Application of dahurian angelica root expansion protein AdEXPA7 in regulation and control of root development

Abstract

The invention provides an application of a dahurian angelica root expansion protein gene AdEXPA7 in regulation and control of root development. According to the application disclosed by the invention, the fact that the AdEXPA7 has a positive regulation effect on the elongation of root cells is determined by utilizing a biotechnology. Expression of an auxin response gene AdARFs can be induced by applying exogenous auxin to treat an angelica dahurica strain, and the auxin response gene AdARF8-2 is combined with an AuxRE cis-element on a promoter of an expansion protein gene AdEXPA7 of the angelica dahurica, so that expression of the AdEXPA7 is positively regulated and controlled. The root system, hypocotyl and root cell length of the arabidopsis thaliana obtained by over-expression of AdEXPA7 are significantly increased. Therefore, through overexpression of AdEXPA7, the invention has important breeding utilization value for promoting growth and development of arabidopsis thaliana roots.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology applications and relates to a gene for an angelica diffusing protein. AdEXPA7 Application in regulating root development. Background Technology

[0002] Angelica dahurica is a perennial, tall herb belonging to the genus Angelica of the Apiaceae family. It typically grows in forests, forest edges, streams, shrublands, and valleys, and both its roots and leaves are used medicinally. As a perennial herb, Angelica dahurica can grow to a height of 1-2.5 meters. Its roots are cylindrical with a yellowish-brown or brown epidermis and a strong odor. The stems are hollow, 2-5 cm in diameter, and purple in color. The leaves are usually ovate or triangular, with petioles up to 15 cm long. The flowers are in compound umbels, 10-30 cm in diameter, with trichomes on the pedicels and pedicels. Literature reports indicate significant differences in the content and spatial distribution of coumarin in the roots of Angelica dahurica from Hangzhou and Qizhou at different developmental stages. Analysis of root tissue sections of Angelica dahurica also shows differences in the type, spatial distribution, and content of coumarin between primary and lateral roots. Through transcriptome and gene expression profiling analysis, six isoprenyltransferase (PTs) coding sequences were screened, and two pentenyltransferases were identified using real-time quantitative PCR and in vitro enzyme activity verification. However, to date, there is limited research on the molecular mechanisms of action in the root development of Angelica dahurica.

[0003] Plant hormones play a crucial role in cell development. Studies have shown that many hormones are involved in regulating plant growth and cell expansion. Among them, auxins are an important class of plant hormones widely found in plants, with indoleacetic acid (IAA) being the most typical representative. Their main functions include promoting cell elongation, inducing tropism, apical dominance, promoting organ formation, and promoting fruit development. In in vitro ovule culture experiments, exogenous application of auxin significantly promoted the initiation of cotton fiber cell development, and targeted expression of auxin biosynthesis genes significantly improved fiber quality. In previous studies, this invention treated Angelica dahurica lines with exogenous auxin, and experimental results showed that low concentrations of auxin significantly activated Angelica dahurica expansion proteins. AdEXPA7 The expression of this protein further promotes root development. Currently, there are no reports on the regulation of root development by the expansor protein EXPA in Angelica dahurica. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide an angelica dilatant protein. AdEXPA7 In the regulation of root development, the full-length ORF nucleotide sequence of the gene and the amino acid sequence of the encoded protein were disclosed. Using this gene as a target gene, the gene was heterologously expressed in the model plant Arabidopsis thaliana by Agrobacterium-mediated flower sac method to clarify its role in plant root development and to cultivate new germplasm for production applications.

[0005] The objective of this invention is achieved through the following technical solution: In a first aspect, the present invention provides an angelica dilatant protein. AdEXPA7 The angelica dilatant protein AdEXPA7 The amino acid sequence is shown in SEQ ID NO.2.

[0006] Secondly, the present invention provides the aforementioned Angelica dahurica dilatant protein. AdEXPA7 The encoding gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] Thirdly, the present invention provides a gene expression cassette comprising the aforementioned encoding gene.

[0008] Fourthly, the present invention provides a recombinant expression vector comprising the aforementioned coding gene.

[0009] Fifthly, the present invention provides a transgenic engineered bacterium comprising the recombinant expression vector described above.

[0010] In a sixth aspect, the present invention provides the application of the coding gene, the gene expression cassette, the recombinant expression vector, or the transgenic engineered bacteria in improving plant root development, wherein improving root development includes at least one of increasing root length, hypocotyl length, and root cell length.

[0011] Furthermore, the plant in question is Arabidopsis thaliana.

[0012] Furthermore, the encoding gene is overexpressed in plants.

[0013] Seventhly, the application of the coding gene, the gene expression cassette, the recombinant expression vector, or the transgenic engineered bacteria in plant breeding to cultivate new Arabidopsis varieties with increased root length, hypocotyl length, and / or root cell length.

