Application of the cassava MeCML42 gene and its encoded protein in regulating cassava salt tolerance

Abstract

This invention discloses the application of the cassava MeCML42 gene or the protein encoded by the MeCML42 gene in regulating cassava salt tolerance. The nucleotide sequence of the MeCML42 gene is shown in SEQ ID NO:1, and the amino acid sequence of the protein is shown in SEQ ID NO:2. This invention utilizes CRISPR / Cas9 technology to transform cassava FEC via Agrobacterium-mediated transformation, editing the cassava mecml42 gene to obtain a mutant. Compared with the control WT, the mutant mecml42 exhibits significantly higher salt tolerance, with a significantly lower degree of inhibition under salt stress compared to the control WT.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to the application of the cassava MeCML42 gene and its encoded protein in regulating cassava salt tolerance. Background Technology

[0002] Cassava (Manihot esculenta Crantz), also known as sweet potato, is a perennial plant belonging to the Euphorbiaceae family and is one of the world's three major tuber crops (cassava, sweet potato, and potato). my country has mature technology for processing cassava into starch and alcohol, and the cost of this process is relatively low. However, my country's cassava production still cannot meet its demand for cassava starch and alcohol, thus requiring large-scale imports of these products annually (Huang Jie et al., 2008). Therefore, increasing my country's cassava planting area and yield would help reduce dependence on imported cassava.

[0003] Cassava is heat-tolerant and tolerant of poor soil, allowing it to thrive even in areas where other crops fail and soil nutrients are scarce (Bull et al., 2011). It also requires relatively low cultivation techniques, making it suitable for areas with less developed agricultural technology. Cassava is classified as a crop "moderately sensitive to salt stress" (Gleadow et al., 2016), making it difficult to grow in saline soils. Therefore, increased salinization and the expansion of saline-alkali soil areas will hinder its cultivation (Carretero et al., 2007). Developing salt-tolerant cassava varieties will facilitate the utilization of saline-alkali land and expand the cassava planting area.

[0004] Gene mutations are often involved in the improvement and domestication of plants. However, the probability of natural plant mutations is low, and superior mutations that meet human needs are even rarer. Therefore, the initial improvement of crops often requires a lot of time and resources. Subsequently, although physical mutagenesis and chemical mutagenesis have been widely used, the probability of producing superior mutant traits through these methods remains low due to their randomness. It is difficult to directly obtain the desired trait mutation, so this method still has the problem of being time-consuming and labor-intensive. Gene editing technology can precisely insert or cut specific sequences of target genes to silence them. With the development of gene sequencing technology, many species have completed whole-genome sequencing. Both of these factors together facilitate the creation of mutants. Compared with traditional breeding methods, gene editing technology is more operable, efficient, direct, and time-saving. At present, gene editing technology is widely used in the fields of plant gene function identification and crop trait genetic improvement (Samanta et al., 2016). Currently, three systems are used in gene editing technology: the zinc finger nuclease system (ZFNs), the TALENs transcription activator effector system, and the CRISPR / Cas system. Among these three gene editing systems, the CRISPR / Cas system has important applications in crop genetic improvement and molecular breeding due to its low cost, high precision, and high speed.

[0005] This invention aims to create salt-tolerant cassava germplasm through transgenic technology and cultivate new salt-tolerant cassava varieties, which is of great significance for increasing the cassava planting area, improving cassava yield, and expanding the arable land area in my country. Summary of the Invention

[0006] The purpose of this invention is to provide the application of the cassava MeCML42 gene and its encoded protein in regulating cassava salt tolerance.

[0007] The above-mentioned objective of the present invention can be achieved by the following technical solution: the application of the cassava MeCML42 gene and its encoded protein in regulating cassava salt tolerance, wherein the nucleotide sequence of the MeCML42 gene is shown in SEQ ID NO:1 and the amino acid sequence of the protein is shown in SEQ ID NO:2.

[0008] Alternatively, salt-tolerant cassava can be obtained by knocking out or downregulating the expression of the MeCML42 gene.

[0009] Optionally, the MeCML42 gene can be knocked out or its expression can be downregulated using CRISPR-Cas9.

