Key gene LiRabA4a for regulating and controlling leaf color of lagerstroemia indica and application of key gene LiRabA4a

Abstract

The invention relates to the technical field of gene engineering application, and particularly discloses a key gene LiRabA4a for regulating and controlling the leaf color of crape myrtle and application thereof. The nucleotide sequence of the gene LiRabA4a is as shown in Seq 1, and the coded amino acid sequence is as shown in Seq 2. The invention relates to cloning of a key gene LiRabA4a for regulating and controlling the leaf color of lagerstroemia indica. Through qRT-PCR analysis, it is found that the LiRabA4a gene is remarkably predominantly expressed in crape myrtle leaves, and the transcriptional level of the LiRabA4a gene is related to the leaf color yellowing degree; the LiRabA4a gene is overexpressed through an agrobacterium tumefaciens-mediated genetic transformation method, so that the chlorophyll content of the crape myrtle can be increased, and the leaf color is greener; liRabA4a gene expression is inhibited, so that the chlorophyll content is reduced, and the leaf color is yellow; the invention further relates to heterologous expression of the LiRabA4a gene in catalpa bungei for remarkably promoting growth and development of catalpa bungei. The LiRabA4a gene provided by the invention can be used for directional genetic improvement of the leaf color of the lagerstroemia indica, can be used for cultivating lagerstroemia indica varieties with different leaf colors, and has an important application value in the aspects of gene engineering and molecular design breeding of the lagerstroemia indica.

Description

Technical Field

[0001] This invention relates to a key gene, LiRabA4a, that regulates the leaf color of crape myrtle and its application, particularly to the application of the LiRabA4a gene in improving the leaf color of crape myrtle, belonging to the fields of plant genetic engineering and biotechnology. Background Technology

[0002] Crape myrtle (Lagerstroemia indica) is a deciduous shrub or small tree belonging to the genus Lagerstroemia in the family Lythraceae. It is an important summer flowering woody plant worldwide, characterized by its long flowering period (up to 3-4 months), vibrant flower colors (including red, pink, purple, and white), and strong adaptability, making it widely used in urban landscaping and garden ornamental purposes. In recent years, variegated crape myrtle has become an important direction in crape myrtle breeding due to its long viewing period (from spring leaf emergence to autumn leaf fall), strong sense of landscape layering, and ability to create large color blocks with leaves instead of flowers during the off-season.

[0003] Currently, research on variegated crape myrtle mainly focuses on phenotypic observation and physiological mechanism analysis, with slow progress in molecular mechanism research, especially in the insufficient discovery and functional analysis of key genes regulating leaf color formation. This makes it difficult to cultivate stable variegated crape myrtle varieties through targeted genetic engineering. We screened a leaf color-related gene from the GL1 dominant bud mutation of crape myrtle with golden leaves. This gene encodes a member of the RabA subfamily of small GTP-binding proteins, homologous to AtRabA4a in Arabidopsis thaliana, and named it LiRabA4a. Small GTP proteins are named for their small molecular weight (20–30 kD) and are ubiquitous in eukaryotes such as yeast, plants, and humans. Based on structure and function, small GTP proteins are divided into five families: Ras, Rho, Rab, Arf, and Ran. The Rab protein family is the largest member of the small GTP superfamily. In plants, the Rab family can be further divided into eight subfamilies: RabA-H. The Rab11 / RabA group is the largest subgroup of the plant Rab family, with 26 genes in Arabidopsis and 17 genes in rice. In current research, leaf color regulation in plants is closely related to chlorophyll metabolism and light signal transduction. Vesicle transport, as a core pathway for the directed transport of intracellular substances, has been found to participate in chloroplast development and chlorophyll synthesis in recent years. However, the role of the small G protein Rab family (key regulators of vesicle transport) in the regulation of Lagerstroemia indica leaf color has not yet been reported. Summary of the Invention

[0004] This invention provides a key gene, LiRabA4a, for regulating the leaf color of crape myrtle and its applications. Overexpression of the LiRabA4a gene via Agrobacterium-mediated genetic transformation increases the chlorophyll content of crape myrtle leaves, resulting in greener leaves; inhibiting LiRabA4a gene expression decreases chlorophyll content, resulting in yellow leaves. Simultaneously, heterologous expression of the LiRabA4a gene in catalpa significantly promotes its growth and development. The LiRabA4a gene provided by this invention can be used for targeted genetic improvement of crape myrtle leaf color, and has important application value in crape myrtle genetic engineering and molecular design breeding.

