A nucleotide sequence, an expression vector and application thereof

Abstract

The application relates to the technical field of genetic engineering, in particular to a nucleotide sequence for improving the differentiation efficiency of Picea abies callus, an expression vector and application thereof, which comprises a coding sequence of an AP2L2 gene, and the expression vector comprises double-strand RNA composed of the nucleotide sequence and its reverse complementary sequence. The application discloses the negative regulation of the AP2L2 gene in the differentiation of Picea abies callus for the first time, and effectively solves the problem of reduced differentiation efficiency by using specific gene silencing technology. The scheme is feasible and has remarkable effect, and is suitable for large-scale breeding production of forest tree varieties.

Description

Technical Field

[0001] This application relates to the field of genetic engineering technology, and in particular to a nucleotide sequence, an expression vector, and its application in improving the differentiation efficiency of plant callus tissue. Background Technology

[0002] As an excellent, fast-growing, and resilient timber species, as well as a suitable tree for ecological landscaping and ornamental purposes, European spruce exhibits significantly greater height growth at 9 years of age than 11-year-old Qinghai spruce (An Sanping, 2018). At 40 years of age, the average height and diameter at breast height (DBH) of European spruce in the Xiaolongshan forest area of ​​Gansu and Yichang, Hubei reach over 18m and 27cm respectively, exceeding the local main species, Qinghai spruce and Masson pine, by more than 20%. It was included in the 2024 List of Improved Forest Tree Varieties on December 30, 2024 (Ma Wenjun, 2025). However, European spruce seedlings grow slowly during the seedling stage, requiring 20-25 years from sowing to seed production and harvesting, and the seed maturity rate is very low, severely limiting its large-scale promotion as a superior variety.

[0003] Somatic cell embryogenesis is one of the important methods for large-scale propagation of superior European spruce varieties. However, in practical applications, embryogenic callus tissue gradually loses its differentiation capacity as the proliferation time increases, resulting in low seedling rates and becoming a major technical bottleneck restricting industrial application. Currently, the molecular mechanisms leading to this phenomenon are not yet clear, especially lacking in-depth research on key regulatory genes.

[0004] The AP2 / ERF family is one of the largest transcription factor families in plants. Its members all contain a conserved AP2 / ERF domain (approximately 60–70 amino acids) and can be divided into five subfamilies based on functional differences: AP2, ERF, DREB, RAV, and Soloists. Existing research indicates that these transcription factors play important regulatory roles in plant growth and development, leaf senescence, and hormone signal transduction. However, functional studies of AP2 / ERF transcription factors in somatic embryogenesis, especially in forest trees and coniferous species, remain relatively scarce, and their regulation of callus differentiation in Picea pulcherrima has not yet been reported. Summary of the Invention

[0005] The purpose of this application is to overcome the bottleneck of reduced callus differentiation rate in European spruce and provide an innovative solution for the breeding of superior tree varieties.

[0006] In a first aspect, this application provides a nucleotide sequence, characterized in that the nucleotide sequence is selected from the coding sequence of the AP2L2 gene, and the length of the nucleotide sequence is 200-500 bp.

[0007] Optionally, the AP2L2 gene is as shown in SEQ ID NO:1.

[0008] Optionally, the nucleotide sequence is selected from the following sequences: 1) The nucleotide sequence shown in SEQ ID NO:2; 2) Nucleotide sequences derived from the nucleotide sequence shown in SEQ ID NO:2 by substitution, deletion, insertion or addition of one or more nucleotides; 3) Contains a nucleotide sequence that has at least 80% homology with SEQ ID NO:2.

[0009] Secondly, this application provides a double-stranded RNA, which is composed of the nucleotide sequence described in the first aspect and its reverse complementary sequence.

[0010] Optionally, the double-stranded RNA can specifically silence the AP2L2 gene through an RNA interference mechanism.

[0011] Thirdly, this application provides a recombinant expression vector containing the nucleotide sequence described in the first aspect or the double-stranded RNA described in the second aspect.

[0012] Fourthly, this application provides a transgenic plant callus, which is obtained by introducing the recombinant expression vector described in the third aspect into the plant callus via Agrobacterium-mediated transformation; wherein the expression level of the AP2L2 gene in the callus is downregulated and its differentiation ability is significantly higher than that of non-transgenic callus.

[0013] Fifthly, this application provides the application of the nucleotide sequence described in the first aspect, the double-stranded RNA described in the second aspect, the recombinant expression vector described in the third aspect, or the transgenic plant callus described in the fourth aspect in improving the differentiation efficiency of plant callus.

[0014] Optionally, the plant is European spruce.

[0015] Sixthly, this application provides a method for improving the differentiation efficiency of plant callus tissue, wherein the transgenic plant callus tissue described in the fourth aspect is cultured on a differentiation medium.

[0016] In summary, this application includes at least one of the following beneficial technical effects: 1. The recombinant expression vector constructed using RNA interference technology can efficiently target the AP2L2 gene and achieve specific silencing. qRT-PCR results showed that the expression level of the AP2L2 gene in transgenic callus tissue was significantly downregulated, confirming the effectiveness of the RNAi vector containing the AP2L2 encoding gene. 2. A breakthrough improvement in the callus differentiation efficiency of European spruce has been achieved, with broad application potential, promoting the breeding of superior tree varieties. Attached Figure Description

[0017] Figure 1 This is the genetic map of the expression vector in this application; Figure 2 This is a diagram of callus differentiation in European spruce (control). Figure 3 This is a diagram of callus differentiation in European spruce callus with gene silencing. Figure 4 This is a PCR identification diagram of gene silencing in European spruce callus. Detailed Implementation

[0018] To provide a clearer understanding of the technical features, objectives, and beneficial effects of this application, the technical solution of the present invention will now be described in detail with reference to the following specific embodiments and accompanying drawings. However, this should not be construed as limiting the scope of implementation of the present invention.

