3) When the corn reaches the V2 stage, keep one seedling in each pot that is growing well and consistently, and begin watering each plant with 100 mL of nutrient solution every day, wherein the nutrient solution formula is the same as that used for seed germination;

[0068] (4) From V4 to V7, 200 mL of nutrient solution was applied to each plant every day;

[0069] (5) From V7 to the silking stage, each plant was irrigated with 400 mL of nutrient solution per day. From the silking stage onwards, 1000 mL of nutrient solution was irrigated per day. A control (6 pots) and low-phosphorus (6 pots, phosphorus concentration was 1 / 50 of the control) treatments were set up. All corn ears were self-pollinated until the final harvest.

[0070] Example 2

[0071] The present invention extracts, purifies, reverse transcribes and performs real-time fluorescence quantitative PCR on RNA of wild-type corn, ZmMYB36 overexpression strains and ZmMYB36 gene knockout strains.

[0072] (I) Extraction of total RNA with Trizol reagent:

[0073] (1) Pre-cool the mortar with liquid nitrogen and quickly grind the sample. Add about 0.1 g of the ground sample into a 2 mL centrifuge tube, quickly add 1 mL of Trizol reagent, shake to mix, and place on ice for 5 min.

[0074] (2) Then, 0.2 mL of chloroform was added, the mixture was shaken vigorously for 1 min, placed on ice for 5 min, and centrifuged at 12,000 rpm and 4°C for 15 min;

[0075] (3) Take the supernatant and transfer it to a new 1.5 mL centrifuge tube. Add the same volume of isopropanol and 1 / 10 volume of sodium acetate (3 M, pH = 5.2). Mix by inverting the tube and store at -20 °C overnight.

[0076] (4) Then centrifuge at 12,000 rpm and 4°C for 15 min, discard the supernatant, add 1 mL of 75% ethanol, invert to suspend the precipitate, centrifuge at 12,000 rpm and 4°C for 5 min, repeat this step once, discard the supernatant, let it air for 15 min, and after the centrifuge tube is air-dried, add 30-50 mL of DEPC water and let it stand for 10 min to dissolve the RNA.

[0077] (ii) RNA purification and reverse transcription: follow the steps of Takara kit.

[0078] (III) Fluorescence quantitative PCR: Fluorescence quantitative PCR was performed using SYBR Premix EX TaqTM (Takara). Four biological replicates were set for each strain and each treatment, and three technical replicates were set for each biological replicate. ZmUbiqutin was used as the internal reference gene, and the comparative CT value method was used to calculate the relative expression level of the gene.

[0079] The PCR reaction system is as follows:

[0080]

[0081] The amplification procedure is as follows:

[0082] Step 1: Pre-denaturation at 95℃ for 30s

[0083] Step 2 Denaturation at 95℃ for 5 seconds

[0084] Step 3 Anneal at 60℃ for 30s

[0085] Step 4: 72℃ extension for 30s

[0086] Step 5 Repeat 39 cycles

[0087] Corn B73 was cultured in full nutrient solution, low nitrogen, low phosphorus, and low potassium nutrient solution, and the whole root sample of corn B73 was harvested at 10 days of growth cycle, and the relative expression of ZmMYB36 gene was analyzed by q-PCR. Figure 3 As shown in the figure on the left, the bar graphs corresponding to CK, LN, and LK are shorter than the bar graph corresponding to LP, indicating that the expression level of ZmMYB36 gene under CK, LN, and LK treatments is lower than that under LP treatment. This result indicates that the ZmMYB36 gene has no response to low nitrogen and low potassium treatments, but is significantly upregulated by about 26 times under low phosphorus induction. Figure 3 As shown in the figure on the right, the expression of ZmMYB36 gene in the control group (CK) remained basically unchanged, while the expression of ZmMYB36 gene in the low phosphorus treatment group (LP) increased rapidly on the 25th day. The results showed that as the phosphorus deficiency time increased, the expression of ZmMYB36 gene increased, and its expression increment increased by 5 times on the 25th day. The results showed that the ZmMYB36 gene had no response to low nitrogen and low potassium treatments, but was significantly upregulated by about 26 times under low phosphorus induction.

[0088] like Figure 5 As shown, the gene expression levels of the overexpression strains were detected by qPCR. Compared with the ZmMYB36 gene expression level of the wild type (WT), the expression level of OE-1 was 3.58, the expression level of OE-2 was 19.55, and the expression level of OE-3 was 1144.5. The results showed that the expression levels of the genes in the ZmMYB36 overexpression strains were upregulated by 3.58, 19.55, and 1144.5 times, respectively.

[0089] Example 3

[0090] This example involves gene gun operation and confocal microscopy observation based on subcellular localization analysis of ZmMYB36 protein.

[0091] (I) Ingredients: Onion inner skin

[0092] (ii) Reagents: 2.5 mol / L CaCl2, 0.1 mol / L spermidine, anhydrous ethanol

[0093] (III) Preliminary preparation: Tear off about 2×2 cm of the inner layer of the onion epidermis, place the torn onion epidermis tissue on the freshly prepared 1 / 2MS culture medium, and culture in the dark for 12 hours.

[0094] (IV) Preparation of DNA-gold powder complex: prepare 1 mL each of 2.5 mol / L CaCl2 and 0.1 mol / L spermidine; vortex the gold powder for 5 min to suspend it in sterile water; pipette 8.5 μL of the gold powder suspension into a 1.5 mL EP tube, and sequentially add 5 μg of plasmid DNA, 17 μL of CaCl2 and 8 μL of spermidine; vortex for 5 min, let stand on ice for 10 min, centrifuge at 3000 rpm for 1 min, and discard the supernatant; add 100 μL of anhydrous ethanol, wash without destroying the precipitate, discard the supernatant, and repeat twice; add 10 μL of anhydrous ethanol and set aside.

