Application of AtGT1 gene in plant root development

Abstract

The invention discloses an application of an AtGT1 gene in plant root development, which is characterized in that the base sequence of the AtGT1 gene is shown as SEQ ID NO.1 in a sequence table, and the AtGT1 gene is used for negative regulation of plant root development. The AtGT1 gene mutant arabidopsis thaliana is obtained for the first time, it is verified that the AtGT1 gene is applied to arabidopsis thaliana to regulate and control growth and development of underground parts of plants, theoretical research of the AtGT1 gene in plant root development is enriched, a theoretical basis is provided for cultivating new varieties of plants with developed root systems, and the AtGT1 gene mutant arabidopsis thaliana has great significance in further understanding of the growth mechanism of the plant root systems. And more genetic resource information can be provided for improving crop root systems on the molecular level.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to the application of AtGT1 gene in plant root development. Background Art

[0002] Roots play a vital role in the development of plants, mainly including fixing plants to the soil, providing mechanical support for plants, absorbing water and nutrients to maintain growth, and establishing beneficial relationships with microbiota. The spatiotemporal structure of roots is called root system architecture (RSA). RSA is plastic in plant growth. This mechanism ensures that plants can adapt to environmental changes and better absorb nutrients and water to maintain photosynthesis in the aboveground plants. With the changes in the environment and the emergence of various biotic and abiotic stresses, good RSA can provide effective protection for plants and maintain normal growth and development of individuals.

[0003] The triple helix transcription factor family is named for its unique highly conserved triple helix domain (helix-loop-helix-loop-helix). It can specifically bind to the GT element (a light-responsive DNA element), so this family is also called the GT family. According to previous studies, the GT family is divided into five subfamilies in plants: GT-1, GT-2, GTγ, SH4 and SIP1. In addition, soybean also contains a subfamily GTδ. Early reports found that the GT family is mainly related to light response. In Arabidopsis, the mRNA expression level of GT-4 is significantly induced by white light. The rice GT-1 family transcription factor Osrml1 is significantly downregulated under continuous light conditions, and the transcription level of Osrml1 also shows rhythmic changes with the alternation of light and dark. Under red light treatment, the transcription level of soybean GT family transcription factor GmGT-2 is significantly downregulated in etiolated seedlings. Studies have found that the GT family plays an important role in the growth and development of plants at different stages. The soybean GmGT-2A and GmGT-2B genes both contain two triple helical domains. Under salt treatment, drought treatment, and low temperature treatment, the activity of Arabidopsis plants overexpressing these two genes was significantly higher than that of the wild type. However, there are few reports on the effects of GT1 family genes on plant root development, and the biological role of GT1 proteins in affecting plant root development is still unclear. Summary of the invention

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

[0005] In one aspect of the present invention, the present invention proposes the application of 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 table SEQ ID NO.1, and the AtGT1 gene is used to negatively regulate plant root development.

[0006] Compared with the prior art, the present invention has the following beneficial effects:

[0007] In the present invention, an AtGT1 gene mutant Arabidopsis was obtained for the first time, verifying the application of the AtGT1 gene in Arabidopsis to regulate the growth and development of the underground part of the plant, enriching the theoretical research on the AtGT1 gene in the development of the plant root system, providing a theoretical basis for breeding new varieties of plants with well-developed root systems, which is of great significance for further understanding the growth mechanism of plant roots, and can also provide more genetic resource information for improving the root system of crops at the molecular level. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0009] Figure 2 This is a diagram of the identification of atgt1 homozygous mutant Arabidopsis thaliana by the three-primer method in Example 2 of the present invention, wherein LP and RP are primers on both sides 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 of the T-DNA insertion position of the atgt1 mutant in Example 2 of the present invention;

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

[0012] Figure 5 The figure shows the comparison of the nutritional growth status of atgt1 mutant plants and control plants in 1 / 2MS medium in Example 2 of the present invention, including (a) root phenotype diagram, (b) lateral root number statistics diagram of WT and atgt1 mutant, and (c) main root length statistics diagram of WT and atgt1 mutant;

[0013] Figure 6 This is a statistical graph of the auxin (IAA) content in the roots of 18-day-old WT (wild type) and atgt1 mutant Arabidopsis thaliana in Example 2 of the present invention. DETAILED DESCRIPTION

[0014] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0015] In the following examples, various processes and methods not described in detail are conventional methods known in the art. The primers used are all indicated when they first appear, and the same primers used thereafter are all the same as the first indicated content.

