A Low-Temperature Inducible Expression Promoter ScMYB6 in Wild Potato and Its Applications

Abstract

This invention relates to the field of plant genetic engineering technology, and discloses a low-temperature induced expression promoter for wild potato. ScMYB6 Its applications. This invention provides a wild potato promoter capable of responding to low-temperature treatment. ScMYB6 The activity of this promoter is regulated by low temperature signals, and it has the function of significantly upregulating gene expression under cold and freezing conditions. Moreover, the promoter activity is higher under freezing conditions than under cold conditions, and it has high application value in the regulation of low temperature response and gene expression in plants.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to a low-temperature induced expression promoter for wild potato. ScMYB6 And its applications. Background Technology

[0002] Temperature, as one of the core environmental factors for plant growth and development, is a key ecological element regulating crop yield and quality. Chill injury (low temperature stress above 0°C) and frost injury (low temperature stress below 0°C) can severely damage various crops, leading to reduced yields and, in extreme cases, even total crop failure. Compared to cultivated potatoes, which are sensitive to low temperatures and lack cold acclimatization ability... Solanum tuberosum ), wild potatoes ( S. commersonii It has strong cold acclimatization and cold resistance capabilities.

[0003] Previous studies have shown that multiple transcription factors play a key role in the plant's response to low temperature stress. Studies in the model plant Arabidopsis thaliana have found that low temperature stress promotes the translocation of the transcription factor RVE8 (REVEILLE8) protein from the cytoplasm to the nucleus, where it activates promoter and gene expression by binding to the cis-acting element EE (Evening Element) in the promoter region of downstream target genes.

[0004] A promoter is a specific DNA sequence located at the start of a gene that controls the expression pattern of the gene it drives, including temporal, spatial, and intensity specificity. Developing promoters from wild germplasm resources that respond to chilling or freezing stress is of great significance for regulating plant responses to low-temperature stress. Summary of the Invention

[0005] The purpose of this invention is to provide a potato promoter that can regulate the upregulation of genes in response to cold or freezing stress.

[0006] To achieve the above objectives, the present invention provides a method for processing wild potatoes (… S. commersonii This invention conducted extensive screening of upstream sequences (promoter regions) of multiple functional genes (CM) and compared them with homologous genes in cultivated potatoes. The findings suggest that... ScMYB6 The upstream sequence of the gene can upregulate gene expression in response to chilling and freezing treatments, and its homologous gene in cultivated potatoes... StMYB6 The promoters do not possess this function. For example, the promoter shown in SEQ ID NO.1 can drive high-level gene expression under cold and frost stress conditions, with expression increasing approximately 3-4 times under cold stress and approximately 5-7 times under frost stress. This promoter can drive the target gene under cold or frost stress conditions, and exhibits stronger initiation activity under frost stress conditions, making the target gene's function more targeted.

[0007] Specifically, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides a low-temperature induced expression promoter for wild potato. ScMYB6 The wild potato low-temperature induced expression promoter ScMYB6 The sequence is shown in SEQ ID NO.1.

[0009] Secondly, the present invention provides an expression cassette comprising a wild-type potato cryogenic expression promoter. ScMYB6 .

[0010] Thirdly, the present invention provides a recombinant expression vector comprising a wild-type potato low-temperature induced expression promoter. ScMYB6 In the recombinant expression vector, the wild potato low-temperature induced expression promoter ScMYB6 It is attached upstream of the gene sequence to be expressed in the vector.

[0011] Fourthly, the present invention provides a host bacterium containing a wild-type potato cryogenic expression promoter. ScMYB6 Contains the low-temperature induced expression promoter of wild potato ScMYB6 The expression cassette or contains a wild potato cryogenic expression promoter ScMYB6 Recombinant expression vectors.

