Application of ZmbZIP89 gene in regulating maize lateral root development and drought resistance

Abstract

This invention relates to the field of plant genetic engineering, specifically to... ZmbZIP89 Application of genes in regulating lateral root development and drought resistance in maize. Overexpression in maize. ZmbZIP89 Genes significantly increased lateral root length, average lateral root length, lateral root density, total root length, root biomass, and lateral root primordium density. ZmbZIP89 In the drought phenotype experiment of overexpression materials, overexpression ZmbZIP89 The survival rate of the gene-expressing plants after drought stress was significantly higher than that of the wild type, and the lateral root length of the overexpressing plants was significantly higher than that of the wild type. In summary, ZmbZIP89 By influencing lateral root development, it regulates water absorption in maize, making the plant more drought-resistant, and is expected to serve as a target gene for improving maize root system and drought resistance.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering, specifically to... ZmbZIP89 Application of genes in regulating lateral root development and drought resistance. Background Technology

[0002] The root system is a vital organ for maize to absorb nutrients and water, and a well-structured root system is crucial for increasing maize yield and coping with various stress conditions. Lateral root formation is a decisive factor in root morphology, responsible for the absorption of the vast majority of water and nutrients. Roots grow underground and possess high environmental plasticity, making phenotypic analysis difficult and hindering genetic improvement through conventional breeding. Identifying key genes controlling lateral root development and using molecular breeding techniques to precisely select and improve maize roots holds immense promise. Therefore, in-depth analysis of the genetic regulatory mechanisms of lateral root development, identification of superior genes and allelic variations, and exploration of their roles in efficient nutrient and water utilization are of great significance for breeding high-yielding, nutrient- and water-efficient new maize varieties.

[0003] In plants, bZIP is a large family of transcription factors that plays an important role in responses to various abiotic stresses. However, current understanding of... ZmbZIP89 Its uses have not been adequately studied, and have not been disclosed. ZmbZIP89 Research on the role of genes in regulating lateral root development and drought resistance. Summary of the Invention

[0004] The purpose of this invention is to provide maize genes. ZmbZIP89 Uses, especially ZmbZIP89 Application of genes in regulating lateral root development and drought tolerance. Overexpression in maize. ZmbZIP89 The gene, lateral root length, average lateral root length, lateral root density, total root length, root biomass, lateral root primordium density, and drought resistance were significantly higher than those of the wild-type ND101.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] corn ZmbZIP89 The application of genes in regulating lateral root development, the aforementioned ZmbZIP89 The gene sequence is as shown in SEQ ID No. 1; or it is generated from the nucleotide sequence shown in SEQ ID No. 1 by adding, substituting, or deleting one or more bases, and encodes control... ZmbZIP89 The nucleotide sequence of a protein.

[0007] Furthermore, the ZmbZIP89 protein is either a) or b) of the following:

[0008] a) A protein consisting of the amino acid sequence shown in SEQ ID No. 2;

[0009] b) Proteins derived from SEQ ID No. 2 by substituting and / or adding and / or deleting one or more amino acid residues of the sequence shown in SEQ ID No. 2.

[0010] Furthermore, regulating maize root development includes increasing maize lateral root length, average lateral root length, lateral root density, total root length, root biomass, and lateral root primordia density.

[0011] This invention also provides ZmbZIP89 The application of genes in regulating drought resistance in maize, the aforementioned ZmbZIP89 The nucleotide sequence of the gene is shown in SEQ ID No. 1; or it is generated by adding, substituting, or deleting one or more bases from the nucleotide sequence shown in SEQ ID No. 1, and encodes the control of maize. ZmbZIP89 The nucleotide sequence of a protein.

[0012] Furthermore, regulating maize drought resistance includes improving maize water use efficiency and increasing maize survival rate after experiencing drought stress.

[0013] Furthermore, the corn ZmbZIP89 Genes regulate water absorption efficiency and drought resistance in maize by influencing lateral root development.

[0014] This invention also provides a method for regulating the development of maize lateral roots, comprising the following steps: ZmbZIP89 The CDS sequence of the gene was constructed into a plant expression vector and transformed into the wild-type maize inbred line ND101 to obtain positive transgenic overexpression plants; the transgenic maize had better root development compared with the wild-type ND101.

[0015] This invention also provides a method for regulating plant water absorption, comprising the following steps: ZmbZIP89 The CDS sequence of the gene was constructed into a plant expression vector and transformed into the wild-type maize inbred line ND101 to obtain positive transgenic overexpression plants; the water uptake efficiency of the transgenic maize after drought stress was significantly improved compared with that of the wild-type ND101 root system.