[0005] To achieve the above-mentioned objectives, the technical solution adopted by the present invention is as follows:

[0006] This invention provides a key gene, LiRabA4a, for regulating the leaf color of crape myrtle. The full-length ORF is 669 bp, and its nucleotide sequence is as described in Seq 1.

[0007] This invention provides a protein encoding LiRabA4a, a key gene regulating the leaf color of crape myrtle, which encodes 222 amino acids, and its amino acid sequence is as described in Seq 2.

[0008] A plant expression vector, pBI121-GFP-LiRabA4a, containing the key gene LiRabA4a that regulates the leaf color of crape myrtle, has been developed. The LiRabA4a gene is promoted by a dual CaMV35S promoter. The vector contains LB and RB sequences, which facilitate the integration of the vector fragment, including the LiRabA4a gene, into the crape myrtle genome.

[0009] A pTRV2 vector containing the key gene LiRabA4a regulating crape myrtle leaf color is disclosed. The vector is initiated by the constitutive strong expression promoter CaMV35S and contains the ppk2 RNA2 sequence of TRV and the interference sequence of the LiRabA4a gene. TRV2:LiRabA4a is mixed with the helper vector TRV1 and then infects crape myrtle leaves to achieve LiRabA4a gene interference.

[0010] Specific applications include: This invention is the first to clone the LiRabA4a gene, a small G protein superfamily gene in Lagerstroemia indica. Using Gateway technology, the gene was recombined into a plant expression vector to construct the overexpression vector pBI121-GFP-LiRabA4a and the silencing vector TRV2:LiRabA4a, obtaining LiRabA4a overexpression and silencing lines, thereby studying the function of the LiRabA4a gene in the regulation of Lagerstroemia indica leaf color. This invention is the first to clone and elucidate the function of the Rab gene in leaf color regulation in Lagerstroemia indica, and also provides the first application of LiRabA4a, a key gene regulating Lagerstroemia indica leaf color.

[0011] Compared with existing technologies, this invention, through qRT-PCR analysis, revealed that the LiRabA4a gene is significantly and predominantly expressed in crape myrtle leaves, and its transcriptional level is correlated with the degree of leaf yellowing. Overexpression of the LiRabA4a gene increases chlorophyll content in crape myrtle leaves, resulting in greener leaves; inhibition of LiRabA4a gene expression decreases chlorophyll content, resulting in yellow leaves. Furthermore, heterologous expression of the LiRabA4a gene in catalpa trees significantly promotes their growth and development. This indicates that the LiRabA4a gene is a key regulatory gene for crape myrtle leaf color and has significant application value in the fields of forestry genetic engineering, ornamental plant variety improvement, and the ornamental plant industry. Attached Figure Description

[0012] The invention will now be described in further detail with reference to the accompanying drawings, but this is not intended to limit the invention.

[0013] Figure 1 Correlation analysis of the transcriptional level of the LiRabA4a gene and leaf color.

[0014] A: Tissue expression characteristics analysis of the LiRabA4a gene; B, C: Phenotypic analysis of different leaf colors of Lagerstroemia indica and transcriptional level analysis of the LiRabA4a gene in its leaves.

[0015] Figure 2 Overexpression of the LiRabA4a gene enhances the chlorophyll content in crape myrtle leaves.

[0016] A: Phenotype of LiRabA4a transgenic crape myrtle plant, Bar = 5cm; B: Phenotype of LiRabA4a transgenic crape myrtle leaf, Bar = 1cm; C: qRT-PCR analysis of the LiRabA4a gene; D: Detection of chlorophyll content in leaves of LiRabA4a transgenic crape myrtle.

[0017] Figure 3 Phenotypic and physiological analysis of VIGS-silenced LiRabA4a gene.