[0012] Fifthly, this invention provides a low-temperature induced expression promoter for wild potato. ScMYB6 Application of regulating the expression of target genes in plants in response to chilling or freezing treatment 。

[0013] Preferably, the application specifically involves: operably linking the target gene downstream of the promoter ScMYB6, so that the promoter ScMYB6 drives the upregulation of the target gene under corresponding cold or freezing stress in plants.

[0014] The present invention contains a promoter ScMYB6 The expression box can be used in the promoter ScMYB6 The downstream of the expression unit can be operatively linked to any target gene sequence to obtain the expression unit.

[0015] The present invention contains a promoter ScMYB6 The recombinant vector can be any vector known in the art, such as a cloning vector, expression vector, integration vector, or transposon.

[0016] The host bacteria described in this invention mainly include Escherichia coli and Agrobacterium.

[0017] The target gene described in this invention can be a functional gene, an antisense gene of a functional gene, or a small RNA gene that can interfere with the expression of a functional gene.

[0018] In the methods described above, the plant is preferably a dicotyledonous plant.

[0019] Cold damage refers to agricultural meteorological disasters caused by crops encountering temperatures above 0°C, sometimes reaching around 20°C, during the growing season, resulting in physiological disorders or damage to reproductive organs.

[0020] Frost damage is a type of agricultural meteorological disaster, caused by freezing inside crops due to temperatures below 0°C.

[0021] Compared with existing technologies, this invention provides a low-temperature induced expression promoter for wild potato. ScMYB6 Its application has the following beneficial effects:

[0022] In existing technologies, promoters used in plant genetic engineering, especially for stress-resistance breeding, are mostly derived from model plants or common crops, such as Arabidopsis thaliana and rice. Their specificity in response to abiotic stress, inducible expression intensity, and tissue specificity are often limited. Furthermore, the activity of these promoters in heterologous species (such as potato) may be weakened or even lost due to species specificity. In contrast, the upstream promoter sequence of the ScMYB6 gene (SEQ ID NO.1) provided in this invention is derived not only from a closely related wild species of potato (… S. commersonii This promoter exhibits greater species compatibility and application potential, and demonstrates significantly stronger stress response characteristics: it upregulates gene expression by approximately 3-4 times under cold damage conditions and by as much as 5-7 times under freezing damage conditions. More importantly, this promoter shows stronger specificity and response intensity to freezing damage, enabling more precise and efficient targeted regulation of stress and avoiding energy waste and potential negative effects caused by the continuous expression of constitutive strong promoters. Therefore, the promoter provided by this invention has significant advancements and practical application value in driving the expression of cold or freezing damage resistance genes and improving crop cold resistance. Attached Figure Description

[0023] Figure 1 This is the analysis result of the expression level of the MYB6 homologous gene in cultivated potato (DM) and wild potato (CM) in response to chilling injury (4℃) and freezing injury (-2℃) treatments in Example 1 of this invention. The expression level of DM at 22℃ was normalized. ACTIN was used as the internal reference gene. Data are the mean ± standard error of three biological replicates. Statistical analysis was performed using Student's Methods. t test, *** represents p<0.001.

[0024] Figure 2The ScRVE8 pair in Embodiment 3 of the present invention is a low-temperature responsive device. ScMYB6 and StMYB6 Analysis results of promoter activity regulation. (A) ScMYB6 and StMYB6 Gene sequence alignment analysis, with blue and yellow indicating regions as ScMYB6 and StMYB6 Collinear regions exist in the starter subregion, indicated by the black arrows. ScMYB6 and StMYB6 (A) Primer positions for promoter cloning. (B) Schematic diagram of vector structure. (C) Immersing infected tobacco leaves in firefly luciferin, imaging with a bioluminescence imaging system, and adding false color. (D) Collecting infected tobacco leaves with a punch, grinding them in liquid nitrogen, and then detecting the activities of LUC and REN using an ELISA reader. The LUC / REN ratio indicates the activity of the reporter gene promoter. Data are the mean ± standard error of 5 biological replicates. The statistical analysis method is one-way ANOVA, and different letters represent p < 0.005.