[0016] This invention also provides a method for improving the drought resistance of corn, comprising the following steps: ZmbZIP89 The CDS sequence of the gene was constructed into a plant expression vector, transformed into the target plant, and positive overexpression transgenic plants were obtained; the transgenic maize showed better drought resistance compared with the target plant.

[0017] Furthermore, the primers used for PCR amplification are:

[0018] OE-F: 5'-GGTGGACGGCGAGGTCGCCG-3';

[0019] OE-R: 5'-TCGGTGACGGGCAGGACCGG-3'.

[0020] Furthermore, the primers used for RT-qPCR are:

[0021] Actin-F: 5'-GTGTCCTGTCCACCCACTCTCT-3';

[0022] Actin-R: 5'-GGAACTCGTTCACATCAACGTTC-3';

[0023] RT-qPCR-F: 5'- CGGTCTCCAGATGGAGGTGT-3';

[0024] RT-qPCR-R: 5'-TGCTTGAGGTGGCTGTTGC-3'.

[0025] Furthermore, ZmbZIP89 The encoded sequence is linked to the corn Ubiquitin promoter driver. ZmbZIP89 -pCombia3300 vector.

[0026] Furthermore, the recombinant plasmid pCombia3300- ZmbZIP89 Recombinant Agrobacterium was obtained by transferring it into Agrobacterium EHA105, and then it was used to infect embryogenic callus tissue of maize ND101. Transgenic plants were then cultured from this tissue.

[0027] The purpose of this invention is to provide maize genes. ZmbZIP89 Uses, especially ZmbZIP89 Applications of genes in regulating lateral root development and drought resistance.

[0028] The regulation refers to the overexpression of the aforementioned substance in maize. ZmbZIP89 The gene significantly increased lateral root length, average lateral root length, lateral root density, total root length, root biomass, and lateral root primordium density. Regarding water absorption, overexpression... ZmbZIP89 The survival rate after drought stress following genetic modification was significantly higher than that of the wild type.

[0029] This invention also provides a transgenic method for cultivating maize root system architecture, comprising the following steps: ... ZmbZIP89The CDS sequence of the gene was constructed into a plant expression vector and transformed into target maize to obtain positive transgenic plants. Compared with the target maize root system, the transgenic maize showed significantly higher overexpression lateral root length, average lateral root length, lateral root density, total root length, root biomass, and lateral root primordium density than the wild type.

[0030] This invention discloses for the first time the corn ZmbZIP89 The gene has a biological function in regulating maize lateral root development. Overexpression of this gene significantly increases lateral root length, average lateral root length, lateral root density, total root length, root biomass, and lateral root primordium density compared to the wild type. ZmbZIP89 The survival rate of the gene-modified variety after drought stress was significantly higher than that of the wild type. This invention provides valuable genetic resources for improving root system architecture and drought resistance, and has important significance for maize breeding and production.

[0031] Beneficial effects

[0032] Longer lateral roots increase the extension and distribution of the root system, helping crops absorb water from the soil more effectively. To verify... ZmbZIP89 Does it affect the drought resistance of corn? We ZmbZIP89 Drought tolerance tests were conducted on the overexpression lines. After 7 days of drought treatment, the wild-type leaves showed significant curling, yellowing leaf tips, and smaller root systems. The lateral root length of the wild-type was 410.1±49.89 cm, while the lateral root lengths of the two overexpression lines were 623.4±42.38 cm and 588.3±13.41 cm, respectively, representing increases of 52.01% compared to the wild-type. P =0.0112) and 43.45% ( P =0.0062); at this time, the net photosynthetic rate, stomatal conductance, and transpiration rate of the overexpression line were significantly higher than those of the wild type, ultimately leading to improved water use efficiency in the overexpression line; after 14 days of drought, rehydration showed that the survival rate of the overexpression line was significantly higher than that of the wild type, indicating that ZmbZIP89 It can enhance the drought resistance of corn seedlings.

