ScAKR2A gene for regulating and controlling low potassium stress resistance of sugarcane and application of ScAKR2A gene

Abstract

The invention discloses an ScAKR2A gene for regulating and controlling low potassium stress resistance of sugarcane and application of the ScAKR2A gene. The ScAKR2A gene plays a negative regulation role in sugarcane, the reduction of the expression level of the ScAKR2A gene can enhance the tolerance of the sugarcane to low potassium stress, and the nucleotide sequence of the ScAKR2A gene is as shown in SEQ ID NO. 1. The invention discloses the sugarcane ScAKR2A gene and the sequence thereof for the first time, and proves that the gene negatively regulates low potassium stress response, the low potassium resistance of sugarcane can be remarkably enhanced by reducing the expression level of the gene, and a directly available gene target and a technical path are provided for molecular breeding of potassium-efficient sugarcane varieties.

Description

A ScAKR2A gene regulating sugarcane tolerance to low potassium stress and its application Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a ScAKR2A gene that regulates sugarcane tolerance to low potassium stress and its application. Background Technology

[0002] Sugarcane is my country's most important sugar crop and energy crop, playing a vital role in ensuring the national sugar supply system and bioenergy production. Sugarcane is a potassium-loving crop with extremely high potassium requirements; potassium deficiency leads to a significant reduction in yield and sugar content. It is estimated that producing one ton of sugarcane requires the absorption of 2-2.5 kg of potassium. However, major sugarcane-producing areas in my country generally face problems such as soil acidification and severe potassium leaching, resulting in low total and available potassium content in the topsoil. Furthermore, my country lacks soluble potassium fertilizer resources, relying on imports for over 50%, and sugarcane's utilization rate of potassium fertilizer is only about 30%. These factors severely restrict the sustainable development of my country's sugarcane industry.

[0003] AKR2A was initially discovered as an interacting protein with 14-3-3. Its subcellular localization is specific, ranging from the cytoplasm to the nucleus, primarily functioning as a molecular chaperone. In Arabidopsis, AKR2A is an essential molecular chaperone for peroxisomal membrane-bound ascorbate peroxidase 3 (APX3), and its function directly affects APX3 synthesis and in vitro biological activity. When plants are subjected to abiotic stress, cells rapidly accumulate large amounts of reactive oxygen species (ROS), and ROS scavenging mechanisms mediated by antioxidant enzymes (such as APX) are crucial for maintaining cellular homeostasis. Studies have shown that under low potassium stress, the expression levels and activities of protective enzymes (SOD, POD, CAT, APX) in sensitive tomato plants decrease with prolonged stress, while ROS and malondialdehyde (MDA) levels increase sharply; while low potassium-tolerant plants effectively mitigate oxidative damage by maintaining the activity of protective enzymes, resulting in relatively less MDA accumulation. This suggests that the expression regulation of protective enzymes such as APX is closely related to plant tolerance to low potassium. In addition, AKR2A is also involved in physiological processes such as chloroplast outer membrane protein transport, low temperature stress tolerance, and flowering regulation.

[0004] Low potassium stress induces the production of large amounts of reactive oxygen species (ROS) in plant root cells, and ROS are involved in regulating the plant's response to low potassium stress. AKR2A can regulate the subcellular localization and activity of the intracellular protective enzyme APX3, suggesting that it may affect plant tolerance to low potassium through the APX3-mediated ROS scavenging pathway. Therefore, in-depth analysis of the biological function and regulatory mechanism of the sugarcane AKR2A gene can not only provide a new perspective for revealing the molecular basis of plant tolerance to low potassium, but also provide theoretical guidance for creating potassium-efficient crop varieties through molecular design breeding, which has important scientific significance and application value for maintaining the healthy and sustainable development of my country's agricultural industry. Summary of the Invention

[0005] The purpose of this invention is to explore in depth the role of the AKR2A gene in sugarcane response to low potassium. + The study aims to investigate the function and molecular mechanisms of stress responses, providing important guidance for the creation of potassium-efficient sugarcane varieties through molecular design breeding.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] On the one hand, the present invention provides a ScAKR2A gene that regulates the tolerance of sugarcane to low potassium stress. The ScAKR2A gene plays a negative regulatory role in sugarcane, and its expression level reduction can enhance the tolerance of sugarcane to low potassium stress. The nucleotide sequence of the ScAKR2A gene is shown in SEQ ID NO.1.