[0014] The beneficial effects of this invention are: (1) This invention, through auxin treatment of plants, for the first time screened and isolated auxin from Angelica dahurica. AdEXPA7 Genes were identified and their expression patterns were systematically analyzed using bioinformatics, clarifying... AdEXPA7 This gene is predominantly expressed in the root system, providing a new key gene for regulating plant root development using genetic engineering techniques. (2) This invention has confirmed through systematic molecular biology experiments that the auxin-responsive gene AdARF8-2 binds to the Angelica dahurica dilatation protein gene. AdEXPA7 AuxRE cis-element (TGTCTC) activation on the promoter AdEXPA7 The expression, AdEXPA7This further affected the root growth and development process, and the analysis... AdEXPA7 The study revealed the key targets and pathways of the regulatory mechanisms that promote root development, laying the foundation for the precise promotion of root development through genetic engineering. (3) This invention constructs an overexpression vector and uses Agrobacterium-mediated heterologous overexpression in Arabidopsis thaliana. AdEXPA7 Detection confirmed that the expression level of this gene was significantly increased in the transgenic lines. Phenotypic analysis showed that, compared with the wild type, the root length, hypocotyl length, and root cell length of the overexpressing lines were all significantly increased, directly confirming the gene's expression level. AdEXPA7 It has the function of promoting the root development of Arabidopsis thaliana, providing an effective way to cultivate new Arabidopsis thaliana varieties with significantly improved root development. Attached Figure Description

[0015] Figure 1 Phenotypic and root indices of Angelica dahurica plants treated with auxin.

[0016] Figure 2 Auxin-responsive genes after auxin treatment AdARFs The level of expression.

[0017] Figure 3 Auxin-responsive genes AdARFs Expression levels of Angelica dahurica in different tissues and organs.

[0018] Figure 4 Transcriptome analysis of Angelica dahurica roots after auxin treatment.

[0019] Figure 5 This study aimed to determine the expression levels of genes involved in cell wall synthesis and polysaccharide metabolism.

[0020] Figure 6 For AdARF8-2 and AdEXPA7 The AuxRE element on the promoter is combined.

[0021] Figure 7 for AdEXPA7 Expression levels of Angelica dahurica in different tissues and organs.

[0022] Figure 8 AdEXPA7 DNA identification of Arabidopsis thaliana overexpression lines.

[0023] Figure 9 AdEXPA7 exist AdEXPA7 Expression levels in transgenic Arabidopsis and WT.

[0024] Figure 10 for AdEXPA7 Phenotypic analysis of heterologous expression in Arabidopsis thaliana. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this invention should be included within the scope of protection of this invention.

[0026] Example 1 AdEXPA7 Gene screening 1. Planting and growing conditions of plant materials Angelica dahurica was grown in a light incubator (16h:8h, light:dark, 24°C:20°C). Wild-type Arabidopsis thaliana was grown in a light incubator (16h:8h, light:dark, 24°C:20°C) for root length determination; and in an incubator (0h:24h, light:dark, 24°C:20°C) for hypocotyl length determination.

[0027] 2. Auxin treatment of Angelica dahurica strains Angelica dahurica plants that had grown normally for 30 days in a light incubator and exhibited uniform growth were divided into an experimental group and a control group, with 12 plants in each group. The experimental group was treated with 10 μM IAA, while the control group was treated with water. Treatments were repeated every 7 days for a total of 4 times. Root samples were taken and root scanners were used to analyze root length, number of root tips, root surface area, root volume, and root diameter in both groups. Experimental data showed that compared to the control, auxin treatment significantly increased root length, number of root tips, root surface area, root volume, root diameter, and root fresh weight (e.g., ...). Figure 1 (As shown in A and B).

[0028] 3. Angelica dahurica auxin-responsive genes AdARFs RT-qPCR analysis RT-qPCR analysis of auxin-responsive genes in the auxin-treated experimental group and the water-treated control group AdARFs level of expression (e.g.) Figure 2 As shown), data were collected from three biological replicates, with two technical replicates for each reaction. Error bars represent the standard deviation (SD) between biological replicates. * and ** are obtained through... t The tests indicate significant differences of 0.05 and 0.01, respectively.

[0029] Genes responding to auxin in different tissues and organs of Angelica dahurica AdARFs Perform quantitative RT-qPCR detection (e.g.) Figure 3(As shown) Data were collected from three biological replicates, with three technical replicates for each reaction. Error bars represent the standard deviation (SD) between biological replicates. Design AdARFs The specific primers are shown in Table 1: Table 1: Primers used for amplification

[0030] 4. Transcriptome analysis was performed on the experimental and control groups. A total of 370 differentially expressed genes (Padjust < 0.05) were found in the roots of Angelica dahurica from the water-treated control group (AdCK) and the auxin-treated experimental group (AdIAA). Volcano plots are used to represent these genes. Among them, 250 differentially expressed genes were upregulated, and 120 differentially expressed genes were downregulated (e.g., ...). Figure 4 As shown in A). GO enrichment analysis of these differentially expressed genes revealed that they were mainly enriched in plant cell wall synthesis, cell wall organization and modification, pectin decomposition and metabolism, and polysaccharide metabolism (e.g., Figure 4 As shown in B).