[0095] (V) Gene gun step: Take 10 μL of the prepared DNA-gold powder complex and evenly apply it on the middle aperture of the bombardment membrane. Place a pressure membrane above the gene gun to bombard the material in the hole. When the pressure in the cabin drops to 0, open the cabin door and take out the material.

[0096] (VI) Cultivation: After bombardment, the onion epidermis was placed in 1 / 2MS culture medium and cultured in a dark incubator at 28-30°C for 22-24 hours, and observed under a confocal microscope.

[0097] Figure 4 The results of subcellular localization analysis of ZmMYB36 protein are shown. As a transcription factor, ZmMYB36 must first be located in the cell nucleus to regulate the transcription level of downstream target genes. The GFP fusion protein of ZmMYB36 was constructed and transferred into onion epidermal cells using a gene gun. It was found that the Merge diagram of ZmMYB36:GFP showed a bright spot located in the cell nucleus. The result showed that ZmMYB36:GFP was specifically located in the cell nucleus, and the negative control GFP was expressed in the entire onion cell. The results showed that ZmMYB36 was located in the cell nucleus. This result provides a basis for the ZmMYB36 gene to regulate the transcription of downstream genes in the cell nucleus.

[0098] Example 4

[0099] This example relates to the determination of phosphorus content in corn.

[0100] A 1 / 10,000 balance was used for weighing, and the phosphorus content in each part was determined by concentrated H2SO4-H2O2 digestion method and vanadium molybdenum yellow colorimetry. All reagents used were high-grade pure reagents.

[0101] The specific operations are as follows:

[0102] (1) After grinding the sample, weigh it on a balance and place it directly in a clean and dried digestion tube. Set up a blank control. Add 5 mL of concentrated sulfuric acid to each tube, shake well, and let it stand overnight.

[0103] (2) Add 4 mL of H2O2 in two portions, mix thoroughly, and boil at 270°C for 10 min.

[0104] (3) After the digestion tube has cooled, add 2 mL of H2O2 and digest at 350°C for 10 min.

[0105] (4) After the digestion tube has cooled, add 2 mL of H2O2 and continue digestion at 350°C for 20 min;

[0106] (5) Thoroughly remove the H2O2, and after the digestion tube has completely cooled, dilute to 50 mL with deionized water and mix by inverting;

[0107] (6) Pipette 5 mL of the liquid to be tested from each tube into a 15 mL imported centrifuge tube, add two drops of 2,4-dinitrophenol indicator, and neutralize with 6 M NaOH until the solution just turns light yellow;

[0108] (7) Add 2 mL of ammonium vanadium molybdate solution, dilute to 10 mL, mix by inversion, and let stand at room temperature for 15 min;

[0109] (8) After the reaction is stable, measure the absorbance at 440 nm using a spectrophotometer;

[0110] (9) Weigh KH2PO4 dried to constant weight at 105°C to prepare a 50 mg / L phosphorus standard solution, then pipette the phosphorus standard solution into a 50 mL volumetric flask, develop the color according to the above steps, set 0, 1 mg / L, 2.5 mg / L, 5 mg / L, 7.5 mg / L, 10 mg / L, and 15 mg / L phosphorus standard solutions, read the corresponding absorbance values, draw a standard curve, and calculate the phosphorus concentration and phosphorus content.

[0111] Homozygous ZmMYB36 CRISPR (gene knockout) strains and OE (overexpression) strains were screened and obtained. Sanger sequencing was used to identify the genotypes of the CRISPR strains, which included CRISPR-1 (insertion of T), CRISPR-2 (deletion of 28 bp), and CRISPR-3 (deletion of 35 bp).

[0112] The phosphorus concentration in roots, ear leaves and grains of wild-type maize, ZmMYB36 CRISPR lines and OE lines was measured. The phosphorus use efficiency in roots, ear leaves and grains of wild-type maize, ZmMYB36 CRISPR lines and OE lines was measured.

[0113] like Fig.11 Analysis of phosphorus concentration and phosphorus use efficiency in different parts of maize wild type, ZmMYB36 CRISPR strain and OE strain during reproductive growth period in sand culture. Root phosphorus use efficiency (kg root g -1P), and found that in the low-P treatment group, the root P utilization efficiencies of the CRISPR strain were 0.62, 0.69, and 0.88, respectively, that of the wild type was 0.78, and that of the OE strain were 1.06, 0.98, and 1.17, respectively.

[0114] Statistical analysis of phosphorus utilization efficiency of ear leaf (kg ear leaf g -1 In the control group, the phosphorus utilization efficiency of the ear leaves of the CRISPR strains was 0.18, 0.16, and 0.17, respectively, that of the wild type was 0.19, and that of the OE strains was 0.28, 0.34, and 0.31, respectively. In the low-phosphorus treatment group, the phosphorus utilization efficiency of the ear leaves of the CRISPR strains was 0.19, 0.20, and 0.23, respectively, that of the wild type was 0.25, and that of the OE strains was 0.33, 0.39, and 0.37, respectively.

[0115] In the control group, the grain phosphorus utilization efficiency of the CRISPR strains was (kg ear g -1 The phosphorus utilization efficiency of the wild type was 0.27, and that of the OE strain was 0.30, 0.29, and 0.30, respectively. In the low-phosphorus treatment group, there was no significant difference in phosphorus utilization efficiency among the CRISPR strain, the wild type, and the OE strain.

[0116] The root phosphorus concentration was statistically analyzed and found that in the control group, the root phosphorus concentrations of the CRISPR strains were 1.54, 1.55, and 1.34, the wild type was 1.69, and the OE strains were 1.30, 1.27, and 1.31. Under low phosphorus treatment, the root phosphorus concentrations of the CRISPR strains were 1.65, 1.46, and 1.15, the wild type was 1.31, and the OE strains were 0.97, 1.02, and 0.86.

