ATAL6 gene and application thereof in regulation and control of plant leaf size development

Abstract

The invention belongs to the field of plant genetic engineering, and particularly relates to an ATAL6 gene and application thereof in regulation and control of plant leaf size development. It is found for the first time that by conducting site-directed mutagenesis on the ATAL6 gene or knocking out or reducing expression of the ATAL6 gene, the gene function is deleted, and the plant leaf area is remarkably reduced. The ATAL6 gene can provide a theoretical basis and a gene source for cultivating new varieties of crops, and has a good practical application value.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering, specifically involving ATAL6 Genes and their application in regulating plant leaf size and development. Background Art

[0002] Leaf organs in higher plants play a crucial role in ensuring normal plant growth. Arabidopsis thaliana, as a model plant, although not directly economically beneficial in practical production, plays an important role in the field of botany due to its small genome, short growth cycle, and abundant seeds. For a long time, in-depth research has been conducted on its leaf development mechanisms, confirming that leaf development is regulated by many genes. These genes determine the size of leaf organs by coordinating cell proliferation and cell expansion. For example, positive regulatory factors... WAIT , AtARGOS and AtGRF It promotes leaf enlargement by regulating cell proliferation, and AtERF4 By promoting cell expansion, leaf enlargement is regulated; among negative regulatory factors... AtTCP4 It affects leaf size by inhibiting cell proliferation, while AtARP1 and AtDRM1 This is achieved by inhibiting cell expansion, leading to smaller leaves. These studies not only reveal the molecular mechanisms regulating plant growth rate but also hold the promise of applying related genes to agricultural production to improve crop yield and resource utilization efficiency.

[0003] Therefore, in-depth exploration and identification of genes related to the regulation of plant leaf size and development, and the use of genetic transformation technology to conduct research and utilization of functional genes are not only of great significance to plant developmental biology and breeding, but also provide valuable gene resources for the innovation of crop germplasm resources and genetic improvement, and have broad application prospects in the field of agricultural production. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides... ATAL6 Genes and their application in regulating plant leaf size and development.

[0005] The present invention adopts the following technical solution: The first aspect of the present invention provides ATAL6 The application of genes in any of the following: (A1) Application in regulating plant leaf size; (A2) Application in cultivating plants with altered leaf size; (A3) Application in molecular breeding for improving plant leaves or in the improvement of germplasm resources related to plant leaves; The ATAL6The nucleotide sequence of the gene is as shown in SEQ ID NO.1 or has 95% or more identity or similarity with the nucleotide sequence shown in SEQ ID NO.1 and expresses the same function as the protein.

[0006] The application can adjust the settings by increasing or decreasing them. ATAL6 It is achieved through the expression level of genes.

[0007] Furthermore, the application may include adjusting... ATAL6 The expression level of genes can be used to increase the size of plant leaves or to cultivate plants with larger leaves.

[0008] Furthermore, the upregulation can be achieved through overexpression. ATAL6 It is achieved through genes.

[0009] Furthermore, the application may include lowering the... ATAL6 The expression level of genes can be used to reduce the size of plant leaves or to cultivate plants with smaller leaves.

[0010] Furthermore, the downregulation can be achieved by suppressing or reducing... ATAL6 This is achieved by adjusting gene expression levels, including using genetic engineering techniques to... ATAL6 A gene is mutated at a specific site, resulting in the loss of its function; or, by knocking out or reducing its function. ATAL6 Gene expression.

[0011] In some embodiments of the present invention, the ATAL6 The gene originates from Arabidopsis thaliana.

[0012] It should be further noted that those skilled in the art can readily employ known methods, such as site-directed mutagenesis (including oligonucleotide primer-mediated site-directed mutagenesis, PCR-mediated site-directed mutagenesis, and cassette mutagenesis) or directed evolution (including error-prone PCR, DNA shuffling, and in vitro random recombination), to modify the present invention. ATAL6 The nucleotide sequence of the gene is mutated. Those artificially modified nucleotide sequences that have 75% or more identity with the ATAL6 protein encoded by this invention, as long as they encode the ATAL6 protein and have the same function as the ATAL6 protein, are all nucleotide sequences derived from and equivalent to the sequences of this invention.