[0018] A: In the plant growth chamber, empty control (TRV:00), golden leaf dominant bud mutant (GL1), VIGS silence (TRV:LiRabA4a) leaf phenotype; B, C: Analysis of LiRabA4a gene transcription level in the leaves of the plants described in (A) (B), chlorophyll content detection (C).

[0019] Figure 4 Heterologous expression of the LiRabA4a gene in Catalpa trees significantly promotes their growth and development.

[0020] A: Phenotype of heterologous LiRabA4a transgenic catalpa, Bar = 5cm; B: Leaf phenotype of heterologous LiRabA4a transgenic catalpa, Bar = 2cm; CE: Net photosynthetic rate (Pn) (C), stomatal conductance (Gs) (D), and transpiration rate (Tr) (E) of heterologous LiRabA4a transgenic catalpa. Detailed Implementation

[0021] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0022] The reagents used in the implementation examples mainly include molecular biology experimental reagents and kits, all of which are commercially available. Unless otherwise specified, the methods provided in the embodiments of this invention are conventional methods.

[0023] Implementation Case 1: Cloning of the LiRabA4a Gene in Lagerstroemia indica

[0024] 1. Extraction of total RNA from Lagerstroemia indica leaves and cDNA reverse mixing

[0025] Fresh leaves of Lagerstroemia indica were collected, rapidly frozen in liquid nitrogen, and total RNA was extracted using the FastPure PlantTotal RNA Isolation Kit (Novizan, RC401), following the kit's instructions. Total RNA was quantified using a One Drop instrument, and integrity was assessed by 1% agarose gel electrophoresis.

[0026] cDNA synthesis was performed according to the instructions of the HiScript III 1st Strand cDNA Synthesis Kit (+gDNAwiper) (Novazia, R312), and the reverse transcription product was stored at -20°C.

[0027] 2. Cloning and sequencing of the LiRabA4a gene

[0028] Primers (F: 5'-AGCTGGAAGAATTGATGGGT-3'; R: 5'-AAGAGGGAGACGGTGAGTGA-3') were designed in the 5' and 3' untranslated regions of the LiRabA4a gene based on the crape myrtle genome information, and the LiRabA4a gene was cloned using map-based cloning.

[0029] PCR amplification was performed using a high-fidelity enzyme (Novizan, P505).

[0030] PCR reaction system: 2×Phanta Max Buffer, 25μL; dNTP Mix (10mM each), 1μL; 100ng / LcDNA, 2μL; F (10μM), 2μL; R (10μM), 2μL; Phanta Max Super-Fidelity DNA Polymerase, 1μL; ddH2O, up to 50μL.

[0031] PCR amplification program: pre-denaturation 95℃ for 3 min, denaturation 95℃ for 15 s, annealing 58℃ for 15 s, extension 72℃ for 1 min, 35 cycles, and final extension 72℃ for 10 min.

[0032] After the reaction, the target band was detected by 1% agarose gel electrophoresis, and the PCR product was recovered using an agarose gel DNA recovery kit according to the manufacturer's instructions. The PCR product was ligated into the pMD19-T vector (Takara, D102A) according to the manufacturer's instructions. The ligation product was then transformed into DH5α E. coli competent cells according to the manufacturer's instructions.

[0033] Using the aforementioned cloning primers, positive transformants were selected for sequencing via PCR (Nanjing Sipujin Biotechnology Co., Ltd.). After sequence alignment, the positive single clones were cultured in a 7:3 mixture with 50% glycerol and stored at -80℃ for later use. Sequencing analysis showed that the coding region of the gene is shown in Seq 1, with an ORF length of 669 bp (including the stop codon), and the encoded amino acid sequence is shown in Seq 2, encoding 222 amino acids.

[0034] Implementation Case 2: Expression Pattern Analysis of the LiRabA4a Gene in Lagerstroemia indica

[0035] The expression patterns of the LiRabA4a gene in different tissues of Lagerstroemia indica, including roots, stems, leaves, flowers, and flower buds, as well as in Lagerstroemia indica germplasm materials with different leaf colors, were analyzed using qRT-PCR. The reagents used were SYBR Premix Ex Taq II (Takara, RR820), and the instrument was an Applied Biosystems Step One Plus™ Real-Time PCR System (AppliedBiosystems, USA). The procedures were performed according to the manufacturer's instructions.

