Nucleotide sequence, expression vector and application thereof

Abstract

The invention relates to the technical field of gene engineering, in particular to a nucleotide sequence for improving the differentiation efficiency of European spruce calluses, an expression vector and application of the nucleotide sequence, the nucleotide sequence is a coding sequence of an AP2L2 gene, and the expression vector comprises double-stranded RNA composed of the nucleotide sequence and a reverse complementary sequence of the nucleotide sequence. The invention discloses the negative regulation effect of the AP2L2 gene in European spruce callus differentiation for the first time, and the industrial problem that the differentiation efficiency is effectively promoted to be reduced through a specific gene silencing technology is solved. The scheme is feasible in operation, remarkable in effect and suitable for large-scale breeding and production of improved forest varieties.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, and in particular to a nucleotide sequence, an expression vector and application thereof in improving plant callus differentiation efficiency. BACKGROUND

[0002] As a good high-resistance fast-growing timber and ecological greening and ornamental tree species, the height growth of 9-year-old Picea abies is significantly higher than that of 11-year-old Picea wilsonii (An, 2018). At 40 years old, the average height and diameter at breast height of Picea abies in the forest area of Xiaolong Mountain in Gansu and Yichang in Hubei reach more than 18 m and 27 cm respectively, which is more than 20% higher than the local main tree species Picea crassiflora and Pinus massoniana, and is selected into the 2024 annual forest tree variety list on December 30, 2024 (Ma, 2025). However, the seedling growth of Picea abies is slow, and it takes 20-25 years from sowing to seed collection, and the seed maturation rate is very low, which seriously limits the large-scale popularization of its good variety.

[0003] Somatic embryogenesis technology is one of the important ways for large-scale breeding of Picea abies varieties, but in practical application, the differentiation ability of embryogenic callus gradually decreases with the prolongation of proliferation time, resulting in low seedling rate, which becomes the main technical bottleneck restricting industrial application. At present, the molecular mechanism leading to this phenomenon is not clear, especially the lack of in-depth research on key regulatory genes.

[0004] The AP2 / ERF family is one of the largest transcription factor families in plants, and its members all contain a conserved AP2 / ERF domain (about 60-70 amino acids). According to the functional differences, it can be divided into five subfamilies: AP2, ERF, DREB, RAV and Soloists. Existing studies have shown that this type of transcription factor plays an important regulatory role in plant growth and development, leaf senescence and hormone signal transduction. However, in forest trees, especially in coniferous species, the function of AP2 / ERF transcription factors in somatic embryogenesis has been relatively scarce, and whether they regulate callus differentiation in Picea abies has not been reported. SUMMARY

[0005] The purpose of the present application is to break through the bottleneck of reduced callus differentiation rate of Picea abies, and to provide an innovative solution for forest tree variety breeding.

[0006] In a first aspect, the present 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) a nucleotide sequence derived from the nucleotide sequence shown in SEQ ID NO: 2 by substitution, deletion, insertion or addition of one or several nucleotides; 3) a nucleotide sequence comprising at least 80% homology with the nucleotide sequence shown in SEQ ID NO: 2.

[0009] In a second aspect, the present application provides a double-stranded RNA consisting of the nucleotide sequence of the first aspect and its reverse complement.

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

[0011] In a third aspect, the present application provides a recombinant expression vector containing the nucleotide sequence of the first aspect or the double-stranded RNA of the second aspect.

[0012] In a fourth aspect, the present application provides a transgenic plant callus obtained by introducing the recombinant expression vector of the third aspect into a plant callus through Agrobacterium-mediated method; wherein the expression amount of the AP2L2 gene in the callus is down-regulated, and the differentiation ability is significantly higher than that of a non-transgenic callus.

[0013] In a fifth aspect, the present application provides the nucleotide sequence of the first aspect, the double-stranded RNA of the second aspect, the recombinant expression vector of the third aspect or the transgenic plant callus of the fourth aspect for use in improving the differentiation efficiency of plant callus.

[0014] Optionally, the plant is Picea abies.

[0015] In a sixth aspect, the present application provides a method for improving the differentiation efficiency of plant callus, which comprises culturing the transgenic plant callus of the fourth aspect in a differentiation medium.

[0016] In summary, the present application includes at least one of the following beneficial technical effects: 1. The recombinant expression vector constructed by the RNA interference technology can efficiently target the AP2L2 gene and achieve specific silencing. The qRT-PCR detection results show that the expression amount of the AP2L2 gene in the transgenic callus is significantly down-regulated, which confirms the effectiveness of the RNAi vector containing the AP2L2 coding gene; 2. The differentiation efficiency of Picea abies callus is significantly improved, which has wide application potential and promotes the breeding of forest tree varieties. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 is the genetic map of the expression vector of the present application; Figure 2 is the differentiation map of Picea abies callus (control); Figure 3 is the differentiation map of Picea abies callus with gene silencing; Figure 4 is the PCR identification map of Picea abies callus with gene silencing. DETAILED DESCRIPTION

[0018] In order to have a clearer understanding of the technical features, objectives and beneficial effects of the present application, the technical solutions of the present application will be described in detail below in conjunction with the following specific examples and the accompanying drawings of the specification, but it should not be understood as limiting the scope of the implementation of the present application.

[0019] The experimental methods in the following examples not specified in the specific conditions, generally in accordance with the conventional conditions, or in accordance with the manufacturer's recommended conditions. The reagents used, if not specified, are commercially available or publicly available reagents.