[0117] In the control group, the phosphorus concentrations of the leaves at the ear position of the CRISPR strain were 5.61, 6.06, and 5.95, respectively, the phosphorus concentration of the leaves at the ear position of the wild type was 5.21, and the phosphorus concentrations of the leaves at the ear position of the OE strain were 3.60, 3.15, and 3.43, respectively. Under low phosphorus treatment, the phosphorus concentrations of the leaves at the ear position of the CRISPR strain were 5.24, 5.09, and 4.49, respectively. The phosphorus concentration of the leaves at the ear position of the wild type was 4.10, and the phosphorus concentrations of the leaves at the ear position of the OE strain were 2.85, 2.70, and 2.73, respectively.

[0118] The grain phosphorus concentration was statistically analyzed and found that in the control group, the grain phosphorus concentrations of the CRISPR strains were 3.80, 3.79, and 3.64, the wild type was 3.64, and the OE strains were 3.31, 3.41, and 3.35. Under low phosphorus treatment, the grain phosphorus concentrations of the CRISPR strains were 3.05, 2.83, and 3.28, the wild type was 3.12, and the OE strains were 3.66, 3.59, and 3.47.

[0119] Under low phosphorus treatment, the root phosphorus concentration and ear leaf phosphorus concentration of the CRISPR line were significantly higher than those of the wild type. The root phosphorus concentration and ear leaf phosphorus concentration of the overexpression line were significantly lower than those of the wild type; under the control treatment, the grain phosphorus concentration of the overexpression line was significantly lower than that of the wild type, while under low phosphorus treatment, its grain phosphorus concentration was significantly higher than that of the wild type. Under low phosphorus treatment, the phosphorus utilization efficiency of the roots and ear leaves of the CRISPR line was significantly lower than that of the wild type. The root phosphorus utilization efficiency and ear leaf phosphorus utilization efficiency of the overexpression line were significantly higher than those of the wild type. The results showed that under control conditions, the phosphorus utilization efficiency of the roots, ear leaves and grains of the OE line was significantly higher than that of the wild type and the CRISPR line.

[0120] Example 5

[0121] (I) Yeast one-hybrid

[0122] (1) Vector construction: With reference to the promoter region sequences of ZmMYB36, ZmPHT1;1, ZmPHT1;2, ​​ZmPHT1;6, ZmGA1, and ZmYIGE1 in the NCBI database, the 200 bp promoter sequences containing P1BS and MRE elements synthesized by a sequencing company were cloned into the pLacZi 2u vector, respectively; the CDS sequences of ZmMYB36, ZmPHR1;1, and ZmPHR1;2 were cloned into the pB42AD vector, respectively, and sequenced for verification.

[0123] (2) Preparation and transformation of yeast competent cells: First, the EGY48 yeast strain taken out from -80℃ was streaked on a YPD plate for culture, and a single clone was picked and shaken in YPD liquid medium until the OD value was 1.5. 5 mL to 200 mL of YPD liquid medium was shaken until the OD value was between 0.4 and 0.6; the cells were collected by centrifugation at room temperature at 4000 rpm for 5 min, and the cells were washed with ddH2O. The cells were collected and repeated three times. After that, the cells were resuspended in 3 mL of freshly prepared 1×TE / LiAc to obtain competent cells; 10 mg / mL salmon sperm DNA was boiled in boiling water for 5 min, placed on ice for 2 min, and repeated once. A 1.5 mL centrifuge tube was taken, 100 ng of plasmid, 10 μL salmon sperm DNA, 100 mL yeast competent cells, and 600 mL PEG / TE / LiAc were added. After oscillation and mixing, the cells were placed in a shaker at 30℃ and cultured at 200 rpm for 30 min; 70 μL of each reaction was added Terminate the transformation with DMSO, incubate in a 42°C water bath for 15 min, and place on ice for 1-2 min; centrifuge, add 200 mL of 1×TE solution to each reaction to resuspend the bacteria, aspirate 100 mL and spread on SD / His-Ura-Trp medium, and culture in a 30°C constant temperature incubator for 2-3 days; pick a single clone and streak it on SD / His-Ura-Trp+X-Gal medium, and culture in a 30°C incubator until the substrate reaction strain turns blue and then take pictures and record.

[0124] The promoter region of the ZmMYB36 gene contains a P1BS element, so it is speculated that ZmMYB36 may be regulated by ZmPHR1s. Fig.12 As shown in the figure, ZmPHR1s can bind to the promoter region of ZmMYB36 containing the P1BS sequence, making the yeast colonies blue. Based on the above results, it was found that both ZmPHR1.1 and ZmPHR1.2 can interact with the P1BS on the promoter of the ZmMYB36 gene in the yeast system. The promoter regions of the ZmPHT1;1, ZmPHT1;2, ​​ZmPHT1;6, ZmGA1 and ZmYIGE1 genes all contain MRE elements, so it is speculated that these genes may be regulated by ZmMYB36. Fig.14 , 16 As shown in Figures 17 and 17, ZmMYB36 can bind to the promoter regions of ZmPHT1;1, ZmPHT1;2, ​​ZmPHT1;6, ZmGA1 and ZmYIGE1 containing MRE elements, making the yeast colonies blue. It can be seen that ZmMYB36 can interact with the MRE on the promoters of the above genes.

[0125] (II) Electrophoretic mobility shift assay (EMSA)

[0126] The CDS sequences of ZmPHR1;1, ZmPHR1;2 and ZmMYB36 were cloned into the PET-30a expression vector. The constructed protein expression plasmid was transformed into the competent E. coli BL21 (DE3), and the purified protein was collected using Ni-NTA-Agarose beads and different concentrations of imidazole elution solution. The promoter regions of ZmMYB36 and ZmNPF5.6 with and without biotin label were synthesized by the company, which contained 40bp sequences of P1BS element and MRE element for EMSA test.

