A larch LaTCTP promoter and its application

Abstract

This invention provides a larch LaTCTP promoter and its applications, belonging to the field of promoter technology. The nucleotide sequence of the larch LaTCTP promoter provided by this invention is shown in SEQ ID NO. 1. The larch LaTCTP promoter of this invention is a constitutive promoter of larch, capable of driving the efficient expression of exogenous genes in plants, providing a theoretical basis for further development and utilization of the larch LaTCTP promoter, and laying the foundation for establishing an efficient expression system for exogenous genes in larch.

Description

Technical Field

[0001] This invention belongs to the field of promoter technology, and in particular relates to a larch LaTCTP promoter and its application. Background Technology

[0002] Larch (Larix spp.) is an important fast-growing timber species for afforestation in northern my country. Due to its complex genetic background and high genome heterozygosity, seed propagation inevitably leads to the segregation of desirable traits. Like all coniferous species, larch presents challenges in genetic manipulation, has a large genome, and is difficult to regenerate, making it unsuitable for direct application to angiosperm model systems such as crops in coniferous research. In model plants like Arabidopsis and tobacco, 35S promoters are typically used to drive target gene expression for gene function studies. However, previous studies have found that 35S is not applicable to larch embryonic cells, whether for stable transformation or transient expression. Therefore, it is necessary to identify strong promoters suitable for larch to achieve overexpression of target genes.

[0003] Translationally controlled tumor proteins (TCTPs) are a family of proteins widely found in eukaryotes and highly expressed in various tissues and organs. Studies have shown that plant TCTP promoters can also serve as constitutive promoters, driving the efficient expression of the exogenous gene GUS in both monocots and dicots. TCTPs are not only related to embryonic development in animals and plants but also closely associated with stress responses. However, there are no reports in this field regarding the endogenous TCTP promoter in larch and whether this promoter can achieve target gene overexpression. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a larch LaTCTP promoter, which, as a constitutive promoter of larch, can drive the efficient expression of exogenous genes in plants.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] The present invention provides a larch LaTCTP promoter, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] Preferably, the promoter is a larch composite promoter.

[0008] Preferably, the expression regulation of the promoter is related to hormone-induced and stress-response-related elements, wherein the hormones include auxin, gibberellin, and salicylic acid, and the stresses include low temperature and drought.

[0009] The present invention also provides an expression vector comprising the promoter described above.

[0010] The present invention also provides an application of the above-mentioned promoter or expression vector in the creation of transgenic plants.

[0011] Preferably, the plants include gymnosperms and angiosperms.

[0012] Preferably, the gymnosperm includes larch.

[0013] Preferably, the angiosperms include Arabidopsis thaliana and tobacco.

[0014] Preferably, the genes include the GUS gene and the GFP gene.

[0015] The beneficial effects of this invention are:

[0016] This invention provides a larch LaTCTP promoter for the first time. As a constitutive promoter for larch, it can drive the efficient expression of exogenous genes in plants, providing a theoretical basis for further development and utilization of larch LaTCTP promoters and laying the foundation for establishing an efficient expression system for exogenous genes in larch. Attached Figure Description

[0017] Figure 1 LaTCTP promoter clone, M is a 250bp DNA ladder, 1 is the first round PCR amplification product, 2 is the second round PCR amplification product, and 3 is the third round PCR amplification product.

[0018] Figure 2 LaTCTP promoter sequence alignment results with NCBIblast;

[0019] Figure 3 pLaTCTP(-2151bp)-GUS vector;

[0020] Figure 4 pLaTCTP(-2151bp)-GFP vector;

[0021] Figure 5 pLaTCTP(-2151bp)-LaSPL3-GFP vector;

[0022] Figure 6 Tissue localization of pLaTCTP in Arabidopsis thaliana;

[0023] Figure 7 Transient expression of GFP driven by pLaTCTP in tobacco;

[0024] Figure 8Staining characteristics of pLaTCTP(-2151bp)-GUS transgenic cell lines;

[0025] Figure 9 Tissue localization of pLaTCTP in somatic embryos at different stages;

[0026] Figure 10 Histological localization of somatic embryos in different groups at different stages. Detailed Implementation

[0027] The present invention provides a larch LaTCTP promoter, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0028] In this invention, the larch LaTCTP promoter is a constitutive promoter. The expression regulation of the larch LaTCTP promoter described in this invention is related to hormone-induced and stress-response-related elements. The hormones preferably include auxin, gibberellin, and salicylic acid, and the stresses preferably include low temperature and drought.

[0029] The present invention also provides an expression vector comprising the promoter described above. The present invention does not specifically limit the type of the expression vector.

[0030] The present invention also provides an application of the above-mentioned promoter or expression vector in the creation of transgenic plants.

