PsBTB gene for regulating low-temperature tolerance of poplar and application of PsBTB gene

Abstract

The invention discloses a PsBTB gene for regulating and controlling low-temperature tolerance of poplar and application of the PsBTB gene, and relates to the technical field of plant genetic engineering. The nucleotide sequence of the coding region of the PsBTB gene is as shown in SEQ ID No.1, or is a nucleotide sequence which is obtained by deleting or adding one or more nucleotides to the nucleotide sequence as shown in SEQ ID No.1 and expresses proteins with the same function. The invention also provides a PsBTB protein obtained by encoding the PsBTB gene, and an amplification primer of the PsBTB protein. According to the invention, the effect and value of the PsBTB gene in cold resistance of the plant are defined, an important basis is provided for improving the cold resistance of the plant by utilizing a molecular biology means in the later period, and theoretical guidance is also provided for subsequent forest molecular design and breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and in particular to a method for regulating the low temperature tolerance of poplar. PsBTB Genes and their applications. Background Art

[0002] Plant growth and development are often affected by stresses such as temperature and salinity. Low temperatures, a particularly damaging abiotic stress, affect plant growth, development, regional distribution, yield, and quality. When exposed to low temperatures, plants trigger a series of physiological responses, activating molecular mechanisms related to cold resistance to improve their tolerance to low temperatures. Improving plant resistance to low temperatures and reducing the damage caused by low temperatures are pressing challenges.

[0003] The BTB (broad-complex, tramtrack, and bric-abrac) domain is a conserved protein-protein interaction motif first discovered in Drosophila. BTB proteins contain one to three BTB domains, as well as other functional domain types, including BTB-Only, BTB-zinc finger, BTB-Kelch, BTB-BACK, BTB-Back-Kelch, Math-BTB, BTB-ANK, BTB-Back, BTB-PHR, and Rho-BTB. BTB proteins have been identified in many plant species, including Arabidopsis, rice, and tomato, and play important roles in plant growth and development, as well as in biotic and abiotic stresses. In Arabidopsis, light, IAA, ABA, and low temperature repress the expression of the BTB-TAZ protein BT2, while cytokinin (CK), methyl jasmonate (Me-JA), and hydrogen peroxide (H2O2) stimulate BT2 gene expression. In tomato, the SlBTB19-SlWRKY2 module negatively regulates CBF-dependent cold tolerance. These studies demonstrate the important role of BTB proteins in regulating plant stress responses.

[0004] Poplars are the main afforestation species in the world, especially in northern China. Populus simonii Carrière) is a major native tree species in my country and is widely distributed in North China. It is drought-resistant, cold-resistant and has strong adaptability. Summary of the Invention

[0005] In order to solve the above technical problems, the purpose of the present invention is to provide a method for regulating low temperature tolerance of poplar. PsBTB Genes and their applications have clarified PsBTBThe role and value of genes in plant cold resistance provide an important basis for the later use of molecular biological methods to improve plant cold resistance, and also provide theoretical guidance for subsequent molecular design breeding of forest trees.

[0006] The technical solution of the present invention to solve the above technical problems is as follows: providing a method for regulating the low temperature tolerance of poplar PsBTB Gene, PsBTB The nucleotide sequence of the gene coding region is as shown in SEQ ID No. 1, or a nucleotide sequence of the nucleotide sequence shown in SEQ ID No. 1 with one or more nucleotides deleted or added and expressing a protein with the same function.

[0007] further, PsBTB The nucleotide sequence of the coding region of the gene is 1083 bp in length, encoding 360 amino acid residues.

[0008] Will PsBTB The gene coding region sequence was fused with the strong promoter CaMV 35S (Cauliflower mosaic virus 35S), cloned into a plant overexpression vector, and overexpression transgenic plants were created through stable genetic transformation.

[0009] The above-mentioned overexpression vector was used to transform Populus argentatus ( Populus alba × P. glandulosa , '84K'), which was overexpressed PsBTB Transgenic plants ( PsBTB -OE-1 and PsBTB -OE-2).

[0010] The present invention also provides a PsBTB protein for regulating low temperature tolerance of poplar. The PsBTB protein is composed of the above PsBTB The gene encoding is obtained, and its amino acid sequence is shown in SEQ ID NO.2.

[0011] The present invention also provides a method for amplifying the above PsBTB The primers for the gene are characterized in that the upstream primer and the downstream primer are shown as SEQ ID NO.3 and SEQ ID NO.4 respectively.