[0033] This invention provides ZmbZIP89 Genes and their applications, overexpression in maize ZmbZIP89 Genes significantly increased lateral root length, average lateral root length, lateral root density, total root length, root biomass, and lateral root primordium density. ZmbZIP89 In drought phenotype experiments of overexpression materials, overexpression ZmbZIP89 The survival rate of the gene-expressing plants after drought stress was significantly higher than that of the wild type, and the lateral root length of the overexpressing plants was significantly higher than that of the wild type. In summary, ZmbZIP89 By influencing lateral root development and thus regulating water absorption in maize, this gene is expected to serve as a target gene for improving maize root systems and enhancing drought resistance. Attached Figure Description

[0034] Figure 1 For two ZmbZIP89 Identification of overexpression transgenic materials (OE1 and OE2). Wherein, (A) represents the overexpression vector; (B) represents... ZmbZIP89 Relative expression levels in overexpression lines and wild-type;

[0035] Figure 2 For two ZmbZIP89 Phenotypic analysis of the overexpression transgenic lines (OE1 and OE2). Wherein, (A) represents... ZmbZIP89 Local root scan images of the overexpression material and wild-type ND101, (B) indicates ZmbZIP89 Root scan images of overexpression material and wild-type ND101, (C) indicates ZmbZIP89 Images of overexpression material and wild-type lateral root primordia staining, (D) indicates ZmbZIP89 Root phenotype analysis of overexpression lines;

[0036] Figure 3 For two ZmbZIP89 Analysis of drought-tolerant phenotypes in overexpressing transgenic lines (OE1 and OE2). Wherein, (A) represents... ZmbZIP89 Overexpression material and wild-type ND101 rehydration phenotype after 14 days of drought, (B) indicates ZmbZIP89 Overexpression material and wild-type ND101 root phenotype after 7 days of drought, (C) indicates ZmbZIP89 Survival rates of overexpressed materials and wild-type ND101 after 14 days of drought followed by rehydration, (D) represents ZmbZIP89 Overexpression material and long lateral root phenotype of wild-type ND101 after 7 days of drought;

[0037] Figure 4 For two ZmbZIP89 Analysis of photosynthetic data of overexpressing transgenic lines (OE1 and OE2) after 7 days of drought. Wherein, (A) represents... ZmbZIP89 Net photosynthetic rates of overexpression material and wild-type ND101 under normal watering, (B) represents ZmbZIP89 Net photosynthetic rates of overexpressed materials and wild-type ND101 after 7 days of drought, (C) represents ZmbZIP89 Stomatal conductance of overexpression material and wild-type ND101 under normal watering, (D) represents ZmbZIP89 Stomatal conductance of overexpressed material and wild-type ND101 after 7 days of drought, (E) represents ZmbZIP89 Transpiration rates of overexpression material and wild-type ND101 under normal watering, (F) represents ZmbZIP89 The transpiration rates of the overexpressed material and wild-type ND101 after 7 days of drought, (G) represents ZmbZIP89 Water use efficiency of overexpression material and wild-type ND101 under normal watering, (H) represents ZmbZIP89Water use efficiency of overexpression material and wild-type ND101 after 7 days of drought. Detailed Implementation

[0038] The technical solutions provided by the present invention will be described in detail below with reference to embodiments, but these embodiments are not limited to the present invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. All experimental materials, reagents, and carriers used in the following embodiments can be obtained commercially.

[0039] Example 1

[0040] ZmbZIP89 Identification of overexpression materials

[0041] Using maize B73 root cDNA as a template, it was obtained by PCR amplification. ZmbZIP89 CDS sequence, ZmbZIP89 The encoded sequence is linked to the corn Ubiquitin promoter driven by... ZmbZIP89 The recombinant plasmid pCombia3300- was placed on the pCombia3300 vector. ZmbZIP89 Recombinant Agrobacterium was obtained by transferring it into Agrobacterium EHA105, which was then used to infect embryogenic callus tissue of maize ND101, and transgenic plants were obtained by cultivation. Two stable genetic positive events were identified by PCR amplification and agarose gel electrophoresis, named OE1 and OE2. Real-time quantitative PCR (RT-qPCR) was used to detect the genetic variation in wild-type and the two positive events. ZmbZIP89 The expression level of [the substance / type] was [high / low]. The results showed that, compared to the wild type, [the expression level was lower / lower]. ZmbZIP89 Expression levels in OE1 and OE2 increased by 14-fold and 15-fold, respectively. Figure 1 The primers used for PCR amplification and RT-qPCR are:

[0042] OE-F: 5'-GGTGGACGGCGAGGTCGCCG-3' (SEQ ID No.3)

[0043] OE-R: 5'-TCGGTGACGGGCAGGACCGG-3' (SEQ ID No. 4)

[0044] Actin-F: 5'-GTGTCCTGTCCACCCACTCTCT-3' (SEQ ID No.5)

[0045] Actin-R: 5'-GGAACTCGTTCACATCAACGTTC-3' (SEQ ID No. 6)

[0046] RT-qPCR-F: 5'-CGGGTCTCCAGATGGAGGTGT-3' (SEQ ID No.7)

[0047] RT-qPCR-R: 5'-TGCTTGAGGTGGCTGTTGC-3' (SEQ ID No. 8)

[0048] Example 2

[0049] ZmbZIP89 Phenotypic analysis of overexpression lines

[0050] This study used hydroponic experiments to identify the root phenotypes of wild-type and overexpression lines. The specific experimental steps are as follows.

