A PsBTB gene that regulates the low-temperature tolerance of poplar and its application

Abstract

This invention discloses a method for regulating the low-temperature tolerance of poplar trees. PsBTB Genes and their applications, involving the field of plant genetic engineering technology; PsBTB The nucleotide sequence of the gene coding region is as shown in SEQ ID No. 1, or a nucleotide sequence as shown in SEQ ID No. 1 with one or more nucleotides deleted or added, expressing the same functional protein. This invention also provides nucleotide sequences derived from the above. PsBTB The gene encodes the PsBTB protein and amplification primers. This invention clarifies... PsBTB The role and value of genes in plant cold resistance provide an important basis for improving plant cold resistance using molecular biology methods in the later stage, and also provide theoretical guidance for subsequent molecular design breeding of forest trees.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to a method for regulating the low-temperature tolerance of poplar trees. PsBTB Genes and their applications. Background Technology

[0002] Plant growth and development are frequently affected by stresses such as temperature and salinity. Low temperature, as a significant abiotic stress, impacts plant growth, development, regional distribution, yield, and quality. When plants are subjected to low temperature stress, a series of physiological responses are induced, activating cold-resistance-related molecular mechanisms to enhance their low-temperature tolerance. Therefore, improving plant resistance to low temperatures and reducing the damage caused by low-temperature stress is an urgent problem to be solved.

[0003] The BTB (broad-complex, tramtrack, and bric-abrac) domain is the first conserved protein-protein interaction motif discovered in Drosophila. BTB proteins contain 1-3 BTB domains, as well as other types of functional domains, including BTB-Only proteins, BTB-zinc finger proteins, BTB-Kelch proteins, BTB-BACK proteins, BTB-Back-Kelch proteins, Math-BTB proteins, BTB-ANK proteins, BTB-Back, BTB-PHR proteins, and Rho-BTB proteins. Currently, BTB proteins have been identified in many plants, such as Arabidopsis, rice, and tomato, playing important roles in plant growth and development, and in responding to biotic and abiotic stresses. In Arabidopsis, light, IAA, ABA, and low temperature inhibit the expression of the BTB-TAZ protein BT2, while cytokinin (CK), methyl jasmonic acid (Me-JA), and hydrogen peroxide (H2O2) stimulate BT2 gene expression. In tomatoes, 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] Poplar is a major afforestation species worldwide, especially in northern China. Among them, the small-leaved poplar (Populus microphylla) Populus simonii Carrière is a major native tree species in my country, widely distributed in North China, and is drought-resistant, cold-resistant, and highly adaptable. Summary of the Invention

[0005] To address the aforementioned technical problems, the purpose of this invention is to provide a method for regulating the low-temperature tolerance of poplar trees. PsBTB Genes and their applications have been clarified PsBTBThe role and value of genes in plant cold resistance provide an important basis for improving plant cold resistance using molecular biology methods in the later stage, 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-mentioned technical problems is as follows: A method for regulating the low-temperature tolerance of poplar trees is provided. PsBTB Gene, PsBTB The nucleotide sequence of the gene coding region is as shown in SEQ ID No. 1, or the nucleotide sequence of SEQ ID No. 1 with one or more nucleotides deleted or added and expressing the same functional protein.

[0007] further, PsBTB The gene has a coding region with a nucleotide sequence length of 1083 bp, 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 overexpressing transgenic plants were created through stable genetic transformation.

[0009] The above overexpression vector was used to transform *Populus silveraefolius* (Silvera spp.) via Agrobacterium-mediated transformation. Populus alba × P. glandulosa (84K) was overexpressed. PsBTB Transgenic plants ( PsBTB -OE-1 and PsBTB -OE-2).

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

[0011] The present invention also provides a method for amplifying the above-mentioned PsBTB The primers for the gene are characterized in that their upstream primer and downstream primer are as shown in 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 the positive regulation of plant resistance to low temperature stress.

[0013] Furthermore, the plant is a 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 the preparation of reagents to enhance plant resistance to low temperature stress.

