AcLTH70 gene for regulating and controlling formation of pineapple leaf thorns and application of AcLTH70 gene

Abstract

The invention belongs to the technical field of molecular biology and genetic engineering, and particularly relates to an AcLTH70 gene for regulating and controlling formation of pineapple leaf thorns and application of the AcLTH70 gene. The pineapple AcLTH70 gene has the effect of regulating and controlling the formation of pineapple leaf thorns, the pineapple variety with thorns, few thorns or no thorns is obtained by regulating and controlling the overexpression or silencing of the AcLTH70 gene, the pineapple variety requirements of different cultivation scenes are met, the pineapple breeding efficiency is further improved, and the pineapple AcLTH70 gene has important industrial application value.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology and genetic engineering technology, specifically relating to an AcLTH70 gene that regulates the formation of pineapple leaf thorns and its application. Background Technology

[0002] Pineapple (Ananas comosus L. Merr., also known as pineapple) is one of the world's most important tropical fruits, serving multiple purposes including fresh consumption, processing, and ornamental value. In pineapple breeding and cultivation, leaf spines are a crucial agronomic trait affecting variety utilization. On one hand, traditional pineapple varieties for fresh consumption (such as Bali and hand-torn pineapple) have leaves densely covered with sharp spines. During planting and management (fertilization, pesticide application, etc.), fruit harvesting, and processing, these spines can easily scratch workers or damage machinery, increasing labor intensity and production costs, and hindering the advancement of mechanized production. On the other hand, many pineapple varieties are ornamental, such as variegated pineapples and flowering pineapples, with vibrant colors and long flowering periods, making them important materials for high-end cut flowers and indoor ornamental plants. Their inflorescences can remain unfaded for over a month under cut flower conditions, possessing high ornamental and economic value. However, currently, most ornamental pineapple varieties widely cultivated in the market have spines on their leaf edges, which not only inconveniences field management and transportation but also affects consumer choice, greatly limiting their horticultural application and industrial promotion. Therefore, analyzing the molecular mechanism of pineapple leaf spine formation and identifying key genes that regulate leaf spine development are of great theoretical and applied value for breeding new spineless or less thorny varieties.

[0003] Currently, research on the genetic basis and regulatory mechanisms of pineapple leaf spine formation is still limited, and no clearly defined functional genes have been reported. Existing breeding methods mainly rely on traditional hybridization and selection, which suffers from problems such as long cycles and poor directionality (easily accompanied by other undesirable traits), making it difficult to efficiently meet the industry's demand for leaf spine trait improvement. With the development of genetic engineering technology, identifying and utilizing key genes regulating leaf spines, and achieving targeted trait improvement through gene editing or overexpression technologies, has become a core direction in crop breeding. Summary of the Invention

[0004] The purpose of this invention is to provide an AcLTH70 gene that regulates the formation of pineapple leaf spines and its application. By regulating the overexpression or silencing of the AcLTH70 gene, pineapple varieties with spines, fewer spines, or no spines can be obtained to meet the needs of pineapple varieties in different scenarios, thereby improving the efficiency of pineapple breeding.

[0005] This invention is achieved through the following technical solution: The base sequence of the AcLTH70 gene that regulates the formation of pineapple leaf thorns in this invention is shown in SEQ ID No.1, and its coding region is 729 bp in length; the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2, and it can encode a protein containing 242 amino acids.

[0006] This invention provides a recombinant expression vector containing the AcLTH70 gene described above. Constructed based on a plant expression vector, this recombinant vector can stably introduce the AcLTH70 gene into pineapple cells, achieving efficient gene expression or silencing, and providing a vector tool for regulating leaf spine traits.

[0007] Furthermore, according to the aforementioned recombinant expression vector, the recombinant expression vector is obtained by inserting the AcLTH70 gene into the multiple cloning site of the plant expression vector pGWB506. The pGWB506 vector contains a strong 35S promoter, a GFP fluorescent tag, and a hygromycin resistance gene. The 35S promoter can drive the efficient expression of the AcLTH70 gene in pineapple, the GFP tag facilitates tracking the gene expression location, and the hygromycin resistance gene can rapidly screen transgenic positive plants, improving the efficiency of vector construction and genetic transformation.