[0008] Furthermore, the amino acid sequence of the protein encoded by the ScAKR2A gene is shown in SEQ ID NO.2.

[0009] On the other hand, the present invention also provides a repressive expression cassette containing an interference fragment of the ScAKR2A gene shown in SEQ ID NO.1, wherein the interference fragment is used to reduce the transcriptional level of ScAKR2A.

[0010] On the other hand, the present invention also provides a CRISPR / Cas9 gene editing vector that targets the coding region or promoter region of the ScAKR2A gene shown in SEQ ID NO.1, for knocking out or inhibiting the expression of ScAKR2A.

[0011] On the other hand, the present invention also provides the application of the ScAKR2A gene in improving the sugarcane's tolerance to low potassium stress by silencing, interfering with, knocking down or knocking out the ScAKR2A gene in sugarcane to improve the sugarcane's tolerance to low potassium stress.

[0012] On the other hand, the present invention also provides the application of the ScAKR2A gene in the breeding of sugarcane varieties resistant to low potassium stress, thereby improving the sugarcane's tolerance to low potassium stress by silencing, interfering with, knocking down or eliminating the ScAKR2A gene in sugarcane.

[0013] On the other hand, the present invention also provides the application of a repressor of the ScAKR2A gene in improving sugarcane tolerance to low potassium, said repressor including RNA interference fragments, antisense oligonucleotides or gene editing vectors.

[0014] Furthermore, the RNA interference fragments include siRNA, miRNA, shRNA, and dsRNA.

[0015] Furthermore, the gene editing vector includes a CRISPR / Cas9 gene editing vector that targets the coding region or promoter region of the ScAKR2A gene shown in SEQ ID NO.1 to knock out or inhibit the expression of ScAKR2A.

[0016] On the other hand, the present invention also provides a method for breeding sugarcane varieties resistant to low potassium stress, wherein the expression or function of the ScAKR2A gene is inhibited in sugarcane by means of silencing, interference, knockdown or knockout, so as to enhance the plant's tolerance to low potassium stress.

[0017] On the other hand, the present invention also provides a transgenic sugarcane plant in which the expression or function of the ScAKR2A gene is silenced, interfered with, knocked down or knocked out, thereby making the plant significantly more tolerant to low potassium stress than the wild type.

[0018] The present invention has the following beneficial effects:

[0019] This invention discloses for the first time the sugarcane ScAKR2A gene and its sequence, confirming that this gene negatively regulates the response to low potassium stress. By reducing its expression level, the sugarcane's tolerance to low potassium can be significantly enhanced, providing a directly usable gene target and technical path for the molecular breeding of potassium-efficient sugarcane varieties. Attached Figure Description

[0020] Figure 1 is an agarose gel electrophoresis image of the PCR product of the sugarcane ScAKR2A gene. In the figure, Marker represents the 2000bp DNA Marker, and ScAKR2A represents the PCR product.

[0021] Figure 2 shows the changes in ScAKR2A protein content in sugarcane root tissue after low potassium treatment.

[0022] Figure 3 shows the phenotypic identification of the yeast mutant CY162 transformed with the sugarcane ScAKR2A gene on culture media with different potassium concentrations. Detailed Implementation

[0023] The present invention will now be described in detail with reference to specific embodiments. The following examples are for illustrative purposes only and are not intended to limit the scope of application of the present invention.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the reagents and materials used are commercially available unless otherwise specified.

[0025] Example 1

[0026] 1. CDS sequence of the sugarcane ScAKR2A gene

[0027] This embodiment provides the CDS sequence of the sugarcane ScAKR2A gene, which is the nucleotide sequence shown in SEQ ID NO.1 of this invention.