[0013] A second aspect of the invention provides a method for cultivating plants with smaller leaves, the method comprising reducing or inhibiting the presence of certain substances in the target plant. ATAL6 The gene expression level was used to obtain plants with leaves smaller than the target plant.

[0014] In some embodiments of the present invention, the reduction or inhibition of the target plant ATAL6 The pathways for adjusting gene expression levels include: using genetic engineering techniques to... ATAL6 A gene is mutated at a specific site, resulting in the loss of its function; or, by knocking out or reducing its function. ATAL6 Gene expression.

[0015] Furthermore, the inhibition or reduction ATAL6 Gene expression levels include those achieved through gene knockdown, gene editing, and / or gene knockout techniques. ATAL6 Nucleic acid molecules with missing or inactivated genes.

[0016] Furthermore, gene knockout technologies (including RNA interference, Morpholino interference, and antisense nucleic acid) are utilized. Gene expression suppression, silencing, or knockout of genes is well known to those skilled in the art, using technologies such as ribozyme technology, gene editing technologies (including zinc finger ribozyme gene editing, TALEN gene editing, and CRISPR gene editing), or gene knockout technologies (including complete gene knockout and conditional gene knockout). For example, targeted proteins can be used to suppress gene expression, silence, or knock out genes. ATAL6 Gene expression can be inactivated or silenced at the post-transcriptional or translational level by shRNA, siRNA, or miRNA encoding the gene. Target genes can also be knocked out using the CRISPR-Cas system, which contains sgRNA and Cas protein.

[0017] Furthermore, nucleic acid molecules that inhibit or reduce the expression of the ATAL6 gene may include (1) double-stranded RNA (dsRNA), small interfering RNA (siRNA), microRNA (miRNA), and short hairpin RNA (shRNA) used in RNA interference technology; (2) antisense RNA (asRNA) and antisense oligonucleotides (AON) used in antisense nucleic acid technology; (3) gRNA and sgRNA used in gene editing technology; and (4) aptamers and ribozymes.

[0018] In some embodiments of the present invention, the method of inhibiting or reducing the expression level of the ATAL6 gene is by inserting a T-DNA sequence into the ATAL6 gene.

[0019] In some embodiments of the present invention, the T-DNA insertion site is at 1325 bp in the ATAL6 gene, which is located on the fourth exon, resulting in the loss of ATAL6 gene function.

[0020] A third aspect of the invention provides a method for cultivating plants with larger leaves, the method comprising increasing the concentration of certain substances in the target plant. ATAL6The gene expression level was used to obtain plants with leaves larger than the target plant.

[0021] Furthermore, the improvement in the target plant ATAL6 Gene expression levels can be achieved through at least one of the following methods: C1) Add the above ATAL6 The copy number of a gene; C2) will the ATAL6 Genes are expressed under the influence of strong promoters; C3) Add the above ATAL6 Gene regulatory elements cause overexpression, including enhancer elements, elements that improve mRNA stability, elements that enhance translation efficiency, and / or elements that enhance protein secretion; C4) Add the above ATAL6 The ribosome binding site of a gene; C5) regarding the above ATAL6 Gene codon optimization; C6) Upregulates gene expression by altering epigenetic modifications such as DNA methylation or histone acetylation.

[0022] Furthermore, the D2) can be achieved by... ATAL6 This can be achieved by replacing the natural promoter of the gene with a strong promoter, or by operatively linking a second promoter to the ATAL6 gene.

[0023] The strong promoters include, but are not limited to, the T7 promoter, CaMV promoter, SV40 promoter, SFFV promoter, ubq promoter, ubi promoter, RBCS promoter, Actin promoter, Emu promoter, CYP450 promoter, Adhl promoter, and pinⅡ promoter.

[0024] The enhancers include, but are not limited to, CMV enhancers, SV40 enhancers, and RSV enhancers.

[0025] In the above method, the improvement of the target plant ATAL6 The expression level of the gene can be achieved by introducing the ATAL6 gene into the target plant.

[0026] In the above method, the ATAL6 The nucleotide sequence of the gene may be as shown in SEQ ID NO.1.