[0020] "RNA interference (RNAi)" refers to some small double-stranded RNA can be efficient, specific to block the expression of specific target genes in vivo, to facilitate the degradation of mature mRNA, so that the biological individual shows a specific gene deletion phenotype. RNA interference is a highly specific mRNA level of gene silencing inhibition.

[0021] Example 1: Construction of Picea abies RNAi-Ap2 vector

[0022] 1.1 Selection of foreign genes

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

[0024] 1.2 Construction of expression vector

[0025] The CDS fragment of 200-500 bp near the start codon of the target gene AP2L2 (the sequence used in this embodiment is shown as SEQ ID NO: 2) was selected, arranged every 20 bases, and subjected to genome BLAST to ensure that it was a specific sequence (avoiding highly conserved regions); then first cleavage and ligation: the original pPFGC5941-Kana plasmid was linearized using the restriction enzyme AscI (3510 bp), purified and recovered, and ligated with the 200-500 bp CDS forward target fragment. After transformation of E. coli and sequencing alignment, the results were correct, and the second cleavage and ligation were performed. Finally, the second cleavage and ligation: the first constructed plasmid was linearized using the restriction enzyme BamHI (4891 bp), purified and recovered, and ligated with the 200-500 bp CDS reverse target fragment. After transformation of E. coli and sequencing, the RNAi vector of the AP2L2 gene fragment (SEQ ID NO: 2) (referred to as RNAi-A or RNAi-Ap2) is shown in Figure 1 .

[0026] Example 2: Construction of transgenic callus

[0027] 2.1 Proliferation of embryogenic callus of Picea abies

[0028] The proliferation medium used MLV medium as the basic medium, supplemented with 9 μM / L 2,4-D and 4.4 μM / L 6-BA, 1 g / L acid hydrolysis casein, 0.5 g / L glutamine, 20 g / L sucrose, 4 g / L gelatin, and the pH of the medium was adjusted to 5.7 before autoclaving. The embryogenic callus induced from the zygotic embryos of Picea abies genotype No. 32 was transferred to the proliferation medium, 5-7 pieces of embryogenic callus were subcultured per medium, subcultured every 14 d, dark incubated, and the temperature was maintained at 24±1℃.

[0029] 2.2 Selection of resistant transgenic callus

[0030] The RNAi-A vector of Example 1 was introduced into the Picea abies callus by Agrobacterium GV3103 mediation, and observed every day until the Picea embryogenic cells started to grow again (usually about 3 days). Then the filter paper was transferred to the solid proliferation medium containing 300 mg / L cefotaxime and the vector screening resistance, and the plate was changed every two weeks, and positive clones appeared after 4-5 weeks.

[0031] Example 3: Differentiation 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 method, as shown in Figure 4 . From Figure 4It can be seen that the expression of AP2L2 in the RNAi-A gene callus is significantly down-regulated.

[0033] The resistant transgenic callus obtained in Example 2 was subjected to differentiation culture for 12 weeks in dark at a temperature of 24±1℃. The 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 gelatin, and the differentiation results are shown in Table 2. Figure 3 The Picea abies callus without RNAi-A was subjected to the same differentiation method, and the differentiation results are shown in Table 2. Figure 2 Compared with the callus of Picea abies (control), Figure 2 Figure 3 the differentiation efficiency of the callus was significantly improved, indicating that the callus with significantly down-regulated AP2L2 expression can significantly improve the differentiation rate of Picea abies callus.

[0034] Of course, the above only describes specific embodiments of the present application and is not intended to limit the scope of the present application. Any equivalent changes or modifications made in accordance with the features and principles described in the patent application of the present application shall be included in the patent application of the present application.​

Claims

1. 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.

2. The nucleotide sequence according to claim 1, wherein, The AP2L2 gene sequence is shown in SEQ ID NO:

1.

3. The nucleotide sequence of claim 1, wherein, The nucleotide sequence is selected from the following sequences: 1) the nucleotide sequence shown in SEQ ID NO: 2; 2) a nucleotide sequence derived from the nucleotide sequence shown in SEQ ID NO: 2 by substitution, deletion, insertion or addition of one or several nucleotides; 3) a nucleotide sequence comprising at least 80% homology with the nucleotide sequence shown in SEQ ID NO:

2.

4. A double-stranded RNA, characterized in that, The double-stranded RNA is composed of the nucleotide sequence as claimed in claim 1 and its reverse complement sequence.

5. The double stranded RNA of claim 4, wherein The double-stranded RNA can specifically silence the AP2L2 gene through the RNA interference mechanism.

6. A recombinant expression vector, characterized in that, The recombinant expression vector contains the nucleotide sequence as claimed in any one of claims 1-3 or the double-stranded RNA as claimed in any one of claims 4-5.

7. A transgenic plant callus, wherein, The recombinant expression vector as claimed in claim 6 is introduced into plant callus by Agrobacterium-mediated method; wherein the expression amount of the AP2L2 gene in the callus is down-regulated, and the differentiation ability is significantly higher than that of non-transgenic callus.

8. Use of the nucleotide sequence as claimed in any one of claims 1-3, the double-stranded RNA as claimed in any one of claims 4-5, the recombinant expression vector as claimed in claim 6 or the transgenic plant callus as claimed in claim 7 in improving the differentiation efficiency of plant callus.

9. Use according to claim 8, characterized in that, The plant is Picea abies.

10. A method for improving the efficiency of plant callus differentiation, characterized by, The transgenic plant callus as claimed in claim 7 is cultured in a differentiation medium. The plant is Picea abies. The transgenic plant callus as claimed in claim 7 is cultured in a differentiation medium.

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