[0025] Figure 3 For the detection in Embodiment 4 of the present invention ScMYB6 The analysis results of promoter response to chilling and freezing injury were obtained by treating tobacco leaves infected with Agrobacterium scMYB6pro:LUC at 22℃, 4℃, and -2℃. The activities of LUC and REN were then measured, and the LUC / REN ratio at 22℃ was normalized. Data are presented as the mean ± standard error of five biological replicates. Statistical analysis was performed using Student's Methods. t test, *** represents p<0.001. Detailed Implementation

[0026] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0028] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. The pGreenII 0800-LUC vector was purchased from Qiyun Bio (https: / / www.qiyunbio.com / productinfo / 1185620.html). Example 1

[0029] Analysis of the response of potato MYB6 expression to chilling and freezing injury

[0030] Wild potato CM material (CIP762476, purchased from the International Potato Center) and cultivated potato DM material (DM1-3516R44, from the Institute of Agricultural Genomics, Chinese Academy of Agricultural Sciences) were cultured for 4 weeks at 22℃ under 16h light / 8h darkness. Young leaves from five plants of each potato species were mixed to create the "22℃" sample. These samples were then transferred to 4℃ and -2℃ for further treatment, and samples were collected at "4℃" and "-2℃". The samples were ground in liquid nitrogen, and RNA was extracted using a plant total RNA extraction kit (Tiangen Biotech, catalog number DP441). Reverse transcription was performed using a first-strand cDNA synthesis kit (Thermo Fisher Scientific, catalog number K1622).

[0031] Designing highly specific [products] using Primer-BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) MYB6 Primer sequences were amplified, and real-time quantitative PCR (RT-qPCR) was performed using the forward primer 5'-ACCAAAGCCCATAAGGATTTCTTGTG-3' and the reverse primer 5'-TCCATCCCCTTCAAGTAATTCACCA-3' to detect the expression of the MYB6 homologous gene in CM and DM materials. ACTIN The specific reaction system used as an internal reference gene is shown in Table 1:

[0032] Table 1

[0033]

[0034] The RT-qPCR reaction program was as follows: 95℃ for 3 min; 95℃ for 10 s; 60℃ for 30 s, for a total of 35 cycles.

[0035] Experimental results show that, at 22℃, the CM material... ScMYB6 and DM materials StMYB6 The expression levels were similar; however, at 4℃, the CM material showed... ScMYB6 The expression increased approximately 3-fold, and at -2°C, it increased approximately 5-fold; in comparison, in DM materials... StMYB6 The expression does not respond to low temperature conditions such as 4℃ and -2℃. Figure 1 The above results prove that wild potatoes contain... ScMYB6 The expression of the substance responded to low temperature, and the upregulation of expression under freezing conditions was greater than that under cold damage conditions. Example 2

[0036] Wild potatoes and cultivated potatoes MYB6Cloning and alignment analysis of upstream gene sequences

[0037] A 4856 bp genome was obtained by PCR amplification from the wild potato CM genome using the forward primer 5'-GGTGGCTGAAAATGGGGAAGA-3' and the reverse primer 5'-ACAACATGGTGCTCTTCCCAT-3'. ScMYB6 The upstream sequence of the gene, verified by sequencing, ScMYB6 The nucleotide sequence upstream of the gene is shown in SEQ ID NO.2.

[0038] A 5021 bp genome was obtained by PCR amplification from the DM genome of cultivated potato using the forward primer 5'-ACCTCCTAATACCAACACAAGCTTC-3' and the reverse primer 5'-ACAACATGGTGCTCTTCCCAT-3'. StMYB6 The upstream sequence of the gene, verified by sequencing, StMYB6 The nucleotide sequence upstream of the gene is shown in SEQ ID NO.3.