[0028] Based on the sequence provided by the full-length transcriptome sequencing results, forward primers (5'-ATGGCTTCCGATGAGAGCAAAAATTCG-3') and reverse primers (5'-TTATAGGAAAGCGTCCTTCTCTAGCAAC-3') were designed to amplify the target gene of the sugarcane ScAKR2A gene. The reagent used was PrimeSTAR HSDNA Polymerase (TaKaRa Code No. R010Q). The PCR amplification reaction system and reaction conditions are shown in Table 1.

[0029] Table 1. CDS sequence amplification reaction system and conditions for the sugarcane ScAKR2A gene.

[0030] Five μL of the PCR product was subjected to 1% agarose gel electrophoresis. The results are shown in Figure 1. The results showed that the amplified product band was approximately 1000 bp in length, which matched the target product length, indicating that the PCR amplification was successful and the PCR product of the target length was obtained. The PCR product was sequenced to obtain the cDNA sequence of the sugarcane ScAKR2A gene, which is the nucleotide sequence shown in SEQ ID NO.1 of this invention.

[0031] 2. The amino acid sequence encoded by the sugarcane ScAKR2A gene

[0032] Based on the full-length cDNA sequence of the sugarcane ScAKR2A gene, it was converted into an amino acid sequence using online software (http: / / www.bioinformatics.org / sms / ). The sequence was determined to encode 323 amino acids, which is the amino acid sequence shown in SEQ ID NO.2 of this invention.

[0033] 3. Prediction of protein domains encoded by the sugarcane ScAKR2A gene

[0034] The domains of the ScAKR2A protein were predicted and analyzed using the protein domain analysis tool of the online analysis database InterPro. The results showed that there are three ANK repeat domains (PF00023) and one PEST domain at the C-terminus of ScAKR2A, which is consistent with the literature and conforms to the structural characteristics of the AKR2A protein.

[0035] 4. Transcriptional expression of sugarcane ScAKR2A gene under low potassium stress

[0036] This embodiment provides a low potassium stress experiment, and the specific experimental procedure is briefly described as follows. Sugarcane cultivar Yuetang 55, cultured at a normal potassium level (3.0 mmol / L) for 20 days, was transferred to a potassium-free nutrient solution for potassium starvation treatment. Root samples were collected at 0 h and 3 days of low potassium stress for total RNA extraction, with three biological replicates for each sample.

[0037] Mass spectrometry analysis was performed on the ScAKR2A protein content in sugarcane root tissue samples after 0 h and 3 d of low potassium treatment. The results showed that, compared with normal potassium nutrition conditions, the ScAKR2A protein content in sugarcane root tissue decreased significantly after 3 days of low potassium treatment, reaching approximately 60% of the normal level (Figure 2). This indicates that sugarcane ScAKR2A mainly participates in the sugarcane response to low potassium stress through protein levels.

[0038] 5. Functional analysis of the sugarcane ScAKR2A gene in potassium ion absorption-deficient yeast

[0039] Using cDNA synthesized from RNA reverse transcribed from root samples of sugarcane cultivar Yuetang 55 under 6 h of low potassium stress as a template, the full-length cDNA of the ScAKR2A gene was cloned. The PCR product was recovered, and the ScAKR2A gene was ligated into the yeast expression vector pGBKT7 (TaKaRa Biotechnology Co., Ltd.) using In-Fusion enzyme.

[0040] The ligation product was transformed into *E. coli* competent cells DH5α, and positive colonies were selected for PCR verification. The verified plasmid carrying the ScAKR2A gene and the empty vector pGADT7 were then transformed into the yeast mutant strain R5421(K). + Competent cells of a defective *Saccharomyces cerevisiae* were selected for positive colonies in SD / -TRP medium. The selected positive yeast cells were cultured overnight in liquid medium until saturation, and the yeast concentration was adjusted to OD0.05. 600=0.8, then serially diluted, and inoculated into SD / -trp medium containing 200 mM, 100 mM, 50 mM and 0 mM KCl respectively, and cultured for 3-5 days to observe yeast growth.