[0127] (1) First, dilute the primers to 100 μM with ddH2O, then use a new 200 μL centrifuge tube to add the forward and reverse primers to dilute the biotin probe to 0.2 μM, and the competitor probe to 10 μM (50×) and 20 μM (100×), and incubate the diluted probes at 96°C for 15 min on a PCR instrument;

[0128] (2) Clean the glue tank and glass plate;

[0129] (3) After the glass plate is dried, install it on a plywood and pour 6% non-denaturing glue;

[0130] (4) Prepare 1 L of 0.5× TBE with high-purity water;

[0131] (5) After the probe is annealed, install the electrophoresis device and add 1 L of 0.5× TBE for pre-electrophoresis for 1 h (constant voltage 80 V);

[0132] (6) Protein-probe binding reaction: prepare the binding reaction system according to the following formula:

[0133] Experimental group: 50 ng fusion protein, 2 μL labeled probe, 6 μL binding reaction buffer, dilute to 20 μL with ultrapure water,

[0134] Competition group: 50 ng fusion protein, 2 μL labeled probe, 2 μL unlabeled competitive probe, 6 μL binding reaction buffer, and dilute to 20 μL with ultrapure water;

[0135] (7) After the binding reaction is completed, add 5 μL of loading buffer, mix thoroughly and then load the sample. The voltage is 90 V. When the indicator reaches 3 / 4 of the gel, stop the electrophoresis.

[0136] (8) Prepare 1.5 L of 0.5× TBE, pre-cool on ice, soak the nylon membrane and 4 layers of filter paper in 0.5× TBE for 10 min in advance, and start transferring the membrane after the electrophoresis is completed. The order of placing the membrane is as follows:

[0137] Black plate, a layer of sponge, a layer of filter paper, glue (cut off the glue hole on the top and drag carefully), nylon membrane, a layer of filter paper, a layer of sponge,

[0138] For white plates, add 1.5L of 0.5×TBE to the electrophoresis tank and transfer the membrane on ice for 1h, with a constant current of 380mA and a voltage of about 80-90V.

[0139] (9) Prepare a clean square culture dish and crosslink the transferred membrane, crosslinking it with the front side facing up for 2 min and the reverse side facing down for 2 min.

[0140] (10) Blocking: Place the UV-crosslinked nylon membrane in a clean dish, add 10 mL of blocking buffer (EMSA kit), and gently shake for 15 min;

[0141] (11) Hybridization: Gently pour off the blocking buffer, add 8 mL of binding / blocking buffer (EMSA kit), and gently shake for 15 min;

[0142] (12) Rinsing: Transfer the nylon membrane to a new plate, add 10 mL of 1× rinsing solution, rinse gently for 5 min, and repeat rinsing three times;

[0143] (13) Equilibration: Transfer the nylon membrane to a new dish, add 15 mL of substrate equilibration buffer, shake gently for 15 min, remove the nylon membrane, and absorb the excess substrate equilibration buffer;

[0144] (14) Luminescence detection: Place in a new dish, add 6 mL of chemiluminescent substrate until the nylon membrane is completely covered, incubate for 5 min, remove the nylon membrane, absorb excess chemiluminescent substrate from the side, wrap with plastic wrap, and expose to the chemiluminescent imaging system for 5 min for development.

[0145] like Fig.12 As shown, the binding of ZmPHR1.1 and ZmPHR1.2 to the P1BS cis-acting element in the promoter region of the ZmMYB36 gene was further confirmed by EMSA experiments. The results showed that ZmPHR1.1 and ZmPHR1.2 could bind to the biotin-labeled P1BS-containing oligonucleotide sequence, affecting the mobility of the ZmPHR1s protein. As the concentration of the non-biotin-labeled probe increased, the signal of the biotin-labeled probe gradually weakened, indicating that ZmPHR1s could bind to the P1BS element in the promoter region of ZmMYB36 in vitro.

[0146] The promoter regions of ZmYIGE1 and ZmNPF5.6 genes contain MRE elements, which may be regulated by ZmMYB36. Fig.16 and 17As shown, EMSA experiments found that ZmMYB36 can bind to biotin-labeled MRE-containing oligonucleotide sequences, affecting the mobility of ZmMYB36 protein. As the concentration of non-biotin-labeled probes increased, the biotin-labeled probe signal gradually weakened, confirming that ZmMYB36 binds to the MRE cis-acting elements in the promoter regions of the ZmYIGE1 and ZmNPF5.6 genes.

[0147] (III) Tobacco dual luciferase assay

[0148] The coding sequences of ZmPHR1;1, ZmPHR1;2 and ZmMYB36 were cloned into the vector pGreen 62-SK, and the promoter sequences of ZmMYB36, ZmGA1 and ZmNPF5.6 (about 2000 bp) were cloned into the vector pGreen II 0800-LUC. After sequencing verification, single clones were picked and the plasmids were extracted and transformed into Agrobacterium tumefaciens GV3101. 62-SK-PHR1s / MYB36 and ProMYB36 / GA1 / NPE5.6-LUC Agrobacterium were mixed in a ratio of 9:1 to infiltrate Nicotiana benthamiana leaves, and pGreen 62-SK empty vector and ProMYB36 / GA1 / NPE5.6-LUC co-transfected tobacco were cultured for 3 days as negative controls. The determination method was based on the dual luciferase kit (Promega), and the GLO-MAX The activities of firefly luciferase (LUC) and Renilla luciferase (REN) were measured using a 20 / 20 luminometer (Promega), and the LUC / REN ratio was calculated. Six biological replicates were set up in the experiment, and the LUC / REN ratio of the negative control was standardized to 1.

[0149] To verify whether ZmPHR1s regulates ZmMYB36 in plants, a dual luciferase activity (LUC / REN) experiment was performed by transient transfection in tobacco. Fig.13 As shown, the LUC / REN ratios measured by 6K-PHR1;1 and 6K-PHR1;2 were higher than those of the control group. The results showed that ZmPHR1.1 and ZmPHR1.2 could promote the expression of ZmMYB36.