[0008] This invention provides a recombinant engineered bacterium, obtained by transforming the aforementioned recombinant expression vector into Agrobacterium GV3101 competent cells. The Agrobacterium GV3101 strain exhibits broad host adaptability and can efficiently introduce the AcLTH70 gene into the pineapple genome by infecting pineapple callus or leaves, making it a highly efficient tool for achieving pineapple genetic transformation.

[0009] This invention provides the application of the AcLTH70 gene in regulating pineapple leaf spine formation. Experiments have shown that this gene has a positive regulatory effect on leaf spine formation: overexpression of the AcLTH70 gene in spineless pineapple varieties (such as upright-leaved varieties) can induce the formation of hooked leaf spines at the leaf margins; conversely, inhibiting the expression of this gene (such as gene silencing) can reduce or eliminate leaf spines in thorny varieties, thus achieving targeted regulation of the leaf spine trait.

[0010] Based on the above applications, specifically: when overexpressing the AcLTH70 gene, the gene is introduced into pineapple cells through recombinant vector-mediated expression, driving efficient gene expression in leaves and promoting leaf spine formation; when inhibiting AcLTH70 gene expression, RNA interference (RNAi) or gene editing (such as CRISPR / Cas9) technology can be used to reduce the in vivo gene expression level, achieving a phenotypic phenotype with reduced or no leaf spines, meeting the needs of different cultivation scenarios (such as the need for spineless varieties for mechanized harvesting, and the need for varieties with specific spine morphology for ornamental applications).

[0011] One method for cultivating pineapple plants with improved leaf spine traits is to use genetic engineering to adjust the expression level of the AcLTH70 gene that regulates pineapple leaf spine formation, including overexpressing the gene to obtain spined pineapple plants or silencing the gene to obtain spineless pineapple plants.

[0012] The primers used for cloning the AcLTH70 gene using genetic engineering techniques are AcLTH70-F and AcLTH70-R; the primer for AcLTH70-F is ATGGCGTCGTCGAGTGGT; and the primer for AcLTH70-R is TGGCACAATGGATCTGGG.

[0013] This invention also provides the application of the above-mentioned recombinant expression vector in the cultivation of new pineapple germplasm with improved leaf spine traits. By transforming pineapple with the recombinant vector containing the AcLTH70 gene through Agrobacterium-mediated transformation, new thorny or spineless germplasm can be cultivated in a targeted manner, shortening the breeding cycle (2-3 years), and the trait is highly targeted, avoiding the problem of undesirable trait linkage in traditional hybridization.

[0014] This invention provides the application of the above-mentioned recombinant engineered bacteria in the genetic improvement of pineapple leaf spine traits. The recombinant engineered bacteria can efficiently mediate the genetic transformation of the AcLTH70 gene, and the operation is simple and the transformation efficiency is stable (positive rate can reach 30%-50%), making it suitable for large-scale cultivation of pineapple plants with improved leaf spines.

[0015] The beneficial effects of this invention are: 1. The pineapple AcLTH70 gene of the present invention can regulate the growth of pineapple leaf spines, and the regulatory effect is significant. Overexpression can induce the production of leaf spines in spineless varieties, and inhibiting expression can eliminate leaf spines in thorny varieties, thus achieving bidirectional targeted regulation.

[0016] 2. The present invention has strong technical operability, mature construction methods for recombinant vectors and engineered bacteria, high genetic transformation efficiency, and is suitable for industrial application.

[0017] 3. This invention can regulate the expression of the AcLTH70 gene in pineapple varieties through genetic engineering, thereby screening and breeding pineapple varieties that can meet different needs such as mechanized harvesting (thornless or less thorny), ornamental (thornless or less thorny), and disease and pest resistance (thorny), thus accelerating the breeding of new varieties and promoting the diversified development of the pineapple industry.

