Use of atgt1 gene in plant root development

Abstract

The application discloses application of an AtGT1 gene in plant root system development, and has the characteristics that a base sequence of the AtGT1 gene is shown in a sequence table SEQ ID NO.1, and the AtGT1 gene is used for negative regulation of plant root system development. The AtGT1 gene mutant Arabidopsis thaliana is obtained for the first time, the AtGT1 gene application in the Arabidopsis thaliana is verified to regulate the growth and development of the underground part of the plant, the theoretical research of the AtGT1 gene in the plant root system development is enriched, a theoretical basis is provided for cultivation of a new plant variety with developed root system, it is of great significance for further understanding of a growth mechanism of the plant root system, and more genetic resource information can be provided for improvement of crop root systems at a molecular level.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to the application of the AtGT1 gene in plant root development. Background Technology

[0002] Roots play a crucial role in plant development, primarily by anchoring the plant to the soil, providing mechanical support, absorbing water and nutrients for growth, and establishing beneficial relationships with the microbial community. The spatiotemporal structure of roots is called root system architecture (RSA). RSA exhibits plasticity during plant growth; this mechanism ensures that plants can adapt to environmental changes, better absorb nutrients and water, and maintain photosynthesis in the above-ground parts of the plant. With environmental changes and the emergence of various biotic and abiotic stresses, a healthy RSA can provide effective protection for plants and maintain normal growth and development.

[0003] The triple-helix transcription factor family is named for its unique, highly conserved triple-helix domain (helix-loop-helix-loop-helix), which specifically binds to GT elements (a type of light-responsive DNA element), hence the name GT family. Previous studies have divided the GT family into five subfamilies in plants: GT-1, GT-2, GTγ, SH4, and SIP1. Additionally, soybean contains a subfamily, GTδ. Early reports indicated that the GT family is primarily associated with light response. In Arabidopsis, the mRNA expression level of GT-4 was significantly induced by white light. The rice GT-1 family transcription factor Osrml1 was significantly downregulated under continuous light conditions, and its transcription level exhibited rhythmic changes with light-dark cycles. Under red light treatment, the transcription level of the soybean GT family transcription factor GmGT-2 was significantly downregulated in etiolated seedlings. These studies have revealed that the GT family plays an important role in plant growth and development at different stages. Both soybean GmGT-2A and GmGT-2B genes contain two triple-helix domains. Arabidopsis plants overexpressing these two genes showed significantly higher root viability than wild-type plants under salt, drought, and low-temperature treatments. However, there are few reports on the effects of GT1 family genes on plant root development, and the biological role of GT1 proteins in influencing plant root development remains unclear. Summary of the Invention

[0004] The purpose of this invention is to provide the application of the AtGT1 gene in plant root development, which can regulate the efficiency of plant root growth and development, enrich the theoretical research on the AtGT1 gene in plant root development, and provide a theoretical basis for breeding new plant varieties with well-developed root systems.

[0005] In one aspect of the present invention, the present invention proposes the application of the AtGT1 gene in plant root development. According to an embodiment of the present invention, the base sequence of the AtGT1 gene is shown in the sequence listing SEQ ID NO.1, and the AtGT1 gene is used to negatively regulate plant root development.

[0006] Compared with the prior art, the beneficial effects of the present invention are:

[0007] This invention yielded the first AtGT1 gene mutant Arabidopsis thaliana, verifying the application of the AtGT1 gene in regulating the growth and development of underground parts in Arabidopsis thaliana. This enriches the theoretical research on the AtGT1 gene in plant root development, provides a theoretical basis for breeding new plant varieties with well-developed root systems, and is of great significance for further understanding the growth mechanism of plant roots. It can also provide more genetic resource information for improving crop roots at the molecular level. Attached Figure Description

[0008] Figure 1 This is a tertiary structure model diagram of the AtGT1 protein in Example 1 of the present invention;

[0009] Figure 2 The diagram shows the identification of the atgt1 homozygous mutant Arabidopsis thaliana using the three-primer method in Example 2 of this invention. LP and RP are primers at both ends of the T-DNA insertion site on the plant genome, BP is a primer on the T-DNA segment, and M represents a marker.