[0012] The present invention also provides the above-mentioned PsBTB Application of genes, PsBTB proteins or primers in positively regulating plant resistance to low temperature stress.

[0013] Furthermore, the plant is poplar.

[0014] Furthermore, the plant is Populus simonii.

[0015] The present invention also provides the above-mentioned PsBTBApplication of genes, PsBTB proteins or primers in preparing reagents for improving plant resistance to low temperature stress.

[0016] The present invention has the following beneficial effects: 1. The present invention utilizes its plant overexpression vector to transform poplar, in order to promote the development of forest molecular breeding technology, provide technical means for cultivating excellent tree species, and lay the foundation for exploring the molecular mechanism of improving plant low temperature tolerance.

[0017] 2. Compared with non-transgenic plants, the overexpressing transgenic plants had higher resistance enzyme activity and significantly lower damage indicators than non-transgenic plants.

[0018] 3. PsBTB The gene plays a key role in improving the low temperature tolerance of poplar trees and has important application value in molecular design breeding of forest trees and the selection of superior varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 For poplar PsBTB Gene cloning electrophoresis diagram; Figure 2 For poplar PsBTB Gene expression levels in different tissues of poplar; Figure 3 for PsBTB Detection diagram of RNA levels in genetically modified poplars; Figure 4 for PsBTB Evaluation results of cold tolerance of transgenic and non-transgenic poplars; A is the Evans blue staining result, B is the conductivity test result, C is the DAB staining result, D is the H2O2 content, E is the NBT staining result, and F is the MDA content; Figure 5 for PsBTB Physiological index detection of transgenic poplar; A is superoxide dismutase activity; B is superoxide dismutase activity; C is proline content. DETAILED DESCRIPTION

[0020] The principles and features of the present invention are described below. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. In the examples, where specific conditions are not specified, conventional conditions or manufacturer-recommended conditions were used. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially.

[0021] Example 1 Poplar PsBTB Gene cloning (1) Using Populus simonii as the material, total RNA was extracted from the leaves of Populus simonii using the Plant Total RNA Extraction Kit (Tiangen). After removing genomic DNA contamination, the total RNA was extracted using PrimeScriptTM The first-strand cDNA was synthesized using the RT reagent Kit (TaKaRa, RR037A). For the specific reaction system, refer to the kit instructions.

[0022] (2) Reference Populus simonii genome data PsBTB Gene sequence, primer sequences were designed (amplified fragments included start and stop codons), wherein the forward primer PsBTB-F sequence is shown in SEQ ID NO.3, specifically: 5'-ATGTCGTCTTCTAGAAGTGG-3', and the reverse primer PsBTB-R sequence is shown in SEQ ID NO.4, specifically: 5'-TTATCGTGCAGTAACCCGAG-3'. Using cDNA as template, PrimeSTAR ® HS DNA Polymerase (TaKaRa) PsBTB The full-length sequence of the gene coding region was amplified.

[0023] (3) Purify the amplified target fragment using a PCR purification kit (OMEGA) and combine the purified product with the cloning vector pMD TM 19-T (TaKaRa). Take 5 μL of the ligation product and transfer it to E. coli DH5α competent cells (E. coli DH5α competent cells purchased from Shenzhen Kangti Life Science Technology Co., Ltd.) through heat shock transformation for blue-white screening. After selecting white clones for expansion culture, take 2 μL of bacterial liquid as a template for PCR detection. Take 5 μL of the amplified product for agarose gel electrophoresis detection. The results show that the single band detected is consistent with PsBTB Gene size is consistent ( Figure 1 M: DNA MarkerDL2000; CK: water as template; 1: PCR identification of positive clones), the positive transformants were sent to Sangon Biotech Co., Ltd. for Sanger sequencing, and finally obtained PsBTB The nucleotide sequence of the gene coding region is 1083 bp in length and can encode 360 ​​amino acid residues. Its nucleotide sequence and amino acid sequence are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0024] Example 2 Poplar PsBTB Tissue expression analysis of genes Plant materials were obtained from various tissues and organs of wild Populus simonii plants grown in a greenhouse for 2 months, including terminal buds, inflorescences, stem segments, unexpanded young leaves at the top of the plant, and mature leaves. Each of the above samples contained three biological replicates. All plant materials were quickly frozen with liquid nitrogen and stored in a -80°C freezer until use. Sample RNA was extracted and reverse transcribed to synthesize cDNA.PsACTIN As an internal reference gene, the primer sequences were 5'-TGTTGCCCTTGACTATGAGCAG-3' and 5'-ACGGAATCTCTCAGCTCCAATG-3', and fluorescence quantitative PCR analysis was performed. PsBTB To characterize the tissue expression characteristics of the gene, primers for amplification were PsBTB-qPCR-F (5'-GTCTAGTAATTTCAAGGCTG-3') and PsBTB-qPCR-R (5'-GGAGACCTTCACATATGGAG-3'). The reaction procedure was as follows: initial denaturation at 94°C for 5 min; 45 cycles of denaturation at 94°C for 20 sec, annealing at 58°C for 20 sec, and extension at 72°C for 20 sec; and a final extension at 72°C for 5 min. Melting curves were obtained using the following procedure: heating from 60°C to 95°C at a rate of 0.06°C / sec (5 acquisitions per°C) until the temperature reached the end of the heating cycle, completing the entire process and terminating the run. The reaction was performed using a 2-well plate. -ΔΔCt The fluorescence quantitative results were analyzed by calculation. Figure 2 shown.