[0018] 4. The discovery of the AcLTH70 gene in this invention provides new insights into the molecular mechanism of pineapple leaf spine formation, and also offers important genetic resources and technical approaches for spineless pineapple breeding and the improvement of new ornamental pineapple varieties. Through the study of... AcLTH70 Gene regulation can produce thornless or less thorny plants while maintaining fruit quality and flower ornamental value, showing significant potential for agricultural and horticultural applications. Attached Figure Description

[0019] Figure 1 shows a comparison of leaf spine phenotypes of different pineapple germplasm resources; from left to right, they are Bali, Ornamental Red Skin, Golden Diamond, Standing Leaf, Hand-Torn, and Sweet Honey varieties. Among them, (A) is a detailed magnified view of the leaf spines, and (B), (C), and (D) are the presence and distribution of leaf spines on the upper, middle, and lower parts of the plant leaves, respectively.

[0020] Figure 2 shows the GWAS analysis results based on the pineapple leaf spine trait; the horizontal axis represents pineapple chromosomes (labeled as Chr01-Chr25), and the vertical axis represents the -log10 (P) value of the association signal (significant association threshold is 6).

[0021] Figure 3 shows the tissue expression heatmap of candidate genes within the leaf spine trait association interval of chromosome Chr07; where the horizontal axis represents the names of the selected candidate genes, and the vertical axis represents different tissue types of pineapple (leaf, fruit, petal, ovule, stamen); the color gradient of the heatmap represents the relative expression level of genes, with orange indicating high expression and green indicating low expression.

[0022] Figure 4 shows a comparison of the leaf spine phenotypes of AcLTH70-overexpressing pineapple and wild-type pineapple in the examples; where (A), (B), and (C) are the plant and leaf phenotypes of wild-type upright-leaf pineapple (thornless variety); and (D), (E), and (F) are the plant and leaf phenotypes of upright-leaf pineapple overexpressing AcLTH70. Detailed Implementation

[0023] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials and reagents used are all commercially available; the experimental operations all follow standard procedures to ensure that the results are reproducible.

[0024] Example 1: Screening and identification of the AcLTH70 gene in pineapple (based on genome-wide association analysis (GWAS)) 1. Determination of Pineapple Germplasm Resources and Leaf Thorn Phenotype The test materials consisted of 187 core pineapple germplasm resources, all of which were grown in our team's pineapple germplasm resource nursery. The leaf spine phenotypes (number of spines, distribution location, density, spine morphology, etc.) varied among different germplasm resources.

[0025] Six representative pineapple varieties—Bali, Ornamental Red Skin, Golden Diamond, Standing Leaf, Hand-Torn, and Sweet Honey—were selected for observation and measurement of leaf spine phenotypic characteristics. Robust suckers of all six varieties, 30-35cm in height, were uniformly planted using a randomized block design with three biological replicates (10 plants per replicate). After the plants reached 12 months of age (the leaf trait stabilization period), leaf spine characteristics were graded and recorded according to the "Pineapple Germplasm Resource Description Specification," including the number of spines, presence / absence (spine present / no spines), spine distribution (upper / middle / lower leaves), and spine morphology (hooked / needle-like). The "leaf spine index" (number of spines per unit length of leaf) was used as phenotypic data for GWAS analysis.

[0026] Figure 1 shows a comparison of the leaf spine phenotypes of six different pineapple germplasm resources. From left to right, they are the Bali, Ornamental Red Skin, Golden Diamond, Vertical Leaf, Hand-Torn, and Sweet Honey varieties. (A) is a magnified detail of the leaf spines, and (B), (C), and (D) show the presence and distribution of leaf spines on the upper, middle, and lower parts of the leaves, respectively. Figure 1 It can be seen that the leaves of the "Liye" variety in the third column are entire and spineless, while the other five varieties, namely Bali, Ornamental Red Skin, Golden Diamond, Hand-Torn, and Sweet Honey, have spines distributed in different parts of their leaves, providing a phenotypic basis for the correlation analysis of leaf spine traits.