[0041] Expression pattern analysis of the ScAKR2A gene under low potassium stress suggests that it may play a role in sugarcane response to low potassium levels. + It plays a key role under stress. This invention utilizes the yeast expression vector pGADT7 to express the ScAKR2A gene in the potassium ion absorption-deficient yeast strain R5421. The results, as shown in Figure 3, indicate that under three potassium ion concentrations of 200 mM, 100 mM, and 50 mM, the yeast strain transformed with ScAKR2A had smaller colonies than the yeast strain transformed with the empty pGBKT7 vector.

[0042] The above results indicate that ScAKR2A can inhibit the potassium ion absorption-deficient yeast strain R5421 from K+ absorption. + Absorption.

[0043] 6. CRISPR / Cas9-mediated ScAKR2A gene knockout and functional verification

[0044] The primer sequences used in this experiment are shown in Table 2.

[0045] Table 2 Primer Information

[0046] Based on the ScAKR2A gene CDS sequence, two specific sgRNA targets were designed using the CHOPCHOP online tool, and corresponding upstream and downstream primers sgRNA1-F / F0 and sgRNA2-R / R0 were synthesized. Two rounds of PCR were performed using KOD high-fidelity enzyme with a 100-fold diluted pBUE411 plasmid as a template and sgRNA1-F, sgRNA2-R, sgRNA1F0, and sgRNA2-R0 as primers. After gel recovery of the PCR products, the gel-recovered products were ligated to the pBUE411 vector using BsaI enzyme, and the ligation products were transformed into competent E. coli DH5α cells and screened using KANA plates. OsU3-FD and TaU3-RD were used as primers for bacterial P detection; after successful sequencing, plasmids were extracted. The correct plasmids were transformed into sugarcane XTT22 via Agrobacterium EAH105, and 20 candidate transgenic sugarcane seedlings were obtained through germplasm and identification screening. DNA was extracted from seedlings, and three stable homozygous lines with ScAKR2A gene knockout were obtained by screening using ScAKR2A gene detection primers, namely cas-2a-1, cas-2a-2, and cas-2a-3.

[0047] ScAKR2A knockout plants and wild-type plants were cultured in a plant nutrient solution (normal potassium concentration 3 mmol / L, low potassium concentration 0.1 mmol / L) for 30 days in a greenhouse environment, with the nutrient solution changed every 3 days. The potassium content in the roots and leaves of sugarcane seedlings was measured. The results showed that under low potassium stress, the potassium ion content in the roots and leaves of ScAKR2A knockout plants was significantly higher than that of wild-type plants, both increasing by more than 40%. Under normal potassium conditions, there was no significant difference in potassium content between the two types, indicating that knocking out the ScAKR2A gene can significantly improve the potassium accumulation capacity of sugarcane under low potassium conditions.

[0048] >SEQ ID NO.1ATGGCTTCCGATGAGAGCAAAAATTCGAAACCAGAAAAGCAGTCTTCAGCAGCTGGTGCAGGCGTACCCAATCCTTTTGATTTTTCTTCTATGAGCAGCTTGCTCAATGACCCATCGATAAGAGAGATGGCGGAGCAAATTGCAAGTGACCCTGTGTTCAACCAGATGGCTGAACAGCTTCAGAAAAGTGCTCAGGGTGCTGGAGAACAGGGTATCCCTGCATTGGATCCTCAACAGTATATGGAAACAATGCAACAAGTCATGCAAAATCCTCAGTTTGTGTCAATGGCAGAGCGTCTCGGCAATGCTCTTATGCAGGATCCCTCTATGTCCAGTATGCTCGAGAACTTGACCAGTCCAGCTCATAAGGAGCAGCTTGAGGAGAGGATGGCCCGTATCAAGGAAGATCCATCCTTGAAGCCAATTCTTGATGAGATAGAGAATGGGGGTCCATCTGCAATGGTGAAGTATTGGAATGACCCTGAGGTTCTTCAAAAGATTGGTCAGGCAATGGGTGTTAACCTTCCCGGAGATTCTAGTGCGTCCACTGTGCTCTCTGGACCCGAAGAGACTGAGGAGGAAGGAGGGGACGACGATGAGTCCATTGTTCACCACACTGCAAGTGTTGGTGATGAAGAGGGTCTGAAGAAAGCTTTAGATGGTGGAGCAGACAAGGATGAAGAAGATGCCGAGGGAAGAAGGGCTTTACATTTTGCATGTGGCTATGGTGAGTTGAAGTGTGCGGAAATCCTCCTGGAGGCGGGGGCTGCAGTTGATGCACTAGATAAGAACAAGAACACTCCGTTGCATTACGCTGCTGGGTATGGTCGGAAGGAATGTGTAGATCTTCTGTTGAAATATGGTGCTGCTGTCACGCTCCAGAATCTGGACGGGAAAACCCCCATCGAGGTTGCGAGGCTCAACAGTCAGGATGAGGTTCTCAAGTTGCTAGAGAAGGACGCTTTCCTATAA