[0031] The pectin esterase gene was identified. AdPE4 , AdPE15 , AdPE24 , AdPE41 , AdPE53 ), pectin acetylesterase gene ( AdPAE9 ), Cellulose synthase A gene ( AdCESA7 ), dilatin A gene ( AdEXPA7 ) and mannosidase gene ( AdMAN6 The TPM values ​​(e.g., in auxin-treated and water-treated Angelica dahurica lines) were respectively... Figure 5 (as shown in A) and level of expression (such as...) Figure 5 (As shown in B). Consistent with transcriptome data, the expression levels of these genes were significantly increased in auxin-treated Angelica dahurica lines compared to water-treated controls. Data were collected from three biological replicates, with two technical replicates for each reaction. Error bars represent the standard deviation (SD) between biological replicates. ** By t The test result indicates a significant difference of 0.01.

[0032] Example 2 AdARF8-2 Construction of overexpression vectors target gene AdARF8-2The full-length ORF sequence was obtained, and recombinant primers were designed using CE Design V1.03 software (see Table 2). PCR amplification was performed using cDNA from the roots of *Angelica dahurica* as a template. After the amplification reaction, 2 μL of 10' Loading buffer was added, followed by agarose gel electrophoresis. The target band was then excised and recovered from the gel; specific steps are detailed in the gel recovery kit instructions. PBI121 is a publicly available vector containing a 35S promoter. The PBI121 vector plasmid was digested with enzymes, and then reacted with the target fragment in a PCR instrument at 37°C for 30 min for recombination. Immediately after 30 min, the mixture was removed and placed on ice. The recombinant product was transformed into competent *E. coli* cells, plated, and incubated upside down at 37°C for 12 h. Single colonies were picked from the plates and placed in liquid LB medium containing 700 μL of the corresponding antibiotic, and incubated at 37°C and 200 rpm for 5–6 h. Using bacterial culture as a template, PCR amplification was performed using universal primers on the vector. After gel running, the positive bacterial culture was sequenced. Plasmids were extracted from bacterial cultures with correct sequencing sequences and transformed into GV3101 Agrobacterium (Qingke Biotechnology, Nanjing).

[0033] Table 2: Primers used for amplification

[0034] Example 3: Verification of AdARF8-2 and AdEXPA7 promoter binding experiment Through the AdEXPA7 Analysis of the upstream 15000 bp promoter revealed an auxin-responsive gene binding element, AuxRE (TGTCTC). Figure 6 (As shown in A). The binding activity of the auxin-responsive gene AdARF8-2 to AuxRE was verified. In the LUC experiment, 35S:: AdARF8-2 As an effector, AuxRE-mini35S:: LUC Constituting the reporting factor. 35S:: LUC As a positive control, the fluorescence signal of tobacco leaves infected with the reporter factor alone was very weak, while the fluorescence signal of tobacco leaves co-transfected with the effector factor and the reporter factor was stronger. The results indicate that AdARF8-2 and... AdEXPA7 AuxRE elements on the promoter are combined (e.g.) Figure 6 (As shown in B). In the Dual-LUC reporting system, at 35S:: AdARF8-2 As an effector factor, 35S:: GUS As an empty vector, six tandemly repeated AuxRE cis-elements were recombined into the mini35S promoter and upstream of the LUC gene to construct a reporter factor (AuxRE-mini35S::). LUCThe REN gene in the reporter factor construction vector serves as an internal reference gene. When the reporter factor and effector factor are co-transfected into tobacco leaves, the LUC / REN ratio significantly increases compared to reporter factor infection alone (e.g., ...). Figure 6 As shown in C), the above experimental results indicate that AdARF8-2 and AdEXPA7 The AuxRE element on the promoter combines positive regulation AdEXPA7 The expression.

[0035] Example 4 RT-qPCR analysis of the Angelica dahurica expansion protein gene AdEXPA7 design AdEXPA7 Specific primers: F: 5'-TTGGTGCTACAAAGGGTCAC-3' (SEQ ID NO. 15) R: 5'-CACCGTTGTTGGAGTCTTGA-3' (SEQ ID NO. 16) Quantitative RT-qPCR was performed to detect the expression level of this gene in different tissues and organs of Angelica dahurica. The results showed that the expression level of this gene differed in roots, stems, leaves, and flowers, with dominant expression in roots (e.g., roots, stems, leaves, and flowers). Figure 7 As shown in the figure, data were collected from three biological replicates, with three technical replicates for each reaction. Error bars represent the standard deviation (SD) between biological replicates. t The test result indicates a significant difference of 0.01.