[0010] Figure 3 This is a diagram showing the T-DNA insertion sites of the atgt1 mutant in Example 2 of the present invention;

[0011] Figure 4 This is a screening diagram of positive Arabidopsis thaliana plants with the atgt1 mutant in Example 2 of the present invention;

[0012] Figure 5 This is a comparison of the vegetative growth status of the atgt1 mutant plant and the control plant in 1 / 2 MS medium in Example 2 of the present invention, wherein (a) root phenotype diagram, (b) statistical diagram of the number of lateral roots of WT and atgt1 mutant, and (c) statistical diagram of the length of the main root of WT and atgt1 mutant.

[0013] Figure 6 This is a statistical chart showing the root auxin (IAA) content of 18-day-old WT (wild type) and atgt1 mutant Arabidopsis thaliana in Example 2 of the present invention. Detailed Implementation

[0014] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0015] In the following embodiments, various processes and methods not described in detail are conventional methods known in the art. All primers used are indicated upon their first appearance, and subsequent use of the same primer will use the same indication as the initial indication.

[0016] Example 1

[0017] Obtaining the AtGT1 gene coding sequence and its amino acid sequence

[0018] The coding sequence and amino acid sequence of the Arabidopsis thaliana AtGT1 gene were retrieved from the Phytozome website. The final sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, as follows:

[0019] Nucleotide sequence:

[0020]

[0021] amino acid sequence:

[0022] MMQLGGGTPTTTTAAATTVTTATAPPPQSNNNDSAATEAAAAAVGAFEVSEEMHDRGFGGNRWPRQETLALLKIRSDMGIAFRDASVKGPLWEEVSRKMAEHGYIRNAKKCKEKFENVYKYHKRTKEGRTGKSEGKTYRFFDQLEALESQS TTSLHHHQQQTPLRPQQNNNNNNNNNNNSSIFSTPPPVTTVMPTLPSSSIPPYTQQINVPSFPNISGDFLSDNSTSSSSSYSTSSDMEMGGGTATTRKKRKRKWKVFFERLMKQVVDKQEELQRKFLEAVEKREHERLVREESWRVQEIAR INREHEILAQERSMSAAKDAAVMAFLQKLSEKQPNQPQPQPQPQQVRPSMQLNNNNQQQPPQRSPPPQPPAPLPQPIQAVVSTLDTTKTDNGGDQNMTPAASASSSRWPKVEIEALIKLRTNLDSKYQENGPKGPLWEEISAGMRRLGFNR NSKRCKEKWENINKYFKKVKESNKKRPEDSKTCPYFHQLDALYRERNKFHSNNNIAASSSSSGLVKPDNSVPLMVQPEQQWPPAVTTATTTPAAAQPDQQSQPSEQNFDDEEGTDEEYDDEDEEEENEEEEGGEFELVPSNNNNNKTTNNL

[0023] Sequence analysis revealed that the AtGT1 gene coding region is 1812 bp in length, encoding 604 amino acids. The protein's molecular weight is 67878.97 Da, and its isoelectric point is 6.3 (https: / / web.expasy.org). Protein prediction (https: / / swissmodel.expasy.org / ) results show that the AtGT1 protein possesses two SANT domains, and its three-dimensional structure exhibits a typical triple-helix structure. Figure 1 ).

[0024] II. Functional Identification of Mutant Arabidopsis thaliana

[0025] Arabidopsis mutant materials are one of the key tools for studying plant gene function. Currently, the main methods for artificially obtaining plant mutants through gene manipulation include Agrobacterium-mediated DNA transfer (T-DNA) insertion technology, EMS chemical mutagenesis technology, and CRISPR / Cas9 gene editing technology. Among these, T-DNA insertion technology is currently the most commonly used method for efficiently obtaining mutant materials of the target gene.