[0016] The present invention has the following beneficial effects:

[0017] 1. This invention utilizes its plant overexpression vector to transform poplar trees, aiming to promote the development of molecular breeding technology for forest trees, provide technical means for cultivating superior tree species, and lay the foundation for exploring the molecular mechanism of improving plant low-temperature tolerance.

[0018] 2. Compared with non-transgenic plants, overexpressing transgenic plants have higher resistance enzyme activity and significantly lower damage indicators than non-transgenic plants.

[0019] 3. PsBTB Genes play a key role in improving the low-temperature tolerance of poplar trees and have important application value in molecular design breeding of forest trees and the selection of superior varieties. Attached Figure Description

[0020] Figure 1 For poplar PsBTB Cloning electrophoresis diagram of a gene;

[0021] Figure 2 For poplar PsBTB Gene expression levels in different tissues of poplar trees;

[0022] Figure 3 for PsBTB RNA level detection graph in genetically modified poplar trees;

[0023] Figure 4 for PsBTB Evaluation results of cold resistance of transgenic and non-transgenic poplar trees; A is Evans blue staining result, B is electrical conductivity test result, C is DAB staining result, D is H2O2 content, E is NBT staining, and F is MDA content.

[0024] Figure 5 for PsBTB Physiological indicators of transgenic poplar trees were detected; A represents superoxide dismutase activity; B represents superoxide dismutase activity; and C represents proline content. Detailed Implementation

[0025] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0026] Example 1

[0027] poplar PsBTB Gene cloning

[0028] (1) Using Populus simonii as material, total RNA was extracted from Populus simonii leaves using a plant total RNA extraction kit (Tiangen). After removing genomic DNA contamination, it was then processed using PrimeScript. TM The RT reagent Kit (TaKaRa, RR037A) was used to synthesize the first strand of cDNA. Refer to the kit instructions for specific reaction systems.

[0029] (2) Referring to the genome data of Populus simonii PsBTB Gene sequence was used to design primer sequences (the amplified fragment contains a start codon and a stop codon). 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 a template, PrimeSTAR was used. ® HS DNA Polymerase (TaKaRa) PsBTB The full-length sequence of the gene coding region was amplified.

[0030] (3) The amplified target fragment was purified using a PCR purification kit (OMEGA), and the purified product was mixed with the cloning vector pMD. TM Ligation was performed using 19-T (TaKaRa). 5 μL of the ligation product was transformed into *E. coli* DH5α competent cells (purchased from Shenzhen Kangti Life Science Technology Co., Ltd.) via heat shock transformation. Blue-white screening was performed, and white clones were selected for amplification culture. 2 μL of the bacterial culture was used as a template for PCR detection. 5 μL of the amplification product was then analyzed by agarose gel electrophoresis. The results showed that the single band detected was consistent with... PsBTB Gene size is consistent ( Figure 1 M: DNA Marker DL2000; CK: Water-based template; 1: Positive clone PCR identification), positive transformants were sent to Sanger sequencing at Sangon Biotech Co., Ltd., and finally obtained PsBTB The nucleotide sequence of the gene coding region is 1083 bp in length and encodes 360 amino acid residues. Its nucleotide sequence and amino acid sequence are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0031] Example 2

[0032] poplar PsBTB Tissue expression analysis of genes

[0033] Plant materials were obtained from various tissues and organs of wild Populus simonii plants grown in a greenhouse for two months, including terminal buds, inflorescences, stem segments, unopened young leaves at the plant tip, and mature leaves. Each sample contained three biological replicates. All plant materials were flash-frozen in liquid nitrogen and stored at -80°C for later use. RNA was extracted from the samples, and cDNA was synthesized by reverse transcription. PsACTIN As an internal reference gene, its primer sequences are 5'-TGTTGCCCTTGACTATGAGCAG-3' and 5'-ACGGAATCTCTCAGCTCCAATG-3', and quantitative real-time PCR was used for analysis. PsBTB The tissue expression characteristics of the gene were determined using PsBTB-qPCR-F (5'-GTCTAGTAATTTCAAGGCTG-3') and PsBTB-qPCR-R (5'-GGAGACCTTCACATATGGAG-3'). The reaction program was as follows: 94 ℃ pre-denaturation for 5 min; 94 ℃ denaturation for 20 sec, 58 ℃ annealing for 20 sec and 72 ℃ extension for 20 sec, for 45 cycles; and a final extension at 72 ℃ for 5 min. Simultaneously, the following melting curve was obtained: During the temperature increase from 60 ℃ to 95 ℃, the temperature was increased at a rate of 0.06 ℃ / sec (5 acquisitions per ℃) until the temperature increase was completed, thus completing the entire program and ending the run. -ΔΔCt The quantitative fluorescence results were analyzed using computational methods. The results are as follows: Figure 2 As shown.