[0036] The results are as follows Figure 1 As shown, the LiRabA4a gene is significantly predominantly expressed in crape myrtle leaves, and the lower the transcription level of the LiRabA4a gene, the deeper the yellowing of the leaves, indicating that the transcription level of the LiRabA4a gene is related to the leaf color of crape myrtle.

[0037] The quantitative primers for the LiEF1α-2 internal reference gene are:

[0038] F-5'-AGCGAGAGCGTGGTATCA-3',

[0039] R-5'-GGTGTCCAGGAGCATCAATG-3'.

[0040] The quantitative primers for the LiANS gene are:

[0041] F-5'-GGACAAGAACGGTATAGG-3',

[0042] R-5'-TAATGTCATACACGAGCAT-3'.

[0043] Implementation Case 3: Construction of LiRabA4a Recombinant Overexpression Vector and Obtaining Overexpression Lines

[0044] 1. Construction of LiRabA4a recombinant overexpression vector

[0045] Based on the sequence information of the LiRabA4a gene and the vector pBI121-GFP, the target gene fragment was inserted into the Kpn I and BamH I sites of the pBI121-GFP vector using recombination to obtain a plant expression vector.

[0046] LiRabA4a gene recombination primers for constructing plant overexpression vectors:

[0047] F-5'-ATTTACGAACGATAGGGTACCATGGCGAATGGAGATGTGAG-3';

[0048] R-5'-CCTTGCTCACCATGGATCCGGAGCAGCCGCAGGACAACA-3'.

[0049] PCR was performed using a high-fidelity enzyme, and the procedure was as described in Example 1. The recombinant reaction was performed according to the instructions of the recombinant kit (Beijing Jinsha, SC612).

[0050] The plant expression vector containing the target gene was introduced into Agrobacterium competent cells EHA105 via a freeze-thaw method. Specific steps: 20 ng of the vector plasmid was added to 100 μL of Agrobacterium EHA105 competent cells; the cells were flash-frozen in liquid nitrogen for 5 min, then incubated in a 37°C water bath for 5 min; 800 μL of LB liquid medium was added, and the cells were shaken at 200 rpm for 4 h at 28°C; the cells were centrifuged at 5000 rpm for 1 min at room temperature, the supernatant was removed, and the remaining cells were resuspended in the supernatant and evenly spread onto LB solid medium containing kanamycin (Kana) (100 mg / L) + rifampin (Rif) (50 mg / L), and incubated in the dark at 28°C for 48-72 h until single colonies appeared.

[0051] Positive transformants were verified by PCR using the LiRabA4a gene recombinant primers used to construct the plant overexpression vector. The positive clone bacterial solution was mixed with 50% glycerol at a 1:1 ratio and stored at -80℃ for later use.

[0052] 2. Genetic transformation of the LiRabA4a gene in Lagerstroemia indica

[0053] After activating the Agrobacterium-positive transformants containing the recombinant overexpression vector of the LiRabA4a gene, the leaves of Lagerstroemia indica were genetically transformed by Agrobacterium-mediated transformation: the third and fourth leaves from the top near the terminal bud of tissue-cultured Lagerstroemia indica seedlings were used as explants, and the LiRabA4a gene was introduced into the Lagerstroemia indica genome by Agrobacterium-mediated transformation.

[0054] Obtaining transgenic plants and analyzing the transcriptional level of the LiRabA4a gene: After obtaining the transformed crape myrtle regenerated plants, leaves were taken, RNA was extracted and reversed into cDNA, and the transcriptional level of the LiRabA4a gene was analyzed using qRT-PCR. Specific procedures are described in Case Study 2.

[0055] like Figure 2 As shown, the LiRabA4a gene was significantly highly expressed in transgenic plants #4 and #6.

[0056] 3. Phenotypic identification of LiRabA4a transgenic Lagerstroemia indica

[0057] like Figure 2 As shown, overexpression of the LiRabA4a gene significantly increased the chlorophyll content in crape myrtle leaves, resulting in greener leaves.