[0027] Furthermore, the improvement in the target plant ATAL6 Gene expression can be achieved by introducing the DNA molecule shown in SEQ ID NO.1 into the target plant.

[0028] The method for cultivating plants with larger leaves may include the following steps: (1) Constructing a system containing the encoding described above ATAL6 Recombinant vectors for gene nucleic acid molecules; (2) Introduce the recombinant vector constructed in step (1) into the target plant; (3) Transgenic plants were obtained through screening and identification.

[0029] Furthermore, the above method may include step (4) after step (3): hybridizing the transgenic plant with the plant to be improved to obtain offspring transgenic plants, wherein the offspring transgenic plants are phenotypically consistent with the transgenic plant (i.e., the transgenic plant as the parent).

[0030] In this invention, in the above-described applications or methods, the plant is any of the following: (B1) Monocotyledonous or dicotyledonous plants; (B2) Grasses or cruciferous plants.

[0031] Furthermore, the plant can be any one or more of crops, such as corn, rice, tomato, potato, peanut, soybean, cotton, tobacco, cucumber, melon, watermelon, Chinese cabbage, rapeseed, bok choy, spinach, and radish.

[0032] In some embodiments of the present invention, the plant is the model plant Arabidopsis thaliana.

[0033] The regulation described in this article can be upregulation (promoting or increasing) or downregulation (inhibiting, reducing or decreasing).

[0034] In this article, the target plant may be one containing... ATAL6 The target plant of genes.

[0035] The article discusses ATAL6 Genes can be either endogenous or exogenous.

[0036] The beneficial effects of the above technical solutions are as follows: This invention is the first discovery ATAL6 Genes positively regulate plant leaf size development. It was found that by... ATAL6 Gene knockout renders the gene non-functional, or site-directed mutation or reduction is achieved through gene editing. ATAL6 Gene expression is significantly reduced, resulting in a substantial decrease in plant leaf area. This invention provides a theoretical basis and gene source for cultivating new high-yielding and superior crop germplasm. Attached Figure Description Figure 1 yes AtAL6 Relative expression levels of genes in wild-type Arabidopsis and mutants; in the figure: WT represents wild-type. atal6-1, atal6-2 To knock out AtAL6 Arabidopsis mutants following the gene; Figure 2 It is the insertion of T-DNA in the al6 mutant. ATAL6 Schematic diagram of gene location; Figure 3 The phenotypes of Arabidopsis thaliana seeds from each group were germinated on normal 1 / 2 MS medium and grown for 10 days. Seedlings with the same growth were then transferred to the substrate and grown for 15 days. In the figure, WT represents the wild type. atal6-1 , atal6-2 To knock out AtAL6 Arabidopsis mutants following the gene; Figure 4 It is wild-type Arabidopsis thaliana, knockout AtAL6 Statistics on leaf phenotype and leaf area of ​​Arabidopsis mutant plants after gene modification.

[0037] In the image: WT represents the wild type; atal6-1 , atal6-2 To knock out AtAL6 Arabidopsis mutants following the gene. Detailed Implementation

[0038] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof. The present invention provides a general and / or specific description of the materials and test methods used in the experiments. Although many materials and operating methods used to achieve the objectives of the present invention are known in the art, the present invention is still described in as much detail as possible herein.

[0039] Purchased T-DNA inserted Arabidopsis mutant seeds (SALK_040877C) from the Arabidopsis mutant library (http: / / www.arashare.cn).

[0040] Example 1 Identification of Arabidopsis mutants ATAL6 Gene expression levels.