[0031] In this invention, the plants preferably include gymnosperms and angiosperms, the gymnosperms preferably include larch, the angiosperms preferably include Arabidopsis thaliana and tobacco, and the genes preferably include the GUS gene and the GFP gene.

[0032] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0033] Unless otherwise specified, the following embodiments are all conventional methods.

[0034] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0035] Example 1

[0036] Cloning and Analysis of the Larch LaTCTP Promoter

[0037] A promoter sequence of approximately 1000 bp was obtained from the larch genome using chromosome walking technology. Based on this, a promoter sequence of approximately 2000 bp was amplified using modified SNM-PCR (patent number: ZL201310452575.2) with gene-specific primers (GSP1-1, GSP1-2, GSP1-3) listed in Table 1. Figure 1 (Left). Due to the short sequence, a second amplification was performed. The gene-specific primers used for the second amplification were GSP2-1, GSP2-2, and GSP2-3, yielding a promoter sequence of approximately 1500 bp. Figure 1 (Right), after splicing, a promoter sequence of about 3000bp was obtained.

[0038] Table 1. LaTCTP promoter cloning gene-specific primers

[0039]

[0040] Using NCBI BLAST to search and compare with the larch genome database, it was found that the sequence obtained in this invention consists of two sequences in the database. Figure 2 This indicates that the LaTCTP promoter obtained by this invention is longer than the sequences in the database and of higher quality than the data in the genome.

[0041] The promoter elements were predicted using PLANTCARE, and the results are shown in Table 2. These results indicate that the regulation of LaTCTP expression may be related to induction by hormones such as auxin (AuxRR-core / TGA-element), gibberellin (TATC-box), and salicylic acid (TCA-element), as well as low temperature response (LTR) and defense resistance (MBS / TC-rich repeats). These factors regulate the expression of downstream genes by acting on the corresponding cis-regulatory elements of the LaTCTP promoter.

[0042] Table 2. Promoter regulatory elements predicted by the LaTCTP5' flanking sequence of larch.

[0043]

[0044] Example 2

[0045] Construction of pLaTCTP(-2151bp)-GUS and pLaTCTP(-2151bp)-GFP vectors

[0046] (1) Primer design

[0047] Primers were designed based on the LaTCTP promoter sequence, as follows:

[0048] Primer 1: 5'-CCC AAGCTTCAATGGTGGAGAGGATTCG-3' (SEQ ID NO.8) (underlined part is the Hind III recognition sequence), primer 2: 5'-GG GGTACC CTTGATGAGCTTGAGATGGAAC-3'(SEQ ID NO.9) (The underlined part is the KpnI recognition sequence).

[0049] (2) PCR amplification

[0050] Genomic DNA was extracted from Japanese larch (Larix spp.) as a template, and PCR amplification was performed using the primer pairs described above. The correctness of the PCR product sequence was confirmed by sequencing.

[0051] (3) Carrier construction

[0052] The PCR product obtained in step 2 was digested with restriction endonucleases Hind III and Kpn I. After gel recovery, it was ligated to the backbone fragments of the RD29A-GFP and RD29A-GUS vectors, which had been digested with the same restriction enzymes, to obtain recombinant plasmids. The recombinant plasmids were identified by double digestion with Hind III and Kpn I. After confirmation, they were sent for sequencing. The recombinant plasmids obtained after sequencing confirmed the insertion of the LaTCTP promoter sequence between Hind III and Kpn I at the multiple cloning site of the backbone vector were named pLaTCTP(-2151bp)-GUS( Figure 3 ) and pLaTCTP(-2151bp)-GFP( Figure 4 ).

[0053] Example 3

[0054] Construction of pLaTCTP(-2151bp)-LaSPL3-GFP vector

[0055] (1) Primer design

[0056] Primers were designed based on the LaSPL3 promoter sequence, as follows:

[0057] Primer 1: 5'-CCC AAGCTT ATGGACGAAGTCGAAGTCAAG-3' (SEQ ID NO.10) (underlined part is the Hind III recognition sequence), primer 2: 5'-GG ACTAGT ATGATTCTTTATTTCCTTGC-3'(SEQ ID NO.11) (The underlined part is the recognition sequence of SpeI).

[0058] (2) PCR amplification

[0059] Total RNA was extracted from Japanese larch (Larix spp.) and reverse transcribed into cDNA. Using cDNA as a template, PCR amplification was performed using the primer pairs described above, and the correctness of the PCR product sequence was confirmed by sequencing.

[0060] (3) The PCR product obtained in step 2 was digested with restriction endonucleases Hind III and PstI. After gel recovery, it was ligated with the large backbone fragment of the pCAMBIA1300 vector that had been digested with the same restriction endonucleases to obtain the recombinant plasmid pCAMBIA1300-pLaTCTP (-2151bp).