[0039] The PCR amplification system (total volume 50 ml) in Example 2 is shown in Table 2:

[0040] Table 2

[0041]

[0042] The PCR amplification program was as follows: 94℃ for 2 min; 98℃ for 10 s; 55℃ for 30 s; 68℃ for 3 min, for a total of 30 cycles; 68℃ for 10 min.

[0043] PCR cloning was obtained ScMYB6 upstream gene sequence and StMYB6 The upstream sequence fragments of the gene were recovered and purified, and the two sequences were ligated into the TOPO vector using the TOPO vector cloning kit (Adley Biotechnology, catalog number CV2101). The reaction system is shown in Table 3.

[0044] Table 3

[0045]

[0046] After the above reaction system was ligated at 25°C for 5 min, E. coli DH5α competent cells were transformed. Single clones were picked for shake culture and plasmid extraction. The plasmids were then sent to the sequencing service center of Sangon Biotech (Shanghai) Co., Ltd. in Kunming, Yunnan Province for sequencing.

[0047] The procedures for transforming E. coli DH5α competent cells and identifying the vector are as follows:

[0048] (1) Prepare LB solid medium containing carbenicillin.

[0049] (2) Melt 100ml of Escherichia coli DH5α competent cells stored in an ultra-low temperature freezer on ice, add 10ml of recombinant product, mix gently, and let stand on ice for 20min; then perform a 1min 42℃ water bath treatment, and immediately let stand on ice for 3min.

[0050] (3) Add 600 ml of LB liquid culture medium and shake in a shaker at 37°C (100 rpm / min) for 40 min.

[0051] (4) Take 100 ml of the bacterial culture recovered in step (3), spread it on LB solid medium containing carbenicillin, and incubate it upside down in a bacterial incubator at 37°C for about 16 hours.

[0052] (5) Select 3 single colonies for shake culture, extract plasmids and then sequence them.

[0053] Wild potatoes were obtained through sequencing. ScMYB6 The nucleotide sequence of the upstream gene is related to that of cultivated potatoes. StMYB6 Compared to the upstream sequence of the gene, ScMYB6 The -2127 to -4520 region in the upstream sequence of the gene and StMYB6 The sequences from -154 to -1932 show high similarity, but their sequence directions are opposite, and StMYB6 There are three large fragment deletions and multiple SNPs or small fragment differences in this region. Figure 2 A). And ScMYB6 The -134 to -2176 region in the upstream sequence of the gene is StMYB6 There are no homologous sequences upstream of the gene, and this region contains two cis-acting elements (EEs) that the cold-response protein RVE8 can bind to. Figure 2 A). The above results indicate that in wild potato CM materials and cultivated potato DM materials... MYB6 Significant differences exist in the upstream sequences of homologous genes, and these differences may determine... ScMYB6 Responding to low temperatures, and StMYB6 Key factors for not responding to low temperatures. Example 3

[0054] Using potatoes ScMYB6 Startup driver LUC Gene expression

[0055] Using the forward primer 5'-TCGACGGTATCGATAGGCATAATCCATCGCTAGCC-3' and the reverse primer 5'-CGCTCTAGAACTAGTGGACAACATGGTGCTCTTCCCAT-3', a 2235bp amplification was obtained from the wild potato CM genome by PCR. ScMYB6 The promoter core sequence was verified by sequencing. ScMYB6 The nucleotide sequence of the promoter is shown in SEQ ID NO.1.

[0056] Using the forward primer 5'-TCGACGGTATCGATAACCTTTAATATGATGTGAACATGTCG-3' and the reverse primer 5'-CGCTCTAGAACTAGTGGACAACATGGTGCTCTTCCCAT-3', a 2192bp amplification was obtained from the DM genome of cultivated potato. StMYB6 The promoter core sequence was verified by sequencing. StMYB6 The nucleotide sequence of the promoter is shown in SEQ ID NO.4.

[0057] To facilitate subsequent ligation with the vector, the PCR product has homologous arms at both ends for recombination reactions.