[0150] The promoter regions of ZmGA1 and ZmNPF5.6 genes contain MRE elements, so it is speculated that these genes may be regulated by ZmMYB36. Fig.15 and 17As shown, the luciferase activity (LUC / REN) experiment was performed by tobacco transient transfection experiment. The results showed that compared with the control group, the LUC / REN ratio measured by 6K-MYB36 was higher, indicating that ZmMYB36 can bind to the MRE elements on the promoter region of ZmGA1 and ZmNPF5.6 genes and upregulate the expression of ZmGA1 and ZmNPF5.6 genes.

[0151] (IV) Chromatin immunoprecipitation-PCR

[0152] (1) Vector ligation: The cloned ZmMYB36 gene coding region sequence was removed from the stop codon using In-Fusion enzyme (Takara) and ligated to the pUC19-35S-HA-RBS (Amp) vector.

[0153] (2) Protoplast preparation and transformation:

[0154] 1. Cultivation of yellow corn seedlings: Cultivate in flower nutrient soil at room temperature and away from light for 10-14 days until the corn grows three leaves, and keep watering during this period.

[0155] 2. Collect and cut leaves and vacuum: prepare enzymatic solution in advance, select leaves with good growth, and quickly cross-cut the leaves with a single-edged blade under weak light conditions. The cut filaments should be about 1 mm wide and quickly placed in a small beaker containing enzymatic solution. Use a vacuum pump to evacuate for 30 minutes to allow the enzymatic solution to fully enter the intercellular spaces of the leaves to promote the dissolution of the cell walls.

[0156] 3. Place the vacuumed enzymatic solution in a shaker at 28°C and 40 rpm and incubate for 3-4 hours.

[0157] 4. Prepare W5, Mmg, WI and 40% PEG solutions.

[0158] 5. Filter the protoplasts: first wet the filter cloth with W5 solution to prevent the formation of bubbles, then filter the protoplasts into a clean and sterilized 50mL round-bottom centrifuge tube, rinse the residue with W5, filter the residue again, and repeat several times to filter out more protoplasts.

[0159] 6. Collect protoplasts: Centrifuge at 100g for 2 minutes and remove the supernatant with a pipette.

[0160] 7. Add an appropriate amount of pre-cooled W5 solution to slowly resuspend the protoplasts and place on ice for 30 minutes, during which time observe the state of the protoplasts.

[0161] 8. Set the acceleration to 1 and the deceleration to 0 at 100g, centrifuge for 2 minutes, collect the protoplasts, add an appropriate amount of pre-cooled Mmg solution according to the required amount for transformation, suspend the protoplasts, and place on ice.

[0162] 9. Protoplast transformation: Each plasmid was transformed in triplicate. First, 300 μL of plasmid (plasmid concentration was 1 μg / μL) was added to a 50 mL Corning tube. Then, 4 mL of protoplast solution was added. After gently mixing, 4.3 mL of 40% PEG was added. The tube was gently inverted to mix. After standing at room temperature for 18 min, twice the volume of 17.2 mL of W5 solution was added to terminate the transformation.

[0163] 10. Protoplast culture: At 100g, set the acceleration to 1 and the deceleration to 0, centrifuge for 2 minutes, collect the protoplasts, add 10mL W5 to resuspend the protoplasts, centrifuge again at the same speed and acceleration and deceleration for 2 minutes, aspirate as much as possible, add 5mL WI solution, gently suspend the protoplasts, and place flat in a 28℃ incubator in the dark for 14-18h.

[0164] (3) Chromatin immunoprecipitation: Protoplasts were collected by centrifugation. Sample cross-linking, nuclear lysis, and ultrasonic fragmentation of DNA were then performed for immunoprecipitation. 40 μL of beads were prepared for each sample and added to the ultrasonic supernatant. The beads were rotated overnight at 4°C on a low-speed shaker to allow the label and beads to fully bind. The next day, the beads were washed, the protein complex was eluted, the cross-linking was removed, and the DNA fragments were purified using the QIAquick PCR purification kit for the ChIP-qPCR experimental steps. Primers were designed based on the promoter sequence of the ZmYIGE1 gene, containing the MRE element fragment (F2), and the fragment without the MRE element (F1) was used as a negative control. The DNA immunoprecipitated from the protoplasts transferred with the empty vector was used as the control group, and the relative enrichment of the DNA immunoprecipitated by the ZmMYB36 tag protein was calculated.

[0165] like Fig.16 As shown, ZmMYB36 ChIP-qPCR revealed that the fragment with MRE (F2) on the ZmYIGE1 promoter was significantly enriched, while the fragment without MRE (F1) had no significant difference, indicating that ZmMYB36 can interact with ZmYIGE1 in vivo.

[0166] The above results show that ZmMYB36 is regulated by the core phosphorus signaling factor ZmPHR1s, and enhances the ability of maize to tolerate low phosphorus through the expression of ZmPHT1;1, ZmPHT1;2, ​​and ZmPHT1;6. In addition, ZmMYB36 can regulate the three genes ZmGA1, ZmYIGE1, and ZmNPF5.6, and promote the increase of maize grain number and ear length by affecting maize hormone levels.

[0167] Example 6

[0168] This example involves observing the phenotypes of the homozygous ZmMYB36 CRISPR strains and OE strains screened under different growth environments.