[0025] Depend on Figure 2 It is known that poplar PsBTB There were significant differences in the expression levels of the gene in different tissues. It was almost not expressed in the terminal buds, inflorescences, and stem segments, but had relatively high expression abundance in young leaves and mature leaves.

[0026] Example 3 Poplar PsBTB Construction of gene plant overexpression vector After the clones identified correctly by sequencing were expanded and cultured, plasmid extraction kit (OMEGA) was used to extract the PsBTB pMD of genes TM 19-T vector plasmid; using GATEWAY technology, the forward primer sequence was designed to be 5'-GGGGACAACTTTGTACAAAAAAGTTGGAATGGAGGAATTTAACAACAC-3', and the reverse primer sequence was 5'-GGCGGCCGCACAACTTTGTACAAGAAAGTTGGGTATTATAGTCTGAAGTTCCTAATAC-3'. Using the plasmid as a template, the amplification PsBTB Full-length sequence of the gene coding region; After purification of the amplified target fragment, the purified product was ligated with the intermediate vector pDNOR207 via BP reaction and transformed into Escherichia coli DH5α competent cells via heat shock transformation for gentamicin resistance screening. After selecting resistant positive single clones for expansion culture, 2 μL of the bacterial solution was aspirated as a template for PCR detection. Positive transformants were sent to Sangon Biotech Co., Ltd. for Sanger sequencing verification. After the positive clones were expanded and cultured, the plasmids were extracted and then subjected to LR reaction with the plant expression vector pMDC32. PsBTB The full-length sequence of the gene coding region was introduced into the plant expression vector pMDC32 and fused with the strong promoter CaMV 35S (Cauliflower mosaic virus 35S). It was transformed into Escherichia coli DH5α competent cells by heat shock transformation for gentamicin resistance screening. After the positive single clone with resistance was selected for expansion culture, 2 μL of bacterial solution was taken as a template for PCR detection. The positive transformants were sent to Sangon Biotech Co., Ltd. for Sanger sequencing verification, and the transformed cells with PsBTB The plant overexpression vector pMDC32-PsBTB contains the full-length sequence of the gene coding region.

[0027] Example 4 Poplar PsBTB Genetic transformation and detection 1. Poplar PsBTB Genetic transformation Will obtain PsBTB The gene plant overexpression vector was transformed into Agrobacterium competent cells GV3101 (Agrobacterium GV3101 competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd.) by electroporation to obtain the recombinant bacteria GV3101-pMDC32-PsBTB.

[0028] The recombinant strain GV3101-pMDC32-PsBTB was transformed into poplar callus tissue by Agrobacterium infection (this method is described in the literature "Shuang-Shuang Wen, Xiao-Lan Ge, Rui Wang, et al. An efficient agrobacterium-mediated transformation method for hybrid poplar 84K ( Populus alba × P. glandulosa) using calli as explants. International Journal of Molecular Sciences, 2022, 23(4): 2216”, the recombinant Agrobacterium was propagated at 28 ℃, and the expanded bacterial solution was used to infect and transform Populus argentatus. After induction culture, the resistant adventitious buds were transferred to a rooting medium containing hygromycin and timentin until roots were induced and complete plants were formed.