[0058] The PCR amplification system (total volume of 50 ml) in Example 3 was the same as that in Table 2 of Example 2. The PCR amplification program was as follows: 94℃ for 2 min; 98℃ for 10 s; 55℃ for 30 s; 68℃ for 1 min, for a total of 30 cycles; 68℃ for 10 min.

[0059] PCR cloning was obtained ScMYB6 promoter ( ScMYB6pro )and [[ID= promoter ( ​ The pGreenII 0800-LUC vector was recovered and purified using [a specific method / method]. ​ III and ​ I was double-digested, and after recovery, it was reacted with... ​ promoters and ​ The purified fragments of the promoter were ligated using a seamless cloning kit (Novizan, catalog number C112). The ligation product was transformed into E. coli DH5α competent cells, and the vectors ScMYB6pro:LUC and StMYB6pro:LUC were obtained through identification.

[0060] The transformation of Escherichia coli DH5α competent cells and the identification of the vector were performed according to the steps in Example 2, with the exception of using LB medium containing kanamycin.

[0061] The correctly sequenced recombinant vectors ScMYB6pro:LUC and StMYB6pro:LUC were transformed into Agrobacterium rhizogenes GV3101 competent cells, respectively. Using Agrobacterium-mediated transformation, ScMYB6pro:LUC and StMYB6pro:LUC were then transformed into tobacco leaves.

[0062] The specific methods for transforming Agrobacterium tumefaciens competent cells, transiently transfecting tobacco leaves, and performing bioluminescent imaging are as follows:

[0063] (1) Thaw two tubes of 50ml Agrobacterium GV3101 competent cells stored in an ultra-low temperature freezer on ice, add 5ml of ScMYB6pro:LUC and StMYB6pro:LUC plasmids respectively, mix gently, let stand on ice for 15min, then transfer to a pre-cooled electroporation cup (model 0.1cm), put it in an electroporator and start the device, set the conditions as follows: capacitance 35μF, resistance 200Ω, voltage 2.5kV.

[0064] (2) Add 800 μl of liquid LB medium to the electroporation cup, transfer the resuspended cells to a 1.5 ml centrifuge tube, and restore the culture at 28°C (150 rpm / min) for 2 h. Then, take 100 μl of bacterial culture and spread it on solid LB medium (containing rifampicin and kanamycin) and incubate it upside down in a bacterial incubator at 28°C for 2 days.

[0065] (3) Pick two single colonies and shake culture them. After extracting the plasmid, use the primers in "Example 2" for PCR identification. Take the correctly identified Agrobacterium carrying ScMYB6pro:LUC and StMYB6pro:LUC for expansion culture. At the same time, shake culture Agrobacterium carrying the control vector 35S:eYFP and the effect factor vector 35S:ScRVE8 respectively. ​ The cloning primers (containing homologous arms for vector construction) are as follows: forward primer 5'-ACGGGGGACTCTAGAGGATCCATGAATGGTTTTGAAAACATG-3', and reverse primer 5'-TTGCTCCATGGTACCGGATCCTATGGTTTGACATGATAGATCA-3'.

[0066] (4) Prepare tobacco leaf infection buffer: Take 200 ml of 1 mol / L MES stock solution, 200 ml of 1 mol / L MgCl2 stock solution and 20 ml of 150 mmol / L acetylsyl syringone stock solution into a 50 mL centrifuge tube, and make up to 20 mL with ultrapure water.

[0067] (5) Centrifuge the cultured Agrobacterium tumefaciens at 3500g for 10 min at room temperature, discard the supernatant, resuspend the cells in the infection buffer obtained in step (4), and adjust the OD. 600 Set to 1.0 and let stand at room temperature for 2 hours.

[0068] (6) Mix equal volumes of Agrobacterium spp. ScMYB6pro:LUC and StMYB6pro:LUC with Agrobacterium spp. 35S:eYFP and 35S:ScRVE8 respectively.