[0027] 2. Genomic DNA extraction and whole-genome resequencing Young leaves of various plant types (3 plants of each plant type were sampled together) were collected, and genomic DNA was extracted using the CTAB method: 0.5g of leaves were ground into powder using liquid nitrogen, and 600μL of 2×CTAB extraction buffer (containing 2% CTAB, 1.4M NaCl, 100mM Tris-HCl pH8.0, 20mM EDTA, and 0.1% β-mercaptoethanol) was added. The mixture was incubated at 65℃ for 1h. An equal volume of chloroform / isoamyl alcohol (24:1) was added, and the mixture was gently inverted for 10min and centrifuged at 12000rpm for 10min. The supernatant was collected and 0.8 volumes of isopropanol were added, and the mixture was precipitated at -20℃ for 30min. The precipitate was washed twice with 75% ethanol, dissolved in TE buffer (containing RNase A), and the purity was determined by NanoDrop (A260 / A280 = 1.8-2.0). The integrity was verified by agarose gel electrophoresis.

[0028] Qualified DNA samples underwent whole-genome resequencing, with the pineapple T2T genome serving as the reference genome.

[0029] 3. SNP typing and GWAS analysis Low-quality reads from the raw sequencing data were filtered using Trimmomatic, aligned to the reference genome using BWA-MEM, and SNPs were detected using GATK. The selection criteria were MAF ≥ 0.05, deletion rate ≤ 0.1%, and Hardy-Weinberg equilibrium P-value ≥ 1e-6 to obtain high-quality SNPs. Leaf spine trait association analysis was performed using TASSEL 5.0 software with a mixed linear model (MLM), incorporating population structure (Q matrix) and phylogenetic relationships (K matrix) to control for false positives.

[0030] The results are as follows Figure 2 As shown, Figure 2 The figure shows the GWAS analysis results based on the pineapple leaf spine trait; the horizontal axis represents pineapple chromosomes (labeled as Chr01-Chr25), and the vertical axis represents the -log10 (P) value of the association signal (significant association threshold is 6). A significant association peak (-log10(P)≥6) was observed in the 359,708-429,207 bp region of chromosome Chr07, containing 19 candidate genes, including: lcfv2_07556 (AcLTH56), lcfv2_07557 (AcLTH57), lcfv2_07558 (AcLTH58), lcfv2_07559 (AcLTH59), lcfv2_07560 (AcLTH60), lcfv2_07561 (AcLTH61), lcfv2_07562 (AcLTH62), lcfv2_07563 (AcLTH63), lcfv2_07564 (AcLTH64), lcfv2_07565 (AcLTH65), and lcfv2_07566 (AcLTH66). lcfv2_07567(AcLTH67), lcfv2_07568 (AcLTH68), lcfv2_07569 (AcLTH69), lcfv2_07570(AcLTH70), lcfv2_07571 (AcLTH71), lcfv2_07572 (AcLTH72), lcfv2_07573(AcLTH73), lcfv2_07574(AcLTH74).

[0031] Further analysis of gene expression within the interval was conducted using transcriptome data from different pineapple tissues, such as... Figure 3 As shown, after filtering out genes with low expression in different tissues (TPM less than 10), 13 genes remained. Figure 3The horizontal axis represents different tissue types of pineapple (sepia, petals, pistil, ovule, pollen, fruit, and leaves), and the vertical axis represents the names of other genes after filtering out low-expression genes; the color gradient of the heatmap represents the relative expression level of genes, with orange indicating high expression and green indicating low expression. Figure 3 The results showed that the AcLTH70 gene was predominantly expressed in leaf tissues, out of three candidate genes (AcLTH57, AcLTH62, and AcLTH70). Further verification of the function of these three candidate genes using an Agrobacterium-mediated stable transformation system for pineapple embryogenic callus revealed that AcLTH57 and AcLTH62 did not yield transgenic positive seedlings. AcLTH10 overexpression-positive pineapple seedlings obtained from the thornless cultivar *AcLTH70* exhibited a leaf spine phenotype. Therefore, based on comprehensive analysis, AcLTH70 was identified as a key candidate gene for regulating pineapple leaf spines.