[0034] Depend on Figure 2 It can be known that poplar trees PsBTB The gene expression levels varied significantly in different tissues, with almost no expression in terminal buds, inflorescences, and stem segments, but relatively high expression levels in young and mature leaves.

[0035] Example 3

[0036] poplar PsBTB Construction of gene overexpression vectors in plants

[0037] After expanding the culture of clones identified by sequencing, plasmids containing [specific compounds] were extracted using a plasmid extraction kit (OMEGA). PsBTB pMD of genes TM19-T vector plasmid; using GATEWAY technology, the forward primer sequence was designed as 5'-GGGGACAACTTTGTACAAAAAAGTTGGAATGGAGGAATTTAACAACAC-3', and the reverse primer sequence was designed as 5'-GGCGGCCGCACAACTTTGTACAAGAAAGTTGGGTATTATAGTCTGAAGTTCCTAATAC-3'. Using the plasmid as a template, amplification was performed. PsBTB Full-length sequence of the gene coding region;

[0038] After purifying the amplified target fragment, the purified product was linked to the intermediate vector pDNOR207 via a BP reaction. The fragment was then transformed into E. coli DH5α competent cells by heat shock transformation for gentamicin resistance screening. Positive single clones with resistance were selected for amplification culture. 2 μL of bacterial culture was used as a template for PCR detection. The positive transformants were sent to Sangon Biotech Co., Ltd. for Sanger sequencing verification.

[0039] After expanding the positive clones through culture, plasmids were extracted and then subjected to an LR reaction with the plant expression vector pMDC32. Following the reaction, [the plasmids were then processed]. PsBTB The full-length coding sequence was introduced into the plant expression vector pMDC32 and fused with the strong promoter CaMV 35S (Cauliflower mosaic virus 35S). The mixture was then transformed into *E. coli* DH5α competent cells via heat shock for gentamicin resistance screening. Resistant positive clones were selected and cultured extensively. Two μL of the bacterial culture was used as a template for PCR detection. Positive transformants were sent to Sanger sequencing verification at Sangon Biotech Co., Ltd., yielding cells containing the gene. PsBTB The plant overexpression vector pMDC32-PsBTB contains the full-length sequence of the gene coding region.

[0040] Example 4

[0041] poplar PsBTB Genetic transformation and detection

[0042] 1. Poplar PsBTB Genetic transformation

[0043] Will get 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 recombinant bacteria GV3101-pMDC32-PsBTB.

[0044] The recombinant strain GV3101-pMDC32-PsBTB was genetically transformed using Agrobacterium-mediated transformation of poplar callus (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”, Recombinant Agrobacterium was propagated at 28 ℃, and the propagated bacterial solution was used to infect and transform Populus alopecuroides. After induction culture, the resistant adventitious shoots were transferred to a rooting medium containing hygromycin and termethin until roots were induced and complete plants were formed.

[0045] 2. Poplar PsBTB Detection of genetically modified plants

[0046] First, after antibiotic screening, 20 resistant transgenic plants were obtained. Then, 7 overexpressing plants were randomly selected. PsBTB Total RNA was extracted from leaf samples of transgenic lines and non-transgenic plants (WT, as a control) and reverse transcribed. Quantitative analysis was performed using PsBTB-qPCR-F and PsBTB-qPCR-R primers to detect RNA levels. PsBTB The relative expression of genes in various transgenic lines, among which... 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-GMO poplar 84K; OE1-OE7 are 7 overexpression genes. PsBTB (Transgenic strains).