[0169] The wild-type B73, ZmMYB36 CRISPR strain and OE strain were planted under normal fertilization treatment in the experimental field. Figure 6 As shown in A, compared with the wild type, the corn ears of the knockout line are significantly smaller than those of the overexpression line. Figure 6 As shown in B, the dry weight of female ears of the wild-type strain was 64 g / plant, the dry weight of female ears of the knockout strain was 41 g / plant, 48 g / plant and 57 g / plant, and the dry weight of female ears of the overexpression strain was 105 g / plant, 96 g / plant and 100 g / plant, respectively. Figure 6 As shown in C, the wild-type grain dry weight is 53 g / plant, the knockout strain grain weights are 31 g / plant, 39 g / plant and 44 g / plant, and the overexpression strain grain weights are 90 g / plant, 78 g / plant and 78 g / plant, respectively. Figure 6 As shown in C, the dry weight of the wild-type spike is 14g / plant, the dry weight of the spike of the knockout strain is 11g / plant, 10g / plant and 15g / plant, and the dry weight of the spike of the overexpression strain is 25g / plant, 20g / plant and 19g / plant. The spike length of the wild-type strain is 10.43cm / plant, the spike length of the knockout strain is 9.22cm / plant, 10cm / plant and 9.72cm / plant, and the spike length of the overexpression strain is 14.25cm / plant, 13.18cm / plant and 14.03cm / plant. The number of grains in the wild-type strain is 233 grains / plant, the number of grains in the knockout strain is 151 grains / plant, 177 grains / plant and 168 grains / plant, and the number of grains in the overexpression strain is 329 grains / plant, 324 grains / plant and 312 grains / plant. When female ears were harvested at maturity and yield-related indicators were statistically analyzed, it was found that the female ear weight, grain weight and number of grains per ear of the CRISPR strain were significantly lower than those of the wild type and overexpression strains.

[0170] Figure 7 The results of plant height phenotype analysis of ZmMYB36 transgenic strains during reproductive growth are shown. The wild type, two CRISPR strains (28bp deletion, 35bp deletion) and two overexpression strains (upregulated by 3.58 times, upregulated by 19.55 times) were selected and cultured in a greenhouse environment with montmorillonite medium. Figure 7The statistical data shown showed that the heights of the knockout strains in the control group were 193.58 cm and 186.75 cm, the height of the wild-type strain was 207.57 cm, and the heights of the overexpression strains were 202.13 cm and 184.55 cm. The heights of the knockout strains in the low-phosphorus treatment were 183.58 cm and 180.08 cm, and the heights of the overexpression strains were 191.8 cm and 172.75 cm. The plant height was measured 43 days after the low-phosphorus treatment, that is, two weeks after the silking period. It was found that the plant height of the CRISPR strain was significantly lower than that of the wild type in both the control and low-phosphorus treatments.

[0171] Figure 8 The aboveground and root phenotype observation results of the ZmMYB36 transgenic strains during maturity are shown. The aboveground results show that the ZmMYB36 CRISPR strains age faster than the wild type and overexpression strains during maturity. The root results show that the roots of the CRISPR strains are significantly smaller than those of the wild type and overexpression strains, while the roots of the overexpression strains are significantly larger than those of the wild type and CRISPR strains.

[0172] Fig. 9 The results of the biomass statistics of the ZmMYB36 transgenic strains at maturity are shown. The aboveground dry weights of the knockout strains in the control group were 80.82 g / plant, 65.22 g / plant and 83.71 g / plant, respectively, the aboveground dry weights of the wild-type strains were 106.4 g / plant, and the aboveground dry weights of the overexpression strains were 99.70 g / plant, 109.21 g / plant and 119.51 g / plant, respectively. The aboveground dry weights of the knockout strains in the low-phosphorus treatment group were 66.70 g / plant, 74.67 g / plant and 83.98 g / plant, respectively, the aboveground dry weights of the wild-type strains were 90.01 g / plant, and the aboveground dry weights of the overexpression strains were 108.06 g / plant, 102.11 g / plant and 116.37 g / plant, respectively.

[0173] In the control group, the root dry weights of the knockout strains were 7.06 g / plant, 6.74 g / plant and 8.03 g / plant, respectively, the root dry weights of the wild-type strains were 15.32 g / plant, and the root dry weights of the overexpression strains were 10.42 g / plant, 14.79 g / plant and 21.84 g / plant, respectively. In the low-phosphorus treatment group, the root dry weights of the knockout strains were 6.98 g / plant, 7.39 g / plant and 9.46 g / plant, respectively, the root dry weights of the wild-type strains were 9.07 g / plant, and the root dry weights of the overexpression strains were 12.82 g / plant, 15.01 g / plant and 19.93 g / plant, respectively.

[0174] In the control group, the root-to-shoot ratios of the knockout strains were 0.082, 0.082 and 0.087, the root-to-shoot ratio of the wild-type strain was 0.129, and the root-to-shoot ratios of the overexpression strains were 0.113, 0.163 and 0.189, respectively. In the low-phosphorus treatment group, the root-to-shoot ratios of the knockout strains were 0.072, 0.087 and 0.087, the root-to-shoot ratio of the wild-type strain was 0.140, and the root-to-shoot ratios of the overexpression strains were 0.116, 0.139 and 0.174, respectively.

[0175] The dry weights of the third leaf under the ear of the knockout lines in the control group were 2.31 g / plant, 2.44 g / plant and 2.57

[0176] g / plant, the dry weight of the third leaf under the ear of the wild-type strain was 3.25 g / plant, and the dry weights of the third leaf under the ear of the overexpression strains were 2.84 g / plant, 3.02 g / plant, and 3.96 g / plant, respectively. In the low-phosphorus treatment group, the dry weights of the third leaf under the ear of the knockout strains were 2.25 g / plant, 2.66 g / plant, and 2.60 g / plant, respectively, the dry weight of the third leaf under the ear of the wild-type strain was 2.31 g / plant, and the dry weights of the third leaf under the ear of the overexpression strains were 3.16 g / plant, 3.48 g / plant, and 3.40 g / plant, respectively.

[0177] The dry weight of leaves at ear position of knockout lines in the control group were 2.93 g / plant, 2.90 g / plant and 3.06 g / plant, respectively.