[0032] The coding region nucleotide sequence (SEQ ID NO.1) of the AcLTH70 gene is as follows: ATGGCGTCGTCGAGTGGTCTGAGGAGCTGCTCCGCCGTCGCGTTCCCGAGACTTCTCTCGTCGTCCAAATCACAATTTGCGGCCTCGCTCGCCATCCCGAACACTTCCTCCGGCCGCGTCTCCATGTCCGCCGAGTGGATGCCCGGCCAGCCCCGCCCTCCCTACCTCGACGGCTCAGCCCCCGGTGATTTTGGGTTCGATCCACTGCGCCTCGGAGAGGTGCCCGAGAACCTGGAGAGGTACAAGGAATCTGAGCTCATCCACTGCAGATGGGCGATGCTCGCTGTGCCAGGGGTCTTGGTGCCGGAGGCGCTGGGGCTCGGGAACTGGGTGAAGGCGCAAGAGTGGGCGGCGATCCCCGGCGGCCAGGCGACCTACTTGGGCAACCCGGTACCATGGGGTACCCTTCCGACCATCCTTATCATCGAGTTCTTCGCCATCGCCTTCGTCGAGCATCAGCGCAGCATGGAGAAGGATCCGGAGAAGAAGAAGTACCCCGGCGGCGCCTTCGACCCCTTGGGATTCTCCAAGGACCCTGAGAAGTTCAAGGAGTACAAGGTCAAGGAAATCAAGAACGGTCGGCTAGCGATCTTGGCCTTCGTGGGTTTCTGTGTGCAGCAGTCGGCGTACCCGGGGACGGGGCCGTTGGAGAACTTGGCGGCCCACCTCGCCGACCCATGGCACAGGAACATCGGCGACGTCATCATCCCCAGATCCATTGTGCCATGA The protein sequence encoded by the AcLTH70 gene (SEQ ID NO.2) is as follows: MASSSGLRSCSAVAFPRLLSSSKSQFAASLAIPNTSSGRVSMSAEWMPGQPRPPYLDGSAPGDFGFDPLRLGEVPENLERYKESELIHCRWAMLAVPGVLVPEALGLGNWVKAQEWAAIP GGQATYLGNPVPWGTLPTILIIEFFAIAFVEHQRSMEKDPEKKKYPGGAFDPLGFSKDPEKFKEYKVKEIKNGRLAILAFVGFCVQQSAYPGTGPLENLAAHLADPWHRNIGDVIIPRSIVP The upstream primer (SEQ ID NO.3) for AcLTH70 gene cloning is as follows: ATGGCGTCGTCGAGTGGT The downstream primers (SEQ ID NO.4) for AcLTH70 gene cloning are as follows: TGGCACAATGGATCTGGG 4. Cloning of the AcLTH70 gene sequence Young leaves (0.5g) of the thorny cultivar Bali were ground into powder using liquid nitrogen. Total RNA was extracted using the Magen HiPure PlantRNA Mini Kit: 750μL Buffer PRC1 was added, vortexed for 15s, and incubated at 55℃ for 3min; centrifuged at 14000g for 5min, and 700μL of the supernatant was filtered through a gDNA filter column and centrifuged at 13000g for 2min; an equal volume of Buffer PRC2 was added to the filtrate, mixed well, and loaded onto a HiPure RNA Mini Column, centrifuged at 12000g for 30s; eluted sequentially with 500μL Buffer RW1 and 500μL Buffer RW2, and RNA was collected with 50μL LEPC water and stored at -80℃.

[0033] cDNA was synthesized using the Thermo Scientific RevertAid RT reverse transcription kit: Genomic DNA removal: 1 μg RNA + 1 μL DNase I + 1 μL 10×Reaction Buffer + ddH2O to 10 μL, incubated at 37°C for 5 min, then at 65°C for 10 min to inactivate DNase I; Reverse transcription: 1 μL LOligo (dT) 18 primer + 4 μL 5×Reaction Buffer + 1 μL RiboLock RNase Inhibitor + 2 μL 10mM dNTP Mix + 1 μL L RevertAid RT, incubated at 42°C for 60 min, then at 70°C for 5 min to terminate the reaction, and stored at -20°C.