[0010] Furthermore, the MeCML42 gene was knocked out using CRISPR-Cas9, including the following steps: a pCAMBIA1301-Cas9-sgRNA editing vector was used to construct a pCAMBIA1301-Cas9-sgRNA-MeCML42 knockout vector; the pCAMBIA1301-Cas9-sgRNA-MeCML42 knockout vector was introduced into cassava using Agrobacterium-mediated transformation; and cassava salt-stress-tolerant MeCML42 gene knockout mutants were screened to obtain the knockout mutants.

[0011] Compared with the prior art, the present invention has the following advantages:

[0012] (1) The present invention creates a mutant cassava mecml42 using CRISPR / Cas9 technology. The salt tolerance of the mutant mecml42 is significantly enhanced compared with the control WT. Under the same salt stress conditions, the growth status of the mutant mecml42 is significantly better than that of the control WT. This result indicates that mecml42 negatively regulates the salt tolerance of cassava.

[0013] (2) Salt stress treatment of potted seedlings of cassava mutants mecml42 and WT revealed that the calcium content of the mecml42 and WT plants was significantly reduced. 2+ The content of Ca increased after salt stress treatment, especially in the roots and stems of mutants. 2+ The accumulation was significantly higher than that of WT, indicating that mecml42 can inhibit cassava Ca. 2+ Accumulation showed that the malondialdehyde (MDA) content in both the mutant mecml42 and WT plants increased under salt stress conditions. However, the MDA content in the roots and stems of the mutant mecml42 was significantly lower than that in WT, indicating that the cassava mutant was less affected by stress and further suggesting that the mutant mecml42 had increased salt tolerance. Attached Figure Description

[0014] Figure 1 This is a simplified diagram of the CRISPR / Cas9 gene editing vector in Example 1;

[0015] Figure 2 This is the genetic transformation process of cassava FEC using the CRISPR / Cas9 gene editing vector in Example 1, where A: SC8 axillary buds; B: somatic embryos (CIM medium); C: FEC (GD medium); D: co-culture of Agrobacterium and FEC (GD medium); E: induction of cotyledon regeneration (MSN medium); F: culture of regenerated cotyledons (CEM medium); GH: root selection (MS medium);

[0016] Figure 3 These are the cassava tissue culture seedlings WT and the mutant mecml42 from Example 1;

[0017] Figure 4 It is the cassava WT and mutant tissue culture seedlings treated with salt stress in Example 1;

[0018] Figure 5 It is the phenotypic analysis of cassava tissue culture seedlings WT and cassava mutant mecml42 treated with salt stress in Example 1, A: Shoot weight; B: Root number; C: Root length; D: Root weight; E: Stem length; F: Leaf number, * indicates 0.01 < P < 0.05, ** indicates 0.001 < P < 0.01, *** indicates 0.0001 < P < 0.001, **** indicates P < 0.0001;

[0019] Figure 6 It is the cassava WT and mutant mecml42 seedlings treated with salt stress in Example 1;

[0020] Figure 7 It is the leaves of cassava WT and mutant mecml42 treated with salt stress in Example 1, A: Cassava WT before salt stress treatment; B: Cassava mutant mecml42 before salt stress treatment; C: Cassava WT after salt stress treatment; D: Cassava mecml42 after salt stress treatment;

[0021] Figure 8 It is the potted seedlings of cassava WT and mutant mecml42 treated with salt stress in Example 1, A: Cassava WT before salt stress treatment; B: Cassava mutant mecml42 before salt stress treatment; C: Cassava WT after salt stress treatment; D: Cassava mecml42 after salt stress treatment;

[0022] Figure 9 It is the determination of MDA content in cassava WT and mutant mecml42 after salt stress treatment in Example 1, ns indicates P > 0.05, * indicates 0.01 < P < 0.05, ** indicates P < 0.01;

[0023] Figure 10 It is the determination of calcium ion content in cassava WT and mutant mecml42 after salt stress treatment in Example 1, ns indicates P > 0.05, * indicates 0.01 < P < 0.05, ** indicates P < 0.01. [[ID=该内容的翻译是实施例1中盐胁迫处理木薯WT和突变体mecml42后,对其MDA含量进行测定,ns表示P>0.05,*表示0.01<P<0.05,**表示P<0.01。]] Specific implementation mode

[0024] Example 1

[0025] 1. Materials and reagents

[0026] 1.1 Plant materials

[0027] The plant materials used in this study are cassava South China No. 8 (Manihot esculenta Crantz.cv.SC8).