[0029] 2. Poplar PsBTB Detection of genetically modified plants First, after antibiotic screening, a total of 20 resistant transgenic plants were obtained. Then, 7 overexpressing PsBTB Total RNA was extracted from leaf samples of transgenic lines and non-transgenic plants (WT, as a control) and reverse transcribed. PsBTB-qPCR-F and PsBTB-qPCR-R were used as quantitative primers to detect the RNA level. PsBTB The relative expression of genes in each transgenic line, PsBTB The expression levels of the gene in the overexpression transgenic lines OE1 and OE2 were 14.3 and 11.1 times higher than those in the non-transgenic plants, respectively ( Figure 3 ; WT: non-transgenic poplar 84K; OE1-OE7 are 7 overexpression PsBTB transgenic lines).

[0030] Example 5 PsBTB Phenotypic observation of transgenic poplar plants 20-day-olds that are in good growth condition and of uniform size PsBTB Tissue culture seedlings of overexpressing transgenic poplars (OE1 and OE2) and non-transgenic poplars (WT) were transplanted into plastic pots filled with artificial soil and cultured in a greenhouse for 2 months. They were then placed in a low temperature environment (4 ℃) for 24 hours, and the 7th to 9th mature leaves from the top were picked for physiological index measurement and biochemical staining analysis. At least 3 biological replicates were set up for each experiment, and each genotype plant contained at least 30 leaves. The results of Evans blue staining and conductivity measurement showed that compared with WT, under normal treatment, there was no significant difference in the staining intensity and conductivity of the leaves of overexpressing transgenic poplars and non-transgenic poplars. Under low temperature stress conditions, the staining of the leaves of overexpressing transgenic plants was lighter than that of non-transgenic poplars, and the conductivity was also significantly lower than that of non-transgenic poplars, indicating that the degree of cell membrane damage in the leaves of overexpressing transgenic plants was significantly lower than that of non-transgenic poplars, and the overexpression PsBTB Enhanced cold resistance of transgenic plants ( Figure 4 A, Figure 4B). Analysis of H2O2 and O2 in leaves using DAB (diaminobenzidine) and NBT (nitroblue tetrazolium) staining - • accumulation. Under normal growth conditions, there was no significant difference in the DAB and NBT staining intensity between the leaves of overexpressing transgenic poplars and non-transgenic poplars. PsBTB The staining of leaves of transgenic and non-transgenic poplars was deepened, but the overexpression PsBTB The H2O2 content of the leaves of transgenic poplars was lighter than that of non-transgenic poplars. Similarly, although the H2O2 content of the leaves of both increased, it was also significantly lower than that of non-transgenic poplars ( Figure 4 C- Figure 4 E). Malondialdehyde (MDA) is an effective indicator of cell membrane oxidative damage. Under normal growth conditions, the MDA content in leaves of overexpressing transgenic poplars and non-transgenic poplars is basically the same. However, under low temperature stress, the MDA level in leaves of overexpressing transgenic poplars is significantly lower than that of non-transgenic poplars ( Figure 4 F).

[0031] Example 6 PsBTB Determination of physiological indicators of transgenic poplar plants Antioxidant enzymes, such as peroxidase (POD) and superoxide dismutase (SOD), are major ROS scavengers and play a key role in ROS homeostasis. PsBTB The POD and SOD activity levels and proline content of transgenic poplars were similar to those of non-transgenic plants. PsBTB The POD and SOD activity levels of transgenic poplars were significantly higher than those of non-transgenic poplars. PsBTB The proline content in the leaves of transgenic poplars was significantly higher than that in non-transgenic plants ( Figure 5 The above results show that overexpression PsBTB Enhanced the cold resistance of transgenic plants.

[0032] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for regulating low temperature tolerance of poplar PsBTB A gene characterized by described PsBTB The nucleotide sequence of the gene coding region is as shown in SEQ ID No. 1, or a nucleotide sequence of the nucleotide sequence shown in SEQ ID No. 1 with one or more nucleotides deleted or added and expressing a protein with the same function.

2. A PsBTB protein for regulating low temperature tolerance of poplar, characterized in that: The PsBTB protein is the one described in claim 1 PsBTB The gene encoding is obtained, and its amino acid sequence is shown in SEQ ID NO.

2.

3. A method for amplifying the PsBTB A primer for a gene, characterized in that The upstream primer and downstream primer are shown as SEQ ID NO.3 and SEQ ID NO.4 respectively.

4. The method according to claim 1 PsBTB Use of the gene, the PsBTB protein described in claim 2 or the primer described in claim 3 in positively regulating plant resistance to low temperature stress.

5. The use according to claim 4, characterized in that The plant is poplar.

6. The method according to claim 1 PsBTB Use of the gene, the PsBTB protein described in claim 2 or the primer described in claim 3 in preparing an agent for improving plant resistance to low temperature stress.

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