[0069] (7) Select healthy 4-week-old tobacco plants, use a 1mL sterile syringe to draw up the suspension, place your fingertip against the front of the leaf, and gently inject the bacterial solution from the back of the leaf to make the local area soaked into a dark green water stain. Place the plant at 25℃ for 2 days.

[0070] (8) Bioluminescence imaging of tobacco leaves: Add 20 mL of luciferase substrate to a culture dish, cut off the tobacco leaves and immerse them in the substrate, place the culture dish in a bioluminescence imaging instrument (Tanon 5200), take a bright field photo, turn off the light, place in darkness for 3 min, and expose for 5 min to perform bioluminescence imaging.

[0071] Experimental results show that ScRVE8 can significantly activate ​ Promoter is active, but cannot be activated. ​ promoter ( ​ This invention, through promoter activity analysis, discovered that the low-temperature-responsive transcription factor ScRVE8 can specifically activate a transcription factor containing two EE cis-acting elements. ​ Promoter. Example 4

[0072] wild potatoes ​ Promoter activity is enhanced under cold and frost stress conditions.

[0073] Agrobacterium carrying ScMYB6pro:LUC and StMYB6pro:LUC obtained in "Example 3" was used to infect leaves of Nicotiana benthamiana. After incubation at 22°C for 2 days, the leaves were transferred to 22°C, 4°C, and -2°C for further treatment. Leaf samples were collected, and bioluminescent signals were detected using a dual-luciferase reporter gene assay kit (Novizan, catalog number DL101-01) and a microplate reader to analyze promoter activity. The specific methods are as follows:

[0074] (1) Use a punch to collect tobacco leaves at 22℃, 4℃ and -2℃ respectively. Take a tobacco leaf disc with a diameter of 8mm and put it into a 1.5mL centrifuge tube. Collect 5 independent samples for each temperature condition.

[0075] (2) Add two small steel balls to a 1.5 mL centrifuge tube containing leaf tissue, pre-cool with liquid nitrogen, and grind into powder.

[0076] (3) Add 50 ml of cell lysis buffer, shake to mix, and let stand at room temperature for 5 min. Then centrifuge at 12000 g for 2 min and take 20 ml of supernatant into a 96-well microplate.

[0077] (4) Add 100 ml of LUC substrate, mix quickly, and immediately use an enzyme-linked immunosorbent assay (ELISA) reader to detect the activity of the LUC reporter gene.

[0078] (5) Add 100 ml of REN activity detection working solution to the above reaction solution, mix quickly and immediately use an enzyme-linked immunosorbent assay (ELISA) reader to detect REN reporter gene activity.

[0079] Experimental results showed that after treatment at 4℃, the relative activity of LUC / REN increased nearly fourfold, reflecting... ​ level of expression and ​ Promoter activity increased nearly fourfold; after treatment at -2℃... ​ Promoter activity increased nearly 7-fold; in comparison, at 4℃ and -2℃, ​ Promoter activity showed no significant difference at 22℃. ​ The above results prove that ​ The promoter activity responds to low temperatures, and the increase in promoter activity under freezing conditions is nearly twice that under cold damage conditions.

[0080] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A low-temperature induced expression promoter for wild potato ScMYB6 Its features are: The wild potato low-temperature induced expression promoter ScMYB6 The sequence is shown in SEQ ID NO.

1.

2. An expression box, characterized in that, The expression cassette contains the wild potato low-temperature induced expression promoter as described in claim 1. ScMYB6 .

3. A recombinant expression vector, characterized in that, The recombinant expression vector contains the wild potato low-temperature induced expression promoter as described in claim 1. ScMYB6 In the recombinant expression vector, the wild potato low-temperature induced expression promoter ScMYB6 It is attached upstream of the gene sequence to be expressed in the vector.

4. A host bacterium, characterized in that, The host bacterium contains the low-temperature induced expression promoter of wild potato as described in claim 1. ScMYB6 The expression cassette of claim 2 or the recombinant expression vector of claim 3.

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