[0034] Using this cDNA as a template, PCR amplification was performed using specific primers (SEQ ID NO.3 and SEQ ID NO.4) in a 2×Hieff Canace® PCR Master Mix. The reaction system (50 μL) consisted of 25 μL of 2×Master Mix, 2 μL of cDNA, 2.5 μL each of 10 μM primers, and 18 μL of ddH2O. The program was as follows: 98℃ pre-denaturation for 3 min, 35 cycles (98℃ for 10 s, 60℃ for 20 s, and 72℃ for 30 s), and final extension at 72℃ for 5 min. The amplified product was recovered from the gel and sequenced, confirming that the sequence was consistent with SEQ ID NO.1, thus obtaining the AcLTH70 gene coding sequence.

[0035] Example 2: Construction of the recombinant vector pGWB506-AcLTH70 1. Vector double enzyme digestion Take the pGWB506 vector (preserved in the laboratory) and double digest it with FastDigest restriction endonucleases BamHI and EcoRI: The reaction system (50 μL) is as follows: 2.5 μg pGWB506 plasmid, 2 μL BamHI, 2 μL EcoRI, 5 μL 10×FastDigest Buffer, and ddH2O to make up to 50 μL. Incubate at 37℃ for 2 h. After electrophoresis, recover the linearized vector (about 10 kb).

[0036] 2. Homologous recombination linkage The AcLTH70 gene fragment was ligated into the linearized pGWB506 vector using the OK Clon DNA ligation kit: 5 μL of linearized vector, 1 μL of AcLTH70 fragment, 1 μL of OK Clon enzyme, and 1 μL of ddH2O. The mixture was incubated at 50 °C for 1 h to obtain the recombinant vector ligation product.

[0037] 3. Transformation and Identification The ligation product was transformed into E. coli DH5α competent cells, plated on LB solid medium containing 50 mg / L kanamycin, and incubated at 37°C for 12-16 h. Single colonies were picked and identified by colony PCR using primers AcLTH70-F (SEQ ID NO.3) and AcLTH70-R (SEQ ID NO.4). Positive clones were sequenced to confirm the correct construction of the recombinant vector, which was named pGWB506-AcLTH70.

[0038] Example 3: Construction of recombinant engineered bacteria GV3101-pGWB506-AcLTH70 1. Preparation of Agrobacterium competent cells Competent cells of Agrobacterium GV3101 were prepared using the freeze-thaw method: GV3101 strain was streaked on LB solid medium containing 50 mg / L rifampicin and cultured at 28°C for 2-3 days; single colonies were picked and inoculated into 5 mL LB liquid medium (containing rifampicin) and cultured at 28°C and 200 rpm for 16 h; 50 mL LB medium was then transferred at a 1:100 ratio and cultured until OD600 = 0.4-0.6; the culture was then incubated on ice for 30 min, centrifuged at 4°C and 4000 g for 10 min, and the supernatant was discarded; the culture was resuspended in 30 mL of pre-chilled Inoue buffer (10.88 g MnCl2·4H2O, 2.20 g CaCl2·2H2O, 18.65 g KCl, 20 mL 0.5 M PIPES pH 6.7, adjusted to 1 L), centrifuged, and then resuspended in 4 mL of Inoue buffer containing 10% glycerol. 50 μL / Dispense into tubes, flash-freeze in liquid nitrogen, and store at -80°C.

[0039] 2. Transformation of Agrobacterium with recombinant vectors Take 1 μg of pGWB506-AcLTH70 recombinant plasmid, add 50 μL of GV3101 competent cells, incubate on ice for 20 min; freeze in liquid nitrogen for 5 min, incubate in water at 37℃ for 5 min; add 1 mL of LB liquid medium, and incubate at 28℃ and 150 rpm for 3 h.

[0040] 3. Screening and Identification Centrifuge at 6000 rpm for 1 min, discard 900 μL of supernatant, resuspend and spread on LB solid medium containing 50 mg / L kanamycin and 50 mg / L rifampin, and incubate at 28 °C for 48 h; pick single colonies and identify them by bacterial PCR using AcLTH70 specific primers (SEQ ID NO. 3, SEQ ID NO. 4); positive colonies are the recombinant engineered bacteria, named GV3101-pGWB506-AcLTH70, and stored at -80 °C with 50% glycerol (final concentration 20%).