[0061] The PCR product obtained in step 2 was digested with restriction endonucleases Hind III and SpeI, and after gel recovery, it was ligated with the large backbone fragment of the pSuper1300-GFP vector that had been digested with the same restriction enzymes to obtain the recombinant plasmid pSuper1300-LaSPL3-GFP.

[0062] The recombinant plasmid pCAMBIA1300-pLaTCTP(-2151bp) was double-digested with restriction endonucleases Xba I and EcoRI. The backbone vector was recovered by gel extraction and ligated with the pSuper1300-SPL3-GFP fragment, which had also been double-digested, to obtain the recombinant plasmid pLaTCTP(-2151bp)-LaSPL3-GFP( Figure 5 ).

[0063] Example 4

[0064] Stable transformation of pLaTCTP(-2151bp)-GUS in Arabidopsis thaliana and GUS staining analysis

[0065] The pLaTCTP(-2151bp)-GUS vector constructed in Example 2 was transformed into Arabidopsis thaliana, yielding T0, T1, and T2 generation seeds. The T2 seeds were seeded on MS plates (containing 25 μg / mL hyg), and seedlings with fully opened cotyledons and normally elongated hypocotyls were stained with GUS solution. Results showed that GUS staining was observed in the cotyledons, hypocotyls, roots, and other organs of the Arabidopsis seedlings. Figure 6 This indicates that LaTCTP is a constitutive expression promoter.

[0066] Example 5

[0067] pLaTCTP drives transient expression of GFP and SPL3-GFP fusion proteins in tobacco.

[0068] The pLaTCTP(-2151bp)-GFP constructed in Example 2 was transiently introduced into tobacco, and the results showed that cocoa-driven GFP expression ( Figure 7The pLaTCTP(-2151bp)-LaSPL3-GFP constructed in Example 3 was transiently used to infect tobacco cells. The results showed that green fluorescence was observed only in the cell nucleus. Figure 7 This indicates that the LaTCTP promoter can drive the expression of exogenous genes in tobacco.

[0069] Example 6

[0070] Stable transformation of pLaTCTP(-2151bp)-GUS in larch and GUS staining analysis

[0071] Through genetic transformation and multiple screenings, 50 C6 transgenic cell lines (8-1, 7-2, 7-3, 7-4, 7-5) were obtained. PCR identification confirmed that the target sequence had been integrated into the genome. Ten transgenic cell lines were randomly selected and stained with GUS staining solution. Different cell lines showed varying degrees of staining. Figure 8 ).

[0072] Transgenic cell lines were transferred to maturation culture medium to induce somatic embryogenesis, and their spatial distribution at different developmental stages was observed. The results showed that GUS-stained sites were distributed at all stages and locations of somatic embryogenesis. Figure 9 This study demonstrates that LaTCTP is a constitutive expression promoter. This invention is the first to achieve tissue localization of an endogenous promoter in larch, consistent with GUS staining results in larch.

[0073] Using the 35S-driven target gene GUS expression as the control group and the pLaTCTP-driven target gene GUS expression of this invention as the experimental group, transgenic cell lines were transferred to maturation culture medium to induce somatic embryogenesis. The spatial distribution of GUS expression at different developmental stages was observed. The results are as follows: Figure 10 As shown. In stable transformed positive larch cell lines, absolute quantitative analysis revealed that the 35S-driven GUS expression copy number was (0.038-2.89) × 10⁻⁶. 3 In approximately 60% of cases, GUS staining was not observable. However, when GUS expression was driven by pLaTCTP in stably transformed larch positive cell lines, absolute quantitative analysis showed that the pLaTCTP-driven GUS expression copy number was (0.138-143) × 10⁻⁶. 3 Only 10% of the samples did not show GUS staining, a difference of 1-2 orders of magnitude.

[0074] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A larch LaTCTP promoter, characterized in that, The nucleotide sequence of the larch LaTCTP promoter is shown in SEQ ID NO.

1.

2. The promoter according to claim 1, characterized in that, The promoter is a larch-based promoter.

3. The promoter according to claim 1, characterized in that, The expression regulation of the promoter is related to hormone-induced and stress-defense response-related elements, including auxin, gibberellin, and salicylic acid, and the stresses include low temperature and drought.

4. An expression carrier, characterized in that, The expression vector includes the promoter as described in claim 1 or 2.

5. The application of the promoter according to any one of claims 1-3 or the expression vector according to claim 4 in the creation of transgenic plants, characterized in that, The plant is larch, Arabidopsis thaliana, or tobacco, and the gene is the GUS gene or the GFP gene.

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