[0028] 1.2 Strains and Vectors

[0029] The competent cells of Escherichia coli DH5α strain and Agrobacterium LBA4404 strain were purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0030] The cassava gene editing vector, pCAMBIA1301-Cas9-sgRNA, was derived from our laboratory and can be constructed using the method described in application number 202111588443.3.

[0031] 1.3 Experimental Reagents and Instruments

[0032] Unless otherwise specified, all experimental reagents and instruments were sourced from commercial channels.

[0033] 1.4 Primers and main culture medium

[0034] Except for the primers listed in the table below, all other primers were either conventionally designed or used as reported in the references.

[0035] Table 1 Primers used in the experiment

[0036]

[0037] CIM medium, GD medium, MSN medium, CEM medium, MS medium, LB medium, YEP medium, etc. are all commercially available media or prepared using existing literature.

[0038] 2. Obtaining the mecml42 mutant

[0039] 2.1 Construction of the editing medium

[0040] The nucleotide sequence of the MeCML42 gene is shown in SEQ ID NO:1, and the amino acid sequence it encodes is shown in SEQ ID NO:2.

[0041] The nucleotide sequence of the MeCML42 gene is as follows:

[0042] ATGACCAAGATAGGAGAATTTGAACTTGTGTTTCGCCACCTGGACAAAAATGGTGACGGGAAGATTTCAGCATCAGAACTGAGTCATCATGTGAGTTTGATAGGAGGGAAGCTGTTCATGAAAGATGCAGAAATGGTGGTTGAGTTATTGGACTCCGATGGAGATGGGTTGTTGGGATTTGATGATCTTGTTAAGCTAATGGAAGGTGGAGGAG AAGAAGAGAAGTTGCAGGATTTAAGAGAAGCTTTTGCAATGTATGACGTAGATAACTGTGGGTTTATAACTCCCAGGAGCTTGAGAAAGATGCTTAAAAAGCTTGGTGAGTCAAAATCTATTGATGAATGTAAAATGATGATAAGTCAGTTTGATCTTAATGGGGATGGTGTTCTTAGCTTCGAAGAATTTAGGGTTATGATGCAGTGA (as SEQ ID NO:1).

[0043] Its encoded amino acid sequence is as follows:

[0044] MTKIGEFELVFRHLDKNGDGKISASELSHHVSLIGGKLFMKDAEMVVEL LDSDGDGLLGFDDLVKLMEGGGEEEKLQDLREAFAMYDVDNCGFITPRSLRK MLKKLGESKSIDECKMMISQFDLNGDGVLSFEEFRVMMQ* (as shown in SEQ ID NO: 2).

[0045] The MeCML42 gene editing target sequence is CATCATGTGAGTTTGATAGGAGG, with a GC content of 40% and the PAM region being AGG. Target site primers were designed based on the vector restriction enzyme sites: the upper primer had the adapter sequence GATT added to its 5' end, and the lower primer had the adapter sequence AAAC added to its 5' end. The gene sequence is shown in Table 1.

[0046] Dilute the primers above and below the target to the working solution concentration, and mix them in equal volumes, as shown in Table 2 below. After thorough mixing, place the mixture in a 98°C water bath for 3 minutes, then turn off the power and allow it to cool naturally to room temperature. Store at -20°C for later use.

[0047] Table 2 Target Primer Annealing System

[0048]

[0049] The gene editing vector pCAMBIA1301-Cas9-sgRNA was digested with the BsaI-HFv2 restriction enzyme from NEB, following the enzyme's instructions. Electrophoresis was performed after digestion to check for complete cleavage; undigested vectors served as controls. The digested products were then recovered using a gel extraction kit from Kangwei Century, following the kit's instructions. The concentration of the recovered linear vector was determined, and ligation was performed using T4 ligase at a linear vector to target primer molar ratio of 5:1, following the manufacturer's instructions.

[0050] The ligation product was transformed into *E. coli* DH5α, and the colonies were screened using plates containing Kana resistance. Growing colonies were selected for PCR detection. Positive colonies were sent for testing, and successfully tested colonies were preserved. The successfully tested bacteria were cultured on a large scale, and the recombinant plasmid pCAMBIA1301-Cas9-sgRNA-MeCML42 was extracted. A simplified diagram of the CRISPR / Cas9 gene editing vector is shown below. Figure 1 As shown, the vector contains the hygromycin (Hyg) resistance gene. The recombinant plasmid was transformed into Agrobacterium LBA4404 and, after testing, used for cassava transgenic applications.