[0041] Example 4: Verification of AcLTH70 gene transformation in pineapple and leaf spine phenotype 1. Pineapple callus induction and transformation Using crown buds of thornless cultivars with upright leaves as explants, the plants were rinsed with running water for 15 min; then sterilized in a clean bench with 75% ethanol for 40 s, 0.1% mercuric chloride (containing 0.1% Tween-20) for 10 min, 2% sodium hypochlorite for 10 min, and rinsed 5 times with sterile water; cut into 0.5-1 cm² pieces and inoculated into MS induction medium (4 mg / L BAP + 0.2 mg / L NAA + 30 g / L sucrose + 7 g / L plant gel, pH 5.8) and cultured in the dark at 26℃ for 10 days to induce callus.

[0042] Recombinant engineered bacteria GV3101-pGWB506-AcLTH70 were streaked onto LB solid medium containing 50 mg / L kanamycin and 50 mg / L rifampin and cultured at 28°C for 48 h. A single colony was inoculated into 5 mL of LB liquid medium (containing the same antibiotics) and cultured at 28°C and 200 rpm for 16 h. The culture was then transferred 1:100 to 50 mL of LB medium and cultured until OD600 = 1.5-1.8. The culture was centrifuged at 4000 rpm for 15 min, resuspended in 25 mL of AAM-AS infection solution (AAM medium + 200 μM acetylsalicylic acid), and the OD600 was adjusted to 1.0. Callus tissue sections were immersed in the infection solution and incubated at 28°C and 150 rpm for 30 min. After aspirating the bacterial culture, the culture was inoculated into MS co-culture medium (4 mg / L BAP + 0.2 mg / L NAA + 200 μM MAS + ... Incubate in 30 g / L sucrose + 7 g / L plant gel (pH 5.8) at 26°C in the dark for 3 days.

[0043] After co-culture, the callus was rinsed 3-5 times with sterile water containing 500 mg / L termethin + 500 mg / L carbenicillin, dried, and then inoculated onto MS antibacterial selection medium (4 mg / LBAP + 0.2 mg / L NAA + 500 mg / L termethin + 50 mg / L hygromycin, pH 5.8); cultured at 26℃ under low light, subcultured every 3-4 weeks, and screened 3 times consecutively; resistant callus was transferred to MS differentiation medium (2 mg / LBAP + 0.2 mg / L NAA + 300 mg / L termethin + 50 mg / L hygromycin, pH 5.8) to induce adventitious shoots; when the shoots were 2-3 cm long, they were inoculated onto MS rooting medium (0.5 mg / L NAA + 300 mg / L termethin, pH 5.8) and cultured for 2-3 weeks.

[0044] 2. Positive Identification and Phenotypic Observation DNA was extracted from the leaves of transgenic seedlings using the CTAB method, and positive lines were identified by PCR using AcLTH70 primers (SEQ ID NO.3, SEQ ID NO.4). The positive lines and wild-type upright-leaved pineapple were transplanted simultaneously into a peat moss:perlite 1:1 substrate and cultured in a 30℃ greenhouse for 3 months. The leaf spine phenotype was observed, and the results are as follows: Figure 4 As shown.

[0045] Figure 4 Images (A), (B), and (C) show the plant and leaf phenotype of a wild-type upright-leaved pineapple (thornless variety). The leaves are entire and thornless, unlike... Figure 1 The results are consistent with those shown in the figures; (D), (E), and (F) show the phenotypes of the upright-leaved pineapple plants (transgenic plants) overexpressing AcLTH70, and the leaves of the overexpressing plants show obvious hook-shaped leaf spines at the leaf margins; (G) shows the upright-leaved pineapple plants (transgenic plants) overexpressing AcLTH70 under soil culture conditions, and the leaf spine phenotypes at the leaf margins of the overexpressing plants are obvious, confirming that the AcLTH70 gene can regulate the formation of pineapple leaf spines.