[0041] 1. RNA was extracted from wild-type Arabidopsis and mutants, respectively, using the following methods: Pre-cool a mortar with liquid nitrogen, weigh 2 g of Arabidopsis thaliana leaves, add liquid nitrogen and grind rapidly. Transfer the powder to a centrifuge tube pre-filled with 1 mL of Trizol, and incubate at room temperature for 30 min to allow for complete lysis. Centrifuge at 12,000 rpm for 20 min at 4°C, and transfer the supernatant to a new RNase-free EP tube. Add 200 μL of chloroform, vortex to mix, and incubate at room temperature for 15 min. Centrifuge at 12,000 rpm for 20 min at 4°C. Take 500 μL of the supernatant and place it in a new EP tube, add 500 μL of pre-cooled isopropanol, and incubate at -20°C for 30 min. Centrifuge at 12,000 rpm for 10 min at 4°C, discard the supernatant, and the precipitate is the total RNA. Add 1 mL of 75% ethanol to the precipitate, gently invert and wash the precipitate, centrifuge at 8,000 rpm for 5 min at 4 °C, repeat twice; discard the supernatant, air-dry the precipitate at room temperature, dissolve the precipitate in 50 μL of DEPC water, and store in an ultra-low temperature freezer at -80 °C for later use.

[0042] Total RNA was obtained from wild-type Arabidopsis and Arabidopsis mutants using this method.

[0043] 2. Reverse transcription PCR Total RNA samples were used as templates for reverse transcription PCR amplification (two-step method). The reverse transcription PCR amplification system is shown in Table 1 and Table 2.

[0044] Table 1. First Step PCR Amplification System

[0045] Reaction conditions: 42℃, 2 min, store at 4℃.

[0046] Table 2. PCR amplification system for the second step

[0047] Reaction conditions: 37℃ for 15 min, 85℃ for 5 s, and store at 4℃.

[0048] Total cDNA from wild-type Arabidopsis and Arabidopsis mutants was obtained using this method. The reagents in Step 1 and Step 2 were obtained from the TAKALA (PrimeScript™ II 1st Strand cDNA Synthesis Kit).

[0049] 3. Real-time quantitative PCR Application of LightCycler ® 480 Real-time PCR Amplification Instrument and Quantitative Real-Time PCR Kit (SYBR) ® Premix EX TaqTM II. TAKARA) was used to detect the total cDNA of wild-type Arabidopsis and Arabidopsis mutants obtained in step 2 of this embodiment using real-time quantitative PCR. The steps were performed according to the LC480 instrument manual and the kit instructions. The design of real-time quantitative PCR primers followed these principles: primer length: 18~22 bp; Tm value: 55~65℃; GC content: 40~60%; product length: 80~250 bp; primer dimers and hairpin structures were avoided.

[0050] The PCR primer sequences are as follows: ATAL6-qF: 5′-TGGCGTCAAGTCTCGCCAAT-3′, ATAL6-qR: 5′-GCACCACAAACCGCACCTTG-3′.

[0051] The PCR reaction system (prepared on ice) is shown in Table 3.

[0052] Table 3 PCR reaction system

[0053] The standard amplification procedure (two-step method) is as follows: Stage 1: Pre-denaturation, 95℃, 30 s, 20℃ / s, 1 Cycle; Stage 2: PCR reaction, 95℃, 5 s, 20℃ / s; 60℃, 20 s, 20℃ / s, 40 cycles; Stage 3: Melting curve analysis: 95℃, 0 s, 20℃ / s; 65℃, 15 s, 20℃ / s; 95℃, 0 s, 0.1℃ / s.

[0054] This method was used to obtain amplified products of total cDNA from wild-type Arabidopsis thaliana and Arabidopsis thaliana mutants, respectively.

[0055] Using the Arabidopsis ACTIN2 gene as an internal reference gene, the relative expression levels in wild-type Arabidopsis and Arabidopsis mutants were compared, specifically as follows: Figure 1 As shown.

[0056] Depend on Figure 1 It can be seen that, compared with wild-type Arabidopsis, the mutant Arabidopsis thaliana... ATAL6 Gene expression levels were significantly reduced, so the two mutant lines in the figure are effective mutants and are named Arabidopsis mutants. atal6-1 and atal6-2, for al6 mutant ATAL6 Gene sequencing, such as Figure 2As shown, a T-DNA insertion occurred at 1325 bp in the genome sequence of this gene, located on the fourth exon, resulting in... ATAL6 Loss of gene function.

[0057] Example 2 Wild-type Arabidopsis seeds and Arabidopsis mutants were collected separately. atal6-1 and atal6-2 After germinating and growing for 10 days on normal 1 / 2 MS medium, the seedlings were transferred to the substrate and grown for 15 days. The area of ​​each individual leaf was measured and statistically analyzed.