[0036] Example 5 AdEXPA7 Construction of overexpression vectors target gene AdEXPA7 The full-length ORF sequence was obtained, and recombinant primers were designed using CE Design V1.03 software (see Table 3). PCR amplification was performed using cDNA from the roots of *Angelica dahurica* as a template. After the amplification reaction, 2 μL of 10' Loading buffer was added, and agarose gel electrophoresis was performed. The target band was then excised and recovered from the gel; specific steps are detailed in the gel recovery kit instructions. The PBI121 vector plasmid was digested with enzymes, and then reacted with the target fragment in a PCR instrument at 37°C for 30 min to perform the recombination reaction. Immediately after 30 min, the mixture was removed and placed on ice. The recombinant product was transformed into competent *E. coli* cells, plated, and incubated upside down at 37°C for 12 h. Single colonies were picked from the plates and placed in liquid LB medium containing 700 μL of the corresponding antibiotic, and incubated at 37°C and 200 rpm for 5–6 h. Using bacterial culture as a template, PCR amplification was performed using universal primers on the vector. After gel running, the positive bacterial culture was sequenced. Plasmids were extracted from bacterial cultures with correct sequencing sequences and transformed into GV3101 Agrobacterium (Qingke Biotechnology, Nanjing).

[0037] Table 3: Primers used for amplification

[0038] Example 6 AdEXPA7 Positive identification and phenotypic analysis of overexpression in Arabidopsis thaliana Arabidopsis thaliana was transformed using the Agrobacterium-mediated flower-pouring method and the results were verified, yielding a 35S promoter-driven [transformation method]. AdEXPA7 Overexpression of Arabidopsis thaliana plants was performed, and DNA was extracted from the transgenic plants for PCR identification (e.g., Figure 8 (As shown). RT-qPCR analysis showed that, compared with the wild type, in AdEXPA7 In Arabidopsis thaliana overexpression, the expression level of this gene is significantly increased (e.g., Figure 9 (As shown). AdEXPA7 After overexpressing Arabidopsis thaliana and wild-type WT plants were cultured under the same normal growth conditions for 9 days, compared with WT, AdEXPA7 Overexpression of Arabidopsis thaliana significantly increased root length (e.g. Figure 10 (As shown in A and B), data were collected from 9 biological replicates. Additionally, AdEXPA7 After overexpressing Arabidopsis thaliana and wild-type WT plants were cultured under the same dark growth conditions for 9 days, the hypocotyl length was observed. The results showed that compared with WT, AdEXPA7 Overexpression of Arabidopsis thaliana significantly increases hypocotyl length (e.g. Figure 10 (As shown in C and D), collected from 6 biological replicates. AdEXPA7 Analysis of root cell length in Arabidopsis thaliana overexpressing the gene and in wild-type WT (wt) plants after 9 days of normal growth showed that, compared with WT, AdEXPA7 Root cell length was significantly increased by overexpression of Arabidopsis thaliana (e.g. Figure 10 (As shown in E and F) Data collected from 6 biological replicates. Scale bar = 50 µm. Error bars represent the standard deviation (SD) between biological replicates. ** By t The test result indicates a significant difference of 0.01.

Claims

1. A type of Angelica dahurica dilatant protein AdEXPA7 Characterized by the presence of angelica dilatant protein AdEXPA7 The amino acid sequence is shown in SEQ ID NO.

2.

2. The Angelica dahurica dilatant protein of claim 1 AdEXPA7 Encoding genes ， Its features are, The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

1.

3. A gene expression cassette, characterized in that, It includes the coding gene as described in claim 2.

4. A recombinant expression vector, characterized in that, It includes the coding gene as described in claim 2.

5. A genetically engineered bacterium, characterized in that, It includes the recombinant expression vector as described in claim 4.

6. The application of the encoding gene of claim 2, the gene expression cassette of claim 3, the recombinant expression vector of claim 4, or the transgenic engineered bacteria of claim 5 in improving plant root development, characterized in that, The improvement of root development includes improving at least one of the following: root length, hypocotyl length, and root cell length.

7. The application according to claim 6, characterized in that, The plant in question is Arabidopsis thaliana.

8. The application according to claim 6, characterized in that, The encoding gene is overexpressed in plants.

9. The application of the encoding gene of claim 2, the gene expression cassette of claim 3, the recombinant expression vector of claim 4, or the transgenic engineered bacterium of claim 5 in plant breeding, characterized in that, To cultivate new Arabidopsis varieties with increased root length, hypocotyl length and / or root cell length.

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