[0046] The AcLTH70 gene of this invention plays a role in regulating the formation of pineapple leaf spines and can be applied to the breeding of new pineapple varieties. Through genetic engineering technology, pineapple varieties that meet industry needs can be bred in a targeted manner. On the one hand, for scenarios requiring spineless traits (such as mechanized harvesting of fresh pineapples and ornamental pineapples), AcLTH70 gene silencing vectors (such as pTCK303-AcLTH70-RNAi) or gene editing vectors (such as pCAMBIA1300-Cas9-AcLTH70) can be transformed into pineapple explant cells (such as callus induced from crown buds and shoot tips). After antibacterial screening, resistance screening, and plant regeneration, spineless pineapple plants with suppressed AcLTH70 gene expression can be obtained. On the other hand, for pineapple varieties that require retaining spines to enhance disease and pest resistance (such as some processing pineapples), AcLTH70 overexpression vectors (pGWB506-AcLTH70) can be transformed into spineless or low-spine pineapple varieties to induce the formation of appropriate leaf spines and improve field resistance.

[0047] This invention identifies the sequence information, recombinant vector, and genetic transformation method of the AcLTH70 gene, providing new gene resources and technical pathways for the targeted improvement of pineapple leaf spine traits. It also lays the foundation for elucidating the molecular mechanism of pineapple leaf spine development. Further research can combine transcriptomics, metabolomics, and other multi-omics technologies to explore downstream target genes regulated by AcLTH70, improve the molecular regulatory network of leaf spine formation, and provide more precise theoretical support for pineapple breeding.

[0048] The terms “comprising,” “including,” or any other variation thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only the elements expressly listed but also other elements not expressly listed or inherent to the process, method, article, or apparatus.

[0049] Although preferred embodiments of the present invention have been described, those skilled in the art, upon learning the basic inventive concept, may make changes and modifications to the present invention without departing from its spirit and claims. All such changes and modifications fall within the protection scope of the present invention. The protection scope of the present invention is defined by the appended claims, and any equivalent substitutions or improvements that do not depart from the essence of the technical solution of the present invention should be included within the protection scope of the present invention.

Claims

1. An AcLTH70 gene that regulates the formation of pineapple leaf spines, characterized in that, The base sequence of this gene is shown in SEQ ID No.

1.

2. A protein encoded by the AcLTH70 gene, which regulates pineapple leaf spine formation as described in claim 1, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. A recombinant expression vector, characterized in that, It contains the AcLTH70 gene as described in claim 1.

4. The recombinant expression vector according to claim 3, characterized in that, The recombinant expression vector was obtained by inserting the AcLTH70 gene into the multiple cloning site of the plant expression vector pGWB506.

5. A recombinant engineered bacterium, characterized in that, The recombinant engineered bacteria is obtained by transforming the recombinant expression vector described in claim 3 into Agrobacterium competent cells GV3101.

6. The application of the AcLTH70 gene for regulating pineapple leaf spine formation as described in claim 1.

7. The application of the AcLTH70 gene regulating pineapple leaf spine formation according to claim 6, characterized in that, The application involves overexpressing the AcLTH70 gene to promote pineapple leaf thorn formation, or inhibiting AcLTH70 gene expression to reduce pineapple leaf thorns or make pineapple leaves thornless.

8. A method for cultivating pineapple plants with improved leaf spine traits, characterized in that, The expression level of the AcLTH70 gene, which regulates the formation of pineapple leaf spines as described in claim 1, can be adjusted through genetic engineering, including overexpressing the gene to obtain thorny pineapple plants or silencing the gene to obtain spineless pineapple plants.

9. The method for cultivating pineapple plants with improved leaf spine traits as described in claim 8, characterized in that, The primers used for cloning the AcLTH70 gene using genetic engineering techniques were AcLTH70-F and AcLTH70-R; the primer for AcLTH70-F was ATGGCGTCGTCGAGTGGT; and the primer for AcLTH70-R was TGGCA. CAATGGATCTGGG.

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