[0058] Implementation Case 4: Obtaining and Phenotypic Analysis of LiRabA4a Gene Silent Lines

[0059] 1. Construction of VIGS vector TRV2:LiRabA4a

[0060] Based on the sequence information of the LiRabA4a gene and the TRV2 vector, the target gene fragment was inserted into the Sac I and EcoRI sites of the TRV2 vector using recombination to obtain a plant expression vector.

[0061] LiRabA4a gene recombination primers for constructing the VIGS vector:

[0062] F-5'-TCTGTGAGTAAGGTTACCGAATTCAGGAGCAGTCGGGGCG-3';

[0063] R-5'-CCGGGCCTCGAGACGCGTGAGCTCGAGCAGCCGCAGGACA-3'.

[0064] PCR was performed using a high-fidelity enzyme, and the procedure was as described in Example 1. The recombinant reaction was performed according to the instructions of the recombinant kit (Beijing Jinsha, SC612).

[0065] The TRV2:LiRabA4a vector was introduced into Agrobacterium competent cells GV3101 using a freeze-thaw method. The procedure was the same as described in Example 3.

[0066] 2. Agrobacterium infection

[0067] Agrobacterium bacterial suspension containing TRV1 and TRV2:LiRabA4a vectors (OD) 600 =0.8) mixed at a 1:1 ratio, and 0.02% Silwet L-77 was added. The mixture was then used to infect Lagerstroemia indica leaves via Agrobacterium injection. The transcriptional level of the LiRabA4a gene was analyzed using qRT-PCR. Specific procedures are described in Case Study 2.

[0068] like Figure 3 As shown, the expression level of the LiRabA4a gene in silenced plants was 30%-40% of that in the control.

[0069] 3. Phenotypic Analysis

[0070] like Figure 3 As shown, the leaves of VIGS-silenced plants turned yellow and the chlorophyll content decreased significantly.

[0071] Implementation Case 5: Heterologous Expression and Phenotypic Analysis of LiRabA4a Gene in Catalpa Genome

[0072] 1. Genetic transformation of the LiRabA4a gene in Catalpa bungei

[0073] After activating the Agrobacterium-mediated transformation of Catalpa embryogenic callus containing the LiRabA4a gene recombinant overexpression vector in Implementation Case 3, the transformation was carried out by Agrobacterium-mediated transformation. Positive detection and transcriptional level analysis were performed by qRT-PCR, with specific procedures following Implementation Cases 2 and 3, to obtain LiRabA4a transgenic Catalpa lines.

[0074] 2. Phenotypic identification of LiRabA4a transgenic Lagerstroemia indica

[0075] The LiRabA4a transgenic catalpa plants were transferred from tissue culture bottles to pots and cultured in soil in a culture room.

[0076] like Figure 4 As shown, the transgenic catalpa lines heterologously expressing the LiRabA4a gene are taller, have larger leaves, and are greener. At the same time, the net photosynthetic rate (Pn), stomatal conductance (Gs), and transpiration rate (Tr) are all significantly higher than the control, indicating that heterologous expression of the LiRabA4a gene in catalpa significantly promotes its growth and development.

Claims

1. A key gene LiRabA4a for regulating leaf color of Lagerstroemia indica and application thereof, characterized in that, The nucleotide sequence of the gene is as described in Seq 1. 2.The key gene LiRabA4a for regulating leaf color of Lagerstroemia indica and application thereof according to claim 1, characterized in that, The gene encodes an amino acid sequence as described in Seq 2.

3. The regulatory key gene LiRabA4a of Lagerstroemia indica according to claim 1 or 2, characterized in that, Overexpression of LiRabA4a gene increases the chlorophyll content of Lagerstroemia indica, and silencing of the gene reduces the chlorophyll content.

4. The regulatory key gene LiRabA4a of Lagerstroemia indica according to claim 1 or 2, characterized in that, Heterologous expression of LiRabA4a gene in Malus halliana significantly promotes its growth and development.

5. The regulatory key gene LiRabA4a of Lagerstroemia indica according to claim 1 or 2, characterized in that, Application of LiRabA4a gene in regulating leaf color of Lagerstroemia indica.

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