[0051] 2.2 Agrobacterium-mediated CRISPR / Cas9 gene editing vector transformation of cassava

[0052] Agrobacterium containing the pCAMBIA1301-Cas9-sgRNA-MeCML42 editing vector was transformed into cassava FECs. The genetically transformed FECs were then transferred to MSN medium (containing 250 mg / L carbenicillin and 15 mg / L hygromycin), and this process was repeated every 14 days until cotyledon formation was induced. The newly grown cotyledons were then transferred to CEM solid medium (with the carbenicillin concentration maintained) and cultured under light (28°C). The medium was changed every 14 days, removing excess tissue each time to avoid affecting cotyledon growth. Once the cotyledons induced shoots, they were transferred to MS solid medium (hygromycin concentration 15 mg / L) and cultured under light (28°C) for root selection. The results of the CRISPR / Cas9 gene-editing vector genetic transformation of cassava FECs are as follows: Figure 2 As shown.

[0053] 2.3 Identification of the Transgenic Effect of Cassava-Resistant Seedlings

[0054] The initially screened transgenic cassava was preliminarily subjected to molecular detection to confirm it as transgenic cassava. Leaves of cassava seedlings that could normally root were selected for leaf PCR detection. The enzyme used was the M5 Super Speed Enzyme MIX from Polymerase Company, the template was the DNA obtained by lysing the leaves with the lysis solution配套 with the M5 Super Speed Enzyme, and the primers were Target Detection - F / R. The specific sequences of the primers are shown in Table 1, and electrophoresis detection was carried out. The identification method for gene editing targets was Sanger sequencing, and the successfully electrophoresed PCR products were sent to Sangon Biotech in Shanghai for sequencing.

[0055] The obtained regenerated cotyledons were transferred to CEM medium for culture. After 30 days, buds were induced from all the regenerated cotyledons, and regenerated plantlet lines were obtained. Among them, the cassava tissue culture seedlings WT and the mutant mecml42 are as Figure 3 shown.

[0056] 2.4 Analysis of the editing types of cassava transgenic seedlings

[0057] According to the Sanger sequencing results, the corresponding edited genes of transgenic cassava were determined. Hi-TOM primers (containing Hi-TOM adapter sequences, Table 1) were designed based on the edited gene sequences. The M5 enzyme was used for amplifying the target gene fragments, and the PCR products were verified by gel electrophoresis detection. The successfully verified PCR was used as the template for the second round of Hi-TOM. The primers and operation procedures for the second round referred to the Hi-TOM system (Liu et al., 2019). Then, the products obtained from the second round of Hi-TOM were subjected to gel electrophoresis detection, and the correct target fragments were excised and recovered. The recovered products were sent to a sequencing company (Beijing Novogene Co., Ltd.) for high-throughput sequencing. The Hi-TOM website (http: / / www.hi-tom.net / hi-tom / ) was used to analyze the sequencing results.

[0058] The product fragments with a recovered band size of 350 bp were subjected to Hi-TOM sequencing to analyze the variation of gene editing targets. The results showed that the MeCML42 gene was successfully edited (Table 3), and mecml42 was a homozygous mutant. There was a T insertion at the 102nd position of the sequence shown in SEQ ID NO:1.

[0059] Table 3 Detection of gene editing types of cassava regenerated resistant lines

[0060] strain Edited gene sequences Editing type WT CATCATGTGAGTTTGATAGGAGG mecml42 CATCATGTGAGTTTGATTAGGAGG Missing T

[0061] Italic letters indicate inserted bases; - indicates deleted bases; the underlined region indicates the PAM region.