[0051] The experimental materials were wild-type ND101 and two ZmbZIP89 Overexpression lines. Specific culture methods: (1) Disinfection: Select seeds with full color and uniform size for each material, remove impurities, rinse repeatedly with clean water, rinse with distilled water, and then completely soak in 10% H2O2 solution for 20 minutes for disinfection. (2) Germination: After taking the seeds out of the 10% H2O2 solution, rinse them with distilled water, and soak them in saturated anhydrous calcium sulfate solution for 6-8 hours. Then, incubate them in the dark at 28℃ and 80% relative humidity using moist germination paper. During this period, pay attention to keeping the paper moist. When you press the seedling paper with your finger, water should seep out slightly. (3) Rolling: After 48 hours, roll the seeds with uniform growth vertically into two layers of brown germination paper (AnchorPaper Company, St Paul, MN, USA) with 8 seeds per roll. Place 6 rolls into a white bucket containing 1L of nutrient solution for culture. The day / night temperatures in the climate chamber were 28 / 22 ℃, the relative humidity was 60%, and the light duration was 14h. The nutrient solution was changed every 3 days. (4) Hydroponics: When the seedlings reached the one-leaf stage, plants with uniform growth were selected, the endosperm was removed, the hypocotyl was wrapped with a sponge, and fixed on a notched disc. The disc was placed in a white bucket containing 2.3 L of nutrient solution, and oxygen was introduced into each white bucket using an air pump. The plants were cultured in an artificial culture room under the same conditions as the previous step. Samples were collected after the phenotype appeared.

[0052] Under hydroponic conditions, the wild type was ND101, and the overexpression lines were OE1 and OE2. The lateral root length of the overexpression lines was increased by 48.2% and 35.0% respectively compared to the wild type, indicating that... ZmbZIP89 It positively regulates maize lateral root length. The average lateral root length of the overexpressing lines was increased by 185.4% and 109.5% compared to the wild type, respectively, indicating that... ZmbZIP89 It positively regulates the average lateral root length of maize. The lateral root density of the overexpression lines was increased by 22.7% and 21.1% compared to the wild type, respectively, indicating... ZmbZIP89 It positively regulates maize lateral root density. The total root length of the overexpression lines was increased by 29.8% and 22.0% compared to the wild type, respectively, indicating that... ZmbZIP89 It positively regulates the total root length of maize. The root dry weight of the overexpression lines was increased by 22.1% and 16.5% respectively compared to the wild type, indicating that... ZmbZIP89 It positively regulates the root dry weight of corn. Therefore, it can be seen that... ZmbZIP89 It affects many root traits of maize.

[0053] Example 3

[0054] ZmbZIP89 Toluidine blue staining of lateral root primordia of overexpression lines

[0055] Toluidine blue staining was used. A 1% toluidine blue solution was diluted with distilled water at a ratio of 1:9 to prepare the staining working solution. The roots to be stained were completely immersed in the staining solution and allowed to stand at 28°C for 30 minutes. After staining, the roots were removed, rinsed with distilled water for 1-2 minutes, and then placed in a 50% ethanol solution overnight for decolorization. The next day, the roots were observed and photographed using a macroscopic zoom microscope.

[0056] Under hydroponic conditions, the wild type was ND101, and the overexpression lines were OE1 and OE2. The lateral root primordium density of the overexpression lines was increased by 19.8% and 25.2% respectively compared to the wild type, indicating that... ZmbZIP89 Positively regulates the density of maize lateral root primordia.

[0057] Example 4

[0058] ZmbZIP89 Drought phenotype analysis of overexpression lines

[0059] The experimental materials were wild-type ND101 and two ZmbZIP89 Overexpression lines. Specific culture methods:

[0060] (1) Germination: Select seeds with full color and uniform size for each material, and soak the seeds in a saturated anhydrous calcium sulfate solution for 6-8 hours. Then, incubate them in the dark at 28℃ and 80% relative humidity using moist germination paper. During this period, keep the seeds moist; when you press the seedling paper with your finger, water should seep out slightly. (2) Sowing: Sow seeds with roots about 1cm long into 30×20×10 black boxes, 15 seedlings per box. The soil medium ratio is nutrient soil: vermiculite = 4:1, and water thoroughly. Seven days after sowing, the drought treatment group is watered for the last time (200ml / pot), while the control group is watered normally. (3) Measurement: Seven days after drought treatment, samples are taken, measured, and photographed. Afterward, continue to maintain drought, re-water at a selected time, collect samples, count the survival rate, and measure the phenotype.