[0058] The culture conditions were as follows: after vertical culture for 10 days in an incubator with a photoperiod of 20 ± 2℃ and 16h (light) / 8h (dark), the cells were transferred to the substrate and placed in a tissue culture room with a photoperiod of 20 ± 2℃ and 16h (light) / 8h (dark) for 15 days.

[0059] Depend on Figure 3 It can be seen that the Arabidopsis mutant atal6-1 and atal6-2 The leaf area of ​​the knockout strain was significantly smaller than that of the wild-type Arabidopsis, indicating that the knockout strain... ATAL6 Significant changes occurred in the development of posterior lobe organs.

[0060] Depend on Figure 4 It can be seen that the Arabidopsis mutant atal6-1 and atal6-2 The leaf area of ​​the wild-type Arabidopsis is significantly smaller than that of the wild-type Arabidopsis. When comparing the leaf area of ​​a single leaf at the same location... atal6-1 and atal6-2 The mutant's leaf area was significantly smaller than that of the wild-type Arabidopsis. This further illustrates... ATAL6 The growth and development of each leaf of the Arabidopsis mutant with significantly reduced gene expression levels showed significant changes.

[0061] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications made within the spirit and principles of the present invention are not permitted. Modifications, equivalent substitutions, improvements, etc., should all be included within the scope of protection of this invention.

Claims

1. ATAL6 The application of genes in any of the following: (A1) Application in regulating plant leaf size; (A2) Application in cultivating plants with altered leaf size; (A3) Application in molecular breeding for improving plant leaves or in the improvement of germplasm resources related to plant leaves; The ATAL6 The nucleotide sequence of the gene is as shown in SEQ ID NO.1 or has 95% or more identity or similarity with the nucleotide sequence shown in SEQ ID NO.1 and expresses the same function as the protein.

2. The application according to claim 1, characterized in that, The application can adjust the settings by increasing or decreasing them. ATAL6 Gene expression levels regulate plant leaf size.

3. The application according to claim 1, characterized in that, The application includes adjusting... ATAL6 The application involves increasing the expression level of the gene to increase plant leaf size or to cultivate plants with larger leaves; or, the application includes downregulating the expression level of the gene. ATAL6 The expression level of genes can be used to reduce the size of plant leaves or to cultivate plants with smaller leaves.

4. A method for cultivating plants with smaller leaves, characterized in that, The method includes reducing or inhibiting the content of [something] in the target plant. ATAL6 The gene expression level was used to obtain plants with leaves smaller than the target plant.

5. The method according to claim 4, characterized in that, The reduction or inhibition of the target plant ATAL6 The pathways for adjusting gene expression levels include: using genetic engineering techniques to... ATAL6 A gene undergoes a site-directed mutation, resulting in the loss of the gene's function.

6. The method according to claim 4, characterized in that, The reduction or inhibition of the target plant ATAL6 The pathways for increasing gene expression include using genetic engineering techniques to insert T-DNA. ATAL6 In genes, repression ATAL6 Gene expression.

7. The method according to claim 4, characterized in that, The plant in question is Arabidopsis thaliana.

8. A method for cultivating plants with larger leaves, characterized in that, The method includes improving the target plant ATAL6 The gene expression level was used to obtain plants with leaves larger than the target plant.

9. The method according to claim 8, characterized in that, The expression level of the ATAL6 gene in the target plant can be increased through at least one of the following methods: C1) Increase the copy number of the ATAL6 gene; C2) The ATAL6 gene is expressed under the drive of a strong promoter; C3) Increase the expression of the ATAL6 gene regulatory elements, including enhancer elements, elements that improve mRNA stability, elements that enhance translation efficiency, and / or elements that enhance protein secretion. C4) Increase the ribosome binding site of the ATAL6 gene; C5) Codon optimization of the ATAL6 gene; C6) Upregulates gene expression by altering epigenetic modifications such as DNA methylation or histone acetylation.

10. The method according to claim 8, characterized in that, The plant in question is Arabidopsis thaliana.

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