[0019] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, all reagents used are commercially available or publicly available.

[0020] RNA interference (RNAi) refers to the efficient and specific blocking of the expression of specific target genes in vivo by small double-stranded RNAs, which promotes the degradation of mature mRNA and causes an organism to exhibit a phenotype of specific gene deletion. RNA interference is a highly specific gene silencing and repression at the mRNA level.

[0021] Example 1: Construction of the European Spruce RNAi-Ap2 vector

[0022] 1.1 Screening of exogenous genes

[0023] Based on transcriptome analysis, existing literature, and quantitative fluorescence verification, the target gene AP2L2 (MA_10434312g0010, SEQ ID NO:1) was screened out.

[0024] 1.2 Construction of expression vector

[0025] A 200-500 bp CDS fragment near the start codon of the target gene AP2L2 was selected (the specific sequence used in this example is shown in SEQ ID NO:2). This fragment was then generated in 20-base sequences and BLASTed in the genome to ensure it was a specific sequence (avoiding highly conserved regions). The first restriction enzyme digestion and ligation were performed: the original pPFGC5941-Kana plasmid was linearized using the restriction enzyme AscI (3510 bp), purified, and ligated to the 200-500 bp CDS fragment in the forward direction. After transformation into *E. coli* and confirmation of correct sequencing alignment, a second restriction enzyme digestion and ligation were performed. Finally, the second restriction enzyme digestion and ligation were performed: the plasmid constructed in the first step was linearized using the restriction enzyme BamHI (4891 bp), purified, and ligated to the 200-500 bp CDS fragment in the reverse direction. A schematic diagram of the RNAi vector (RNAi-A or RNAi-Ap2) containing the AP2L2 gene fragment (SEQ ID NO:2) after transformation into *E. coli* and confirmation of correct sequencing is shown below. Figure 1 As shown.

[0026] Example 2: Construction of transgenic callus

[0027] 2.1 Proliferation of embryogenic callus from European spruce

[0028] MLV medium was used as the basal medium for proliferation, supplemented with 9 μM / L 2,4-D and 4.4 μM / L 6-BA, 1 g / L acid-hydrolyzed casein, 0.5 g / L glutamine, 20 g / L sucrose, and 4 g / L gel per molecule. The pH of the medium was adjusted to 5.7 before autoclaving. Embryogenic callus induced from zygotes of the European Spruce 32 genotype was transferred to the proliferation medium. 5-7 embryogenic callus were subcultured per medium, every 14 days, in the dark at 24 ± 1 °C.

[0029] 2.2 Screening of resistant transgenic callus

[0030] The RNAi-A vector from Example 1 was transferred into European spruce callus via Agrobacterium GV3103. Observations were made daily until spruce embryogenic cells began to grow again (generally around 3 days). Then, filter paper was transferred to solid proliferation medium containing 300 mg / L cefotaxime and the vector for selecting resistance. The medium was changed every two weeks, and positive clones appeared in 4-5 weeks.

[0031] Example 3: Differentiation and culture of transgenic callus

[0032] The silencing effect of the target gene AP2L2 in the transgenic callus obtained in Example 2 was detected by qRT-PCR. Figure 4 As shown. From Figure 4It can be seen that the expression level of AP2L2 in the RNAi-A callus tissue of this gene is significantly downregulated.

[0033] The resistant transgenic callus obtained in Example 2 was cultured for differentiation for 12 weeks in the dark at a temperature maintained at 24±1℃. Differentiation culture was performed using MLV medium + 60 μM / L ABA + 1 g / L acid-hydrolyzed casein + 0.5 g / L glutamine + 60 g / L sucrose + 6 g / L gel permeate. The differentiation results are as follows: Figure 3 As shown. European spruce callus without RNAi-A was differentiated using the same method, and the differentiation results are shown below. Figure 2 As shown. Compared to Figure 2 (Comparison) Figure 3 The callus differentiation efficiency was significantly improved, indicating that callus with significantly downregulated AP2L2 expression can significantly improve the differentiation rate of European spruce callus.

[0034] Of course, the above description is only a specific embodiment of this application and is not intended to limit the scope of the invention. All equivalent changes or modifications made in accordance with the features and principles described in the claims of this invention should be included in the scope of the claims of this invention.

Claims

1. A transgenic European spruce callus, characterized in that, The recombinant expression vector was introduced into the callus of *Picea spruce* via Agrobacterium-mediated transformation. The AP2L2 gene expression level in the callus was downregulated, and its differentiation capacity was significantly higher than that of non-transgenic callus. The recombinant expression vector contained double-stranded RNA, which consisted of the nucleotide sequence shown in SEQ ID NO:2 and its reverse complementary sequence.

2. A method for improving the callus differentiation efficiency of European spruce, characterized in that, Transgenic European spruce callus as described in claim 1 was cultured using a differentiation medium.

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