[0016] Example 1

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

[0018] The coding sequence and amino acid sequence of the Arabidopsis thaliana AtGT1 gene were queried 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 a total of 604 amino acids. The protein molecular mass is 67878.97 Da and the isoelectric point is 6.3 (https: / / web.expasy.org). Protein prediction (https: / / swissmodel.expasy.org / ) showed that the AtGT1 protein has two SANT domains, and the protein three-dimensional structure shows that the AtGT1 protein has a typical triple helical structure ( Figure 1 ).

[0024] 2. Functional identification of mutant Arabidopsis

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

[0026] 1. Acquisition and identification of the atgt1 mutant Arabidopsis thaliana

[0027] The mutant with AtGT1 gene knockout by T-DNA technology was purchased from the Arabidopsis mutant library and named atgt1 mutant. DNA was extracted from leaves of 20-day-old atgt1 mutants. According to the three-primer method, three different types were screened and identified in the offspring lines of AtGT1 gene knockout mutants, including homozygous mutants, heterozygous mutants and wild types. There were 15 homozygous lines, 1 heterozygous line and 3 wild-type (WT) Arabidopsis lines. Figure 2 As shown. Among them, the AtGT1 gene function is lost in the homozygous mutant and can be stably inherited, so subsequent experiments can be carried out; the heterozygous offspring will still separate and be unstable; the AtGT1 gene function is not lost in the wild type (that is, the AtGT1 gene knockout fails), and it contains the AtGT1 gene.

[0028] Sequencing of identified homozygous and heterozygous lines: The insertion position of T-DNA in atgt1 mutants was determined through the Arabidopsis mutant website (http: / / signal.salk.edu / cgi-bin / tdnaexpress) and DNA sequencing methods, such as Figure 3 As shown in Figure 1, the T-DNA insertion site is located at 99 bp on the second exon of the AtGT1 gene.

[0029] The rosette leaves of the wild-type Arabidopsis thaliana atgt1 mutant and the atgt1 mutant identified as a homozygous strain were used for RNA extraction. Based on the fluorescence quantitative results, two mutant strains with significantly lower AtGT1 gene expression levels than the wild-type were screened out from the homozygous atgt1 mutant and named: atgt1-1 and atgt1-3.

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

[0031] 2. Analysis of root growth of atgt1 mutant Arabidopsis

[0032] Arabidopsis seedlings with uniform growth on 1 / 2MS medium were selected for transplanting. Photos were taken 5 days later and the number of lateral roots and taproot length were counted. Figure 5 As shown, compared with WT (3.75±1.25), the number of lateral roots of mutants atgt1-1 and atgt1-3 were 15.14±2.86 and 13.14±2.14, respectively, with significant differences and a significant increase of about 247%. Compared with the taproot length of WT (5.99±0.43cm), the taproot length of atgt1-1 and atgt1-3 mutants was 10.55±0.19cm and 10.27±0.17cm, a significant increase of about 56.7%. The results showed that the mutant atgt1 promoted the elongation of the main root of Arabidopsis, indicating that the AtGT1 gene can promote the development of plant root architecture.

[0033] 3. Analysis of the physiological basis of atgt1 mutant Arabidopsis

[0034] Figure 6 This is a comparison chart of the effects of atgt1 mutants on the physiological indicators of Arabidopsis. The roots of 18-day-old WT and atgt1-1 mutant Arabidopsis were collected, and the auxin content was measured and analyzed. The results showed that the auxin content in the WT root system was 1.224nmol / L, while the auxin content in the atgt1-1 mutant root system was 1.409nmol / L, a significant increase of about 13.2%. The results show that the loss of AtGT1 gene function will lead to increased auxin synthesis in the underground part of the plant.

[0035] In the present invention, an AtGT1 gene mutant Arabidopsis thaliana was obtained for the first time, verifying that the AtGT1 gene can promote the growth and development of plant roots, enriching the theoretical basis of the AtGT1 gene in the research on plant root growth and development, providing a theoretical basis for breeding new varieties of other plants with well-developed root systems, and having important guiding significance for discovering and identifying functional genes for crop root development.

[0036] The above contents are merely examples and explanations of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in a similar manner. As long as they do not deviate from the structure of the present invention or exceed the scope defined by the claims, they shall all fall within the protection scope of the present invention.

Claims

1. Application of AtGT1 gene in plant root development, characterized by: The base sequence of the AtGT1 gene is shown in the sequence table SEQ ID NO.1, and the AtGT1 gene is used for negatively regulating plant root development.

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