[0178] The dry weight of ear leaves of the wild-type strain was 4.04 g / plant, and the dry weight of ear leaves of the overexpression strains was 3.74 g / plant, 3.84 g / plant, and 4.01 g / plant, respectively. In the low-phosphorus treatment group, the dry weight of ear leaves of the knockout strains was 3.20 g / plant, 3.42 g / plant, and 3.36 g / plant, respectively, the dry weight of ear leaves of the wild-type strain was 3.31 g / plant, and the dry weight of ear leaves of the overexpression strains was 4.06 g / plant, 3.78 g / plant, and 4.15 g / plant, respectively.

[0179] The dry weight of the third leaf on ear of the knockout lines in the control group was 2.62 g / plant, 2.51 g / plant and 2.84 g / plant, respectively.

[0180] g / plant, the dry weight of the third leaf on the ear of the wild-type strain was 3.41g / plant, and the dry weights of the third leaf on the ear of the overexpression strains were 3.03g / plant, 3.56g / plant, and 3.37g / plant, respectively. In the low-phosphorus treatment group, the dry weights of the third leaf on the ear of the knockout strains were 2.43g / plant, 2.40g / plant, and 2.69g / plant, respectively, the dry weight of the third leaf on the ear of the wild-type strain was 2.92g / plant, and the dry weights of the third leaf on the ear of the overexpression strains were 3.27g / plant, 3.14g / plant, and 3.38g / plant, respectively.

[0181] The stem dry weights of the knockout lines in the control group were 22.62 g / plant, 22.52 g / plant, and 22.17 g / plant, respectively.

[0182] The stem dry weight of the wild-type strain was 33.98 g / plant, and the stem dry weight of the overexpression strains was 28.08 g / plant, 30.95 g / plant, and 37.52 g / plant, respectively. The stem dry weight of the knockout strains in the low-phosphorus treatment group was 20.77 g / plant, 18.63 g / plant, and 21.51 g / plant, respectively, the stem dry weight of the wild-type strain was 26.12 g / plant, and the stem dry weight of the overexpression strains was 31.03 g / plant, 29.37 g / plant, and 35.05 g / plant, respectively.

[0183] The dry weights of other parts of the knockout strains in the control group (excluding the six parts of the ear leaf, the third leaf under the ear, the third leaf above the ear, the stalk, the female ear, and the root) were 23.37 g / plant, 23.77 g / plant, and 26.28 g / plant, respectively, the dry weights of other parts of the wild-type strains were 32.77 g / plant, and the dry weights of other parts of the overexpression strains were 30.77 g / plant, 30.75 g / plant, and 35.90 g / plant, respectively. The dry weights of other parts of the knockout strains in the low-phosphorus treatment group were 24.18 g / plant, 23.97 g / plant, and 26.48 g / plant, respectively, the dry weights of other parts of the wild-type strains were 28.89 g / plant, and the dry weights of other parts of the overexpression strains were 31.79 g / plant, 29.87 g / plant, and 34.51 g / plant, respectively.

[0184] At maturity, the roots and different parts of the aboveground parts (ear leaves, the third leaf on the ear, the third leaf under the ear, the stalk, the female ear, and the remaining parts) were harvested and their biomass was measured. It was found that the aboveground and root biomass of the CRISPR strain were significantly lower than those of the wild type, and the aboveground and root biomass of the OE strain were significantly higher than those of the wild type, regardless of whether it was under the control or low-phosphorus treatment. The root-to-crown ratio was statistically found to be significantly lower in the CRISPR strain than in the wild type, and the root-to-crown ratio of the OE strain was significantly higher than that of the wild type under the control and low-phosphorus conditions. In addition, the biomass of different parts of the aboveground parts was statistically found to be significantly lower in the CRISPR strain than in the wild type, and the biomass of different parts of the OE strain was significantly higher than that of the wild type. Fig.10 Figure 2 shows the results of female ear phenotype analysis of ZmMYB36 transgenic strains during the sand culture period. Fig.10The corn ear phenotype diagram shown shows that compared with the wild type, the corn ears of the knockout strains in the control group are significantly smaller than those of the wild type and overexpression strains. In the control group, the wild type grain weight was 32.99 g / plant, the knockout strain grain weights were 23.59 g / plant, 20.88 g / plant and 26.69 g / plant, and the overexpression strain grain weights were 33.05 g / plant, 30.56 g / plant and 33.9 g / plant, respectively. In the low phosphorus treatment group, the wild type grain weight was 21.75 g / plant, the knockout strain grain weights were 24.01 g / plant, 22.14 g / plant and 23.19 g / plant, and the overexpression strain grain weights were 32.63 g / plant, 29.97 g / plant and 32.71 g / plant, respectively.

[0185] In the control group, the dry weight of the wild-type cob was 7.16 g / plant, the dry weight of the cob of the knockout strain was 4.72 g / plant, 4.41 g / plant and 5.53 g / plant, and the dry weight of the cob of the overexpression strain was 7.30 g / plant, 7.12 g / plant and 9.02 g / plant. In the low-phosphorus treatment group, the dry weight of the wild-type cob was 5.26 g / plant, the dry weight of the cob of the knockout strain was 4.83 g / plant, 5.38 g / plant and 4.65 g / plant, and the dry weight of the cob of the overexpression strain was 8.64 g / plant, 6.97 g / plant and 8.52 g / plant.

[0186] In the control group, the wild-type spike length was 9.1 cm / plant, the knockout strain spike lengths were 7.84 cm / plant, 7.76 cm / plant, and 9.25 cm / plant, respectively, and the overexpression strain spike lengths were 10.22 cm / plant, 10.5 cm / plant, and 10.08 cm / plant, respectively. In the low-phosphorus treatment group, the wild-type spike length was 8.14 cm / plant, the knockout strain spike lengths were 7.80 cm / plant, 8.93 cm / plant, and 9.12 cm / plant, respectively, and the overexpression strain spike lengths were 10.16 cm / plant, 10.00 cm / plant, and 11.47 cm / plant, respectively.