[0062] 2.5 Preliminary analysis of the salt tolerance phenotype of cassava mutant mecml42 tissue culture seedlings

[0063] Genetically edited cassava strains of different lines were propagated and cultured. After obtaining a certain number of gene-edited cassava strains of different lines with the same growth status, salt stress was applied. Three MS media were prepared: MS medium without NaCl, and MS medium with NaCl concentrations of 20 mM and 40 mM, respectively. The obtained transgenic cassava tissue culture seedlings of different lines and unedited transgenic cassava tissue culture seedlings were cultured in tissue culture bottles containing these three MS media. The cassava tissue culture seedlings of different lines were propagated in three bottles of MS medium containing different NaCl concentrations. Three cassava stem cuttings were inserted into each bottle, each stem cutting containing only one bud and with similar morphology. The culture was carried out at room temperature (28℃) under light. After two months, the growth status of the transgenic tissue culture seedlings of different lines was observed, with unedited transgenic cassava tissue culture seedlings as a control. Preliminary statistical analysis of the morphology of the cassava tissue culture seedlings was performed, and the lines with the best growth status were selected for subsequent experiments.

[0064] The results are as follows Figure 4 As shown, both the untreated cassava mutant and the control WT grew normally; however, under 20 mM NaCl stress, the growth of the mutant mecml42 was significantly better than that of the control WT; and under 40 mM NaCl stress, the growth of mecml42 was superior to that of the control WT. This indicates that the cassava mutant mecml42 has good tolerance to salt stress.

[0065] To further analyze the salt tolerance of the mecml42 mutant and the effect of gene mutation on plant growth, this study statistically analyzed various growth indicators (A: aboveground part weight; B: root number; C: root length; D: root weight; E: stem length; F: leaf number) of cassava WT and the mutant after salt stress treatment, and used data analysis software to perform significance analysis on the nine sets of replicate data. Figure 5 Without stress treatment, the cassava mutant mecml42 showed no significant difference from WT in any of its indicators except for root length, which was significantly lower.

[0066] When the salt stress concentration was 20 mM, the cassava mutant mecml42 showed significantly or extremely significantly higher values ​​than the control WT in several aspects, except for the number and weight of fibrous roots, which were similar to or slightly higher than WT. Overall, the negative impact of salt stress on the mutant mecml42 was not obvious; in fact, the root growth was better than in the untreated case, and the overall biomass also increased compared to the untreated case.

[0067] When the salt stress concentration reached 40 mM, the growth of both the cassava mutant mecml42 and the control WT was significantly inhibited. However, the cassava mutant mecml42 had a better phenotype, and the aboveground growth indicators of the mutant mecml42 were significantly or extremely significantly higher than those of the control WT.

[0068] Ultimately, after comprehensive evaluation, it was determined that the cassava mutant mecml42 has significant salt tolerance.

[0069] 3. Physiological and biochemical analysis and agronomic trait identification of cassava mutant mecml42

[0070] 3.1 Analysis of potential off-target sites in cassava mutant mecml42 plants

[0071] To further clarify that the enhanced salt tolerance of the mecml42 mutant is related to the mecml42 mutation, rather than due to off-target editing of other genes, the target sites of the cassava mutant with the best salt tolerance obtained in the above experiment were compared and analyzed on the analysis website (CRISPR-P v2.0 (hzau.edu.cn)) to identify potential off-target sites of the mecml42 gene. Using transgenic cassava DNA as a template, the target fragment (including potential off-target sites) was amplified. The obtained PCR products were sent to a sequencing company for Sanger sequencing. The gene sequences near the PAM region of the potential target sites were observed and analyzed, and the corresponding sequences of SC8 were used to determine whether off-target effects existed.

[0072] This study analyzed potential off-target sites. The results showed that there are a total of 12 potential off-target sites for this target in the cassava genome. Among them, only five potential off-target sites—Off-target-2, Off-target-4, Off-target-5, Off-target-8, and Off-target-10—are located in the CDS region. These five off-target scores are low, with the highest not exceeding 0.4. Moreover, theoretically, there should be no off-target phenomenon when there are base differences between the target and the PAM core region (0-8 bp upstream). The other potential off-target sites are all located in non-coding regions, and theoretically, off-target effects in these regions would not affect other functional genes.

[0073] Five primers were designed to detect potential off-target sites located in the CDS region. Using DNA from mutant mecml42 and SC8 as templates, PCR amplification of the potential off-target site regions was performed. Sanger sequencing analysis revealed that the five potential off-target sites of mutant mecml42 were similar to the peak values ​​of SC8. Figure 1 The result indicates that the DNA at these potential sites was not edited, and there is no off-target problem.