[0026] 1. Obtaining and identifying the mutant atgt1 Arabidopsis thaliana

[0027] A mutant strain with the AtGT1 gene knocked out using T-DNA technology was purchased from an Arabidopsis mutant library and named the atgt1 mutant. DNA was extracted from leaves of the 20-day-old atgt1 mutant. Using a three-primer method, three different types were identified in the offspring lines of the AtGT1 gene knockout mutant: homozygous mutant, heterozygous mutant, and wild-type mutant. Fifteen homozygous lines, one heterozygous line, and three wild-type (WT) Arabidopsis lines were identified. Figure 2 As shown in the figure, the AtGT1 gene in the homozygous mutant loses its function and can be stably inherited, allowing for further experiments; the offspring of the heterozygous mutant will still segregate and are unstable; the AtGT1 gene in the wild type does not lose its function (i.e., knocking out the AtGT1 gene failed), and it contains the AtGT1 gene.

[0028] Sequencing was performed on the identified homozygous and heterozygous lines: the insertion site of T-DNA in the atgt1 mutant was determined using the Arabidopsis mutant website (http: / / signal.salk.edu / cgi-bin / tdnaexpress) and DNA sequencing methods, such as... Figure 3 As shown, the T-DNA insertion site is located at 99 bp in the second exon of the AtGT1 gene.

[0029] RNA was extracted from rosette leaves of wild-type Arabidopsis thaliana atgt1 mutant and atgt1 mutant lines identified as homozygous. Based on quantitative fluorescence results, two mutant lines with significantly lower AtGT1 gene expression levels than wild-type were screened from the homozygous atgt1 mutant lines and named atgt1-1 and atgt1-3.

[0030] like Figure 4 As shown, after the three Arabidopsis strains WT, atgt1-1 and atgt1-3 were cultivated to maturity, seeds were harvested for subsequent experiments and functional verification.

[0031] 2. Root growth analysis of the mutant atgt1 Arabidopsis thaliana

[0032] Arabidopsis seedlings with uniform growth on 1 / 2 MS medium were selected for transplanting. Photographs were taken 5 days later, and the number of lateral roots and the length of the taproot were recorded. Figure 5 As shown, compared to WT (3.75±1.25), the number of lateral roots in mutants atgt1-1 and atgt1-3 were 15.14±2.86 and 13.14±2.14, respectively, showing a significant difference and an increase of approximately 247%. Compared to the taproot length of WT (5.99±0.43 cm), the taproot lengths of the atgt1-1 and atgt1-3 mutants were 10.55±0.19 cm and 10.27±0.17 cm, respectively, representing a significant increase of approximately 56.7%. These results indicate that the atgt1 mutant promotes taproot elongation in Arabidopsis, suggesting that the AtGT1 gene can promote root architecture development in plants.

[0033] 3. Physiological basis analysis of atgt1 mutant Arabidopsis thaliana

[0034] Figure 6 This figure compares the effects of the atgt1 mutant on physiological parameters in Arabidopsis thaliana. Roots of 18-day-old *Strombus haematococcus* (WT) and atgt1-1 mutant *Arabidopsis thaliana* were collected, and auxin content was measured and analyzed. The results showed that the auxin content in the roots of the WT mutant was 1.224 nmol / L, while the auxin content in the roots of the atgt1-1 mutant was 1.409 nmol / L, a significant increase of approximately 13.2%. These results indicate that loss of function of the AtGT1 gene leads to increased auxin synthesis in the underground parts of the plant.

[0035] This invention provides the first AtGT1 gene mutant Arabidopsis thaliana, verifying that the AtGT1 gene can promote the growth and development of plant roots, enriching the theoretical basis of AtGT1 gene in the study of plant root growth and development, providing a theoretical basis for breeding new varieties of other plants with well-developed roots, and having important guiding significance for the discovery and identification of functional genes for crop root development.

[0036] The above description is merely an example and illustration of the present invention. Those skilled in the art can make various modifications or additions to the specific embodiments described, or use similar methods to replace them, as long as they do not deviate from the structure of the present invention or exceed the scope defined in the claims, all of which should fall within the protection scope of the present invention.

Claims

1. The application of the AtGT1 gene in plant root development, characterized by: The base sequence of the AtGT1 gene is shown in SEQ ID NO.1 of the sequence listing. The AtGT1 gene is used to negatively regulate the root development of plants, and the plant is Arabidopsis thaliana.

Images