[0049] >SEQ ID NO.2MASDESKNSKPEKQSSAAGAGVPNPFDFSSMSSLLNDPSIREMAEQIASDPVFNQMAEQLQKSAQGAGEQGIPALDPQQYMETMQQVMQNPQFVSMAERLGNALMQDPSMSSMLENLTSPAHKEQLEERMARIKEDPSLKPILDEIENGGPSAMVKYWND PEVLQKIGQAMGVNLPGDSSASTVLSGPEETEEEGGDDDESIVHHTASVGDEEGLKKALDGGADKDEEDAEGRRALHFACGYGELKCAEILLEAGAAVDALDKNKNTPLHYAAGYGRKECVDLLLKYGAAVTLQNLDGKTPIEVARLNSQDEVLKLLEKDAFL*

[0050] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope 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 ScAKR2A gene that regulates sugarcane tolerance to low potassium stress, characterized in that, The ScAKR2A gene plays a negative regulatory role in sugarcane, and its reduced expression level can enhance the sugarcane's tolerance to low potassium stress. The nucleotide sequence of the ScAKR2A gene is shown in SEQ ID NO.

1.

2. The ScAKR2A gene as described in claim 1, characterized in that, The amino acid sequence of the protein it encodes is shown in SEQ ID NO.

2.

3. A suppressive expression cassette, characterized in that, It contains an interference fragment of the ScAKR2A gene shown in SEQ ID NO.1, which is used to reduce the transcriptional level of ScAKR2A.

4. A CRISPR / Cas9 gene editing vector, characterized in that, It targets the coding or promoter region of the ScAKR2A gene shown in SEQ ID NO.1 to knock out or inhibit the expression of ScAKR2A.

5. The application of the ScAKR2A gene according to claim 1 in improving the tolerance of sugarcane to low potassium stress, characterized in that, Improve sugarcane tolerance to low potassium stress by silencing, interfering with, knocking down or eliminating the ScAKR2A gene described in sugarcane.

6. The application of the ScAKR2A gene as described in claim 1 in the breeding of sugarcane varieties resistant to low potassium stress, characterized in that, Improve sugarcane tolerance to low potassium stress by silencing, interfering with, knocking down or eliminating the ScAKR2A gene described in sugarcane.

7. The application of the ScAKR2A gene repressor as described in claim 1 in improving sugarcane's tolerance to low potassium, characterized in that, The inhibitory factors include RNA interference fragments, antisense oligonucleotides, or gene editing vectors.

8. The application according to claim 7, characterized in that, The gene editing vector includes a CRISPR / Cas9 gene editing vector that targets the coding region or promoter region of the ScAKR2A gene shown in SEQ ID NO.1 to knock out or inhibit the expression of ScAKR2A.

9. A method for breeding sugarcane varieties resistant to low potassium stress, characterized in that, In sugarcane, the expression or function of the ScAKR2A gene can be suppressed by silencing, interfering with, knocking down, or knocking out, in order to enhance the plant's tolerance to low potassium stress.

10. A transgenic plant, characterized in that, The expression or function of the ScAKR2A gene in the plant genome was silenced, interfered with, knocked down, or removed, thereby making the plant significantly more tolerant to low potassium stress than the wild type.

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