[0187] In the control group, the 100-grain weight of the wild type was 23.00 g / plant, the 100-grain weight of the knockout strains was 20.54 g / plant, 20.38 g / plant and 17.75 g / plant, and the 100-grain weight of the overexpression strains was 21.21 g / plant, 20.00 g / plant and 21.89 g / plant. In the low-phosphorus treatment group, the 100-grain weight of the wild type was 22.61 g / plant, the 100-grain weight of the knockout strains was 19.02 g / plant, 21.16 g / plant and 18.07 g / plant, and the 100-grain weight of the overexpression strains was 21.88 g / plant, 20.61 g / plant and 22.29 g / plant.

[0188] In the control group, the harvest index (ratio of grain weight to dry weight above ground) of the wild type was 0.27, the harvest indexes of the silent strains were 0.22, 0.18 and 0.30, and the harvest indexes of the overexpression strains were 0.33, 0.30 and 0.29, respectively. In the low phosphorus treatment group, the harvest index of the wild type was 0.31, the harvest indexes of the silent strains were 0.27, 0.28 and 0.22, and the harvest indexes of the overexpression strains were 0.31, 0.29 and 0.29, respectively.

[0189] In the control group, the number of wild-type grains was 156, the number of knockout lines was 114, 111 and 153, and the number of overexpression lines was 168, 167 and 162. In the low-phosphorus treatment group, the number of wild-type grains was 108, the number of knockout lines was 125, 104 and 135, and the number of overexpression lines was 149, 150 and 167.

[0190] In the control group, the number of grains per row of the wild type was 11, the number of grains per row of the knockout lines was 7, 6 and 13, and the number of grains per row of the overexpression lines was 13, 14 and 14. In the low phosphorus treatment group, the number of grains per row of the wild type was 8, the number of grains per row of the knockout lines was 7, 8 and 9, and the number of grains per row of the overexpression lines was 13, 13 and 13.

[0191] In the control group, the number of ear rows of the wild type was 13, the number of ear rows of the knockout lines were 11, 11 and 12, and the number of ear rows of the overexpression lines were 13, 12 and 12. In the low phosphorus treatment group, the number of ear rows of the wild type was 11, the number of ear rows of the knockout lines were 10, 11 and 12, and the number of ear rows of the overexpression lines were 12, 12 and 12.

[0192] according to Fig.10 It can be seen that when the female ears were harvested at maturity, the grain weight and ear axis of CRISPR were significantly lower than those of the wild type under the control condition; the grain weight and ear axis of the overexpression strain under low phosphorus treatment were significantly greater than those of the wild type; regardless of the control or low phosphorus treatment, the 100-grain weight and harvest index of the CRISPR strain were significantly lower than those of the wild type. Under the control condition, the grain number, ear length, number of grains per row, and number of rows per ear of the CRISPR strain were significantly lower than those of the wild type, while the grain number, ear length, and number of grains per row of the OE strain under low phosphorus treatment were significantly higher than those of the wild type.

[0193] The above phenotypic observation results showed that under greenhouse sand culture conditions, the root and aboveground biomass of the ZmMYB36 CRISPR strain was significantly smaller than that of the wild type and overexpression strains. When the female ears were harvested at maturity, it was found that the grain weight and ear axis of the CRISPR strain under the control condition were significantly smaller than those of the wild type; the grain weight and ear axis phenotype of the overexpression strain under low phosphorus treatment were significantly greater than those of the wild type.

[0194] Table 1

[0195]

[0196]

[0197] It should be noted that the above-mentioned specific embodiments are exemplary, and those skilled in the art can come up with various solutions inspired by the disclosure of the present invention, and these solutions also belong to the disclosure scope of the present invention and fall within the protection scope of the present invention. Those skilled in the art should understand that the present invention specification and its drawings are illustrative and do not constitute a limitation of the claims. The scope of protection of the present invention is defined by the claims and their equivalents. The present invention specification contains multiple inventive concepts, such as "preferably", "according to a preferred embodiment" or "optionally", all of which indicate that the corresponding paragraph discloses an independent concept, and the applicant reserves the right to file a divisional application based on each inventive concept. Throughout the text, the feature guided by "preferably" is only an optional method and should not be understood as a must-have setting. Therefore, the applicant reserves the right to abandon or delete the relevant preferred features at any time.

Claims

1. ZmMYB36 The application of the gene in improving the tolerance of corn to low phosphorus stress is characterized in that: Said ZmMYB36 The nucleotide sequence of the gene is shown in SEQ ID NO.1, wherein: ZmMYB36 The ZmPHR1 pathway regulates ZmPHT1;1 , ZmPHT1;2 and ZmPHT1;6 Genes improve plant tolerance to low phosphorus, the application comprising ZmMYB36 The gene is overexpressed in maize.

2. ZmMYB36 The application of the gene in increasing corn ear weight and ear length is characterized in that: Said ZmMYB36 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the application comprises ZmMYB36 The gene is overexpressed in maize.

3. ZmMYB36 The application of the gene in increasing the number of corn kernels is characterized in that: Said ZmMYB36 The nucleotide sequence of the gene is shown in SEQ ID NO.1, wherein: ZmMYB36 Genes are regulated by ZmGA1 Gene, ZmYIGE1 Gene, ZmNPF5.6 Genes involved in corn hormone levels and physiological processes to increase corn kernel number and ear length, and the application includes ZmMYB36 The gene is overexpressed in maize.

4. ZmMYB36 Application of gene in preparing transgenic corn, said ZmMYB36 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the application comprises ZmMYB36 The gene is overexpressed in maize.

5. A method for cultivating a grass plant resistant to low phosphorus stress, characterized in that: The expression level of the ZmMYB36 gene is increased by genetic engineering, wherein the nucleotide sequence of the ZmMYB36 gene is shown in SEQ ID NO.1.

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