[0074] 3.2 Hardening-off and transplanting of cassava mutant mecml42 and transgenic unedited plants

[0075] The unedited cassava genetically modified plants were obtained by infecting cassava FEC with Agrobacterium tumefaciens and transferring the seedlings into an empty vector, followed by hardening-off and transplanting.

[0076] 3.3 Preliminary analysis of salt tolerance in cassava mutant seedlings mecml42

[0077] To further determine whether the selected cassava mutant mecml42 is salt-tolerant, a preliminary experiment was conducted using cassava mutant mecml42 seedlings with consistent growth status and after hardening, as well as transgenic unedited cassava seedlings (WT). The NaCl concentrations for salt stress treatment were initially selected as 0 mM, 130 mM, and 160 mM for the cassava seedlings, while the control was treated with water. Three seedlings of each of the two lines were used for each concentration. Phenotypic observations were conducted every 7 days after salt stress treatment, which lasted for a total of 7 days.

[0078] The results are as follows Figure 6 As shown, the growth of the cassava mutants mecml42 and WT was consistent before and after treatment with water. When the salt treatment concentration was 130 mM, the growth of both mecml42 and WT was not significantly negatively affected after 7 and 14 days of treatment, with only a small number of lower leaves turning yellow. When the treatment concentration reached 160 mM, some leaves of the WT cassava began to turn yellow after 7 days of salt stress treatment. After 14 days of salt stress treatment, the control WT began to show a large number of leaves withering and falling off, while the mecml42 cassava mutant only had a small number of leaves withering and falling off after 14 days of salt stress treatment. This indicates that the mecml42 cassava mutant has a stronger tolerance to salt stress than the control WT.

[0079] Leaves of the mutant mecml42 and the control WT (transgenic unedited cassava) that are in the same condition and mature are selected and punched. When punching, the midrib of the leaf should be avoided. Then, the leaves of the mutant mecml42 and WT obtained by punching are immersed in H2O and 150mM NaCl solutions, respectively. Ten leaves are placed for each treatment. Phenotypic analysis is performed after 5 days of treatment.

[0080] The obtained perforated leaves were soaked in water and a 150 mM NaCl solution for 5 days, respectively. No significant difference was observed in the leaf changes of the cassava mutant mecml42 and WT when soaked in water. Figure 7 (Figures A and B in the image); cassava WT leaves soaked in NaCl solution become thinner, lighter in color, and more easily broken. Figure 7 (Figure C) , while the leaves of the cassava mutant mecml42, which was also immersed in NaCl solution, did not show significant changes (Figure C). Figure 7 (See Figure D). In conclusion, cassava scape mecml42 exhibits stronger salt tolerance.

[0081] 3.4 Analysis of physiological and biochemical results of cassava mutant mecml42 after salt stress treatment

[0082] Potted seedlings of the mecml42 cassava mutant and transgenic unedited cassava (WT) with consistent and good growth were subjected to salt stress treatment. The NaCl concentration of the stress treatment was 200 mM. Potted seedlings of cassava treated with water were used as a control. Five cassava seedlings were treated for each line. After 7 days of treatment, tissue samples were taken from roots, stems and leaves. The malondialdehyde (MDA) content in fresh samples of each plant tissue was determined using the malondialdehyde content determination kit from Suzhou Keming Biotechnology Co., Ltd. The calcium content in each plant tissue was determined by flame photometry using the company's calcium content determination kit. For specific steps, please refer to the instruction manual.

[0083] The results showed that after 7 days of salt stress treatment, the phenotype of potted seedlings of the cassava mutant mecml42 did not show significant changes, but the lower leaves of the control cassava WT plants had already turned yellow and curled significantly. Figure 8 Samples were taken from the roots, stems, and leaves of both treated and untreated cassava potted seedlings to determine subsequent physiological and biochemical indicators.

[0084] MDA content is an indicator of the degree of cell membrane lipid peroxidation. Higher MDA content indicates greater stress damage to the plant. Therefore, by measuring MDA content, the degree of stress impact on the plant can be determined. This study measured the MDA content in three different tissues of cassava WT and the mutant mecml42: roots, stems, and leaves.