[0047] Example 5

[0048] PsBTB Phenotypic observation of transgenic poplar plants

[0049] 20-day-old animals that are growing well and are of uniform size PsBTBTransgenic poplar seedlings (OE1 and OE2) and non-transgenic poplar (WT) were transplanted into plastic pots filled with artificial soil and cultured in a greenhouse for two months. Afterward, they were treated at low temperature (4 ℃) for 24 h. The 7th-9th mature leaves from the top were harvested for physiological index determination and biochemical staining analysis. Each experiment had at least three biological replicates, and each genotype plant contained at least 30 leaves. Evans blue staining and conductivity measurements showed no significant difference in staining intensity and conductivity between overexpressing and non-transgenic poplar leaves under normal treatment compared to WT. Under low-temperature stress, the leaves of overexpressing transgenic plants showed lighter staining and significantly lower conductivity than non-transgenic poplars, indicating that the degree of cell membrane damage in the leaves of overexpressing transgenic plants was significantly lower than that in non-transgenic poplars. PsBTB It enhanced the cold resistance of the transgenic plants. Figure 4 A, Figure 4 B). 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 DAB and NBT staining intensity between leaves of overexpressing transgenic poplar and non-transgenic poplar. Under low temperature stress, overexpression... PsBTB Both transgenic and non-transgenic poplar leaves showed increased staining, but overexpression... PsBTB Genetically modified poplars are shallower than non-genetically modified poplars. Similarly, although the H2O2 content in the leaves of both types of poplars is increased, it is still significantly lower than that of non-genetically modified poplars. Figure 4 C- Figure 4 E). Malondialdehyde (MDA) is an effective indicator of oxidative damage to cell membranes. Under normal growth conditions, the MDA content in the leaves of overexpressing transgenic poplar and non-transgenic poplar is essentially the same. However, under low-temperature stress, the MDA level in the leaves of overexpressing transgenic poplar is significantly lower than that in non-transgenic poplar. Figure 4 F).

[0050] Example 6

[0051] PsBTB Physiological indicators of transgenic poplar plants

[0052] Antioxidant enzymes, such as peroxidase (POD) and superoxide dismutase (SOD), are major ROS scavengers and play a crucial role in ROS homeostasis. Studies have found that under normal growth conditions, overexpression of... PsBTB The POD and SOD activity levels and proline content of transgenic poplar plants were similar to those of non-transgenic plants. However, under low-temperature stress, overexpression... PsBTB The POD and SOD activity levels of transgenic poplar were significantly higher than those of non-transgenic poplar. Meanwhile, overexpression... PsBTBThe proline content in the leaves of genetically modified poplar plants was significantly higher than that in non-genetically modified plants. Figure 5 The above results indicate that overexpression PsBTB It enhanced the cold resistance of the transgenic plants.

[0053] 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 within the protection scope of the present invention.

Claims

1. PsBTB Application of gene and PsBTB protein in positive regulation of poplar low-temperature stress resistance; PsBTB The nucleotide sequence of the gene coding region is shown in SEQ ID No. 1; the PsBTB protein is composed of the... PsBTB The gene is encoded, and its amino acid sequence is shown in SEQ ID NO.

2.

2. PsBTB Application of gene and PsBTB protein in the preparation of reagents to improve the low-temperature stress resistance of poplar; PsBTB The nucleotide sequence of the gene coding region is shown in SEQ ID No. 1; the PsBTB protein is composed of the... PsBTB The gene is encoded, and its amino acid sequence is shown in SEQ ID NO.

2.

3. As described in claim 1 PsBTB The application of the gene and PsBTB protein in the positive regulation of poplar low-temperature stress resistance is characterized by, Used to amplify PsBTB The primers for the gene include upstream and downstream primers, as shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

4. The method according to claim 2 PsBTB The application of the gene and PsBTB protein in the preparation of reagents to improve the low-temperature stress resistance of poplar trees is characterized by, The amplification of PsBTB The primers for the gene include upstream and downstream primers as shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

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