[0061] Seven days after drought treatment, the wild-type plants exhibited significant leaf curling, yellowing leaf tips, and smaller root systems. The lateral root length of the wild-type plants was 410.1±49.89 cm, while the lateral root lengths of the two overexpression lines were 623.4±42.38 cm and 588.3±13.41 cm, respectively, representing increases of 52.01% compared to the wild-type. P =0.0112) and 43.45% ( P =0.0062); at this time, the net photosynthetic rate, stomatal conductance, and transpiration rate of the overexpression line were significantly higher than those of the wild type, ultimately leading to improved water use efficiency in the overexpression line; after 14 days of drought, rehydration showed that the survival rate of the overexpression line was significantly higher than that of the wild type, indicating that ZmbZIP89 It can enhance the drought resistance of corn seedlings.

[0062] ZmbZIP89 The nucleotide sequence (SEQ ID No. 1)

[0063]

[0064] ZmbZIP89 Amino acid sequence of the protein (SEQ ID No.2)

[0065] MAQLPPRAPGGAAPQDHWSAAGEFLGFAAARRGAHRRSASDSAAFLEALPMEDVIGGDGFDRLDDDQLMSMFSDVDAPAVSAGDAAQLMDVADADADADDDGSAASSRAAADGVAADPKRVKRILANRQSAQRSRVRKLQYISELERSVTGLQMEVSALSPRVAFLDHQRSLLTVGNSHLKQRIAALAQDKIFKDAHQEALKKEIERLRHVYQQQQQQQQIKVATTGGADIAAAASMQARQELLACEGAAIR*。

Claims

1. ZmbZIP89 The application of genes in the positive regulation of maize lateral root development is characterized by, The ZmbZIP89 The gene sequence is shown in SEQ ID No.

1. ZmbZIP89 The amino acid sequence of the protein is shown in SEQ ID No.

2.

2. The application according to claim 1, characterized in that, The positive regulation of maize lateral root development includes increasing maize root biomass.

3. ZmbZIP89 The application of genes in the positive regulation of drought resistance in maize is characterized by, The ZmbZIP89 The nucleotide sequence of the gene is shown in SEQ ID No.

1.

4. The application according to claim 3, characterized in that, The positive regulation of corn drought resistance includes: improving corn water use efficiency and improving corn survival rate after experiencing drought stress.

5. A method for promoting the development of lateral roots in maize, characterized in that, Includes the following steps: Will ZmbZIP89 The CDS sequence of the gene was constructed into a plant expression vector, transformed into target plants, and stably inherited positive plants were identified by PCR amplification and agarose gel electrophoresis. Positive overexpression transgenic plants were obtained by real-time quantitative PCR. The transgenic maize showed better lateral root development compared to the target plant. ZmbZIP89 The gene sequence is shown in SEQ ID No.

1.

6. A method for improving the drought resistance of maize, characterized in that, Includes the following steps: Will ZmbZIP89 The CDS sequence of the gene was constructed into a plant expression vector, transformed into target plants, and stable genetically positive plants were identified by PCR amplification and agarose gel electrophoresis. Positive overexpression transgenic plants were obtained by real-time quantitative PCR. The transgenic maize exhibited better drought resistance compared to the target plant. ZmbZIP89 The gene sequence is shown in SEQ ID No.

1.

7. The method according to claim 5 or 6, characterized in that, The primers used for PCR amplification are: OE-F: 5'-GGTGGACGGCGAGGTCGCCG-3'; OE-R: 5'-TCGGTGACGGGCAGGACCGG-3'.

8. The method according to claim 5 or 6, characterized in that, The primers used for RT-qPCR are: Actin-F: 5'-GTGTCCTGTCCACCCACTCTCT-3'; Actin-R: 5'-GGAACTCGTTCACATCAACGTTC-3'; RT-qPCR-F: 5'- CGGTCTCCAGATGGAGGTGT-3'; RT-qPCR-R: 5'-TGCTTGAGGTGGCTGTTGC-3'.

9. The method according to claim 5 or 6, characterized in that, Will ZmbZIP89 The encoded sequence is linked to the corn Ubiquitin promoter driver. ZmbZIP89 On the -pCombia3300 vector; the recombinant plasmid was placed... ZmbZIP89 -pCombia3300 was transferred into Agrobacterium EHA105 to obtain recombinant Agrobacterium, which was then used to infect embryogenic callus tissue of maize ND101, and then cultured to obtain transgenic plants.

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