[0085] like Figure 9 As shown, there were no significant differences in MDA content among the tissues before salt stress treatment. After salt stress treatment, the MDA content in the roots and stems of both the control WT and the mutant mecml42 increased significantly, and the MDA content of the control WT was significantly higher than that of the mutant mecml42. This result indicates that the roots and stems of the mutant mecml42 suffered significantly less damage from salt stress than the control WT, and the mutant mecml42 had stronger salt tolerance than the control WT.

[0086] When plants are subjected to salt stress, the calcium in their bodies... 2+ The content will change, and MECML42 is a calcium ion sensor that can be activated by the calcium ions present. 2+ The study aimed to infer the mechanism of plant response to salt stress by analyzing changes in calcium content and distribution. This study used a flame method to determine the calcium content in various tissues of cassava. 2+ The ion content, the results are as follows Figure 10 As shown. After salt stress treatment, the calcium levels in various tissue sites of the control WT and the mutant mecml42 were [data missing]. 2+ The contents of all increased significantly, Ca 2+ It is mostly concentrated in the roots and leaves of cassava, especially in the roots, where Ca... 2+ The increase was the greatest, and the Ca of the mutant mecml42 was the highest.2+ The increase was greater than the control WT. It is speculated that the cassava mutant mecml42 may accumulate more Ca through absorption. 2+ To lower Na + Damage to cassava, therefore, is most directly and severely affected by salt stress in the root system (Ca). 2+ The accumulation of WT was greater than that of the control group WT.

[0087] Therefore, this invention has discovered through experiments that:

[0088] 1) In this study, CRISPR / Cas9 technology was used to transform cassava FEC through Agrobacterium-mediated transformation and edit the cassava mecml42 gene to obtain a mutant. Compared with WT, the mutant mecml42 showed significantly greater salt tolerance and was significantly less inhibited by salt stress than the control WT.

[0089] 2) In this study, under low salt stress (NaCl concentration of 20 mM), the aboveground biomass of the mecml42 mutant tissue culture seedlings was not significantly different from that of the untreated seedlings. The newly grown stems were longer than those of the untreated seedlings, and the root biomass was significantly increased compared with that before treatment, indicating that the mecml42 mutant has good salt tolerance in tissue culture bottles. With the increase of salt stress concentration, the inhibitory effect on plants also increased.

[0090] 3) In this application, after the cassava mutant mecml42 potted seedlings were treated with 200mM NaCl salt stress, the MDA content increased, and the MDA content in the control WT plants increased even more. In particular, the MDA content in the roots of the control WT plants was significantly higher than that in the mutant mecml42, indicating that the mutant mecml42 was less affected by salt stress. The MDA content in the roots and stems of the mecml42 mutant was significantly lower than that in WT, indicating that the mecml42 mutant was more tolerant to salt stress.

[0091] 4) Ca in the cassava mutant mecml42 after salt stress treatment and the control WT 2+ The content of all substances was significantly increased, with Ca in the cassava mutant mecml42 showing a significant increase. 2+ The accumulation of Ca in the roots of the treated mutant was higher than that in the control WT. 2+ The accumulation of calcium in the cassava mutant mecml42 was significantly greater than that in the untreated control (WT) than in the control group (WT). 2+ The concentration increased, and mecml42 is a calcium ion receptor. The plant salt tolerance SOS pathway requires the joint participation of multiple calcium ion receptors for regulation. Therefore, it is speculated that the mecml42 gene may be involved in the SOS pathway.

[0092] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of the cassava MeCML42 gene or the protein encoded by the MeCML42 gene in regulating cassava salt tolerance, characterized in that, The nucleotide sequence of the MeCML42 gene is shown in SEQ ID NO:1, and the amino acid sequence of the protein is shown in SEQ ID NO:2; Highly salt-tolerant cassava was obtained by knocking out or downregulating the expression of the MeCML42 gene.

2. The application according to claim 1, characterized in that, The MeCML42 gene was knocked out or its expression was downregulated using CRISPR-Cas9.

3. The application according to claim 2, characterized in that, The knockout of the MeCML42 gene using CRISPR-Cas9 includes the following steps: constructing a pCAMBIA1301-Cas9-sgRNA-MeCML42 knockout vector using the pCAMBIA1301-Cas9-sgRNA editing vector; introducing the pCAMBIA1301-Cas9-sgRNA-MeCML42 knockout vector into cassava using Agrobacterium-mediated transformation; and screening to obtain cassava salt-stress-tolerant MeCML42 gene knockout mutants.

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