Cassava MeCML42 gene and application of protein coded by cassava MeCML42 gene in regulation and control of salt tolerance of cassava

Abstract

The invention discloses application of a cassava MeCML42 gene or a protein coded by the MeCML42 gene in regulation and control of salt tolerance of cassava, the nucleotide sequence of the MeCML42 gene is as shown in SEQ ID NO: 1, and the amino acid sequence of the protein is as shown in SEQ ID NO: 2. According to the present invention, the CRISPR / Cas9 technology is adopted to perform agrobacterium-mediated transformation on cassava FEC and edit cassava mecml42 gene to obtain the mutant, and compared with WT, the mutant mecml42 has significant salt tolerance, and the salt stress inhibition degree is significantly less than that of the control WT;

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and in particular relates to application of cassava MeCML42 gene and protein encoded by the gene in regulating the salt tolerance of cassava. Background Art

[0002] Cassava (Manihot esculenta Crantz), also known as cassava, South Asian yam, and manioc, is a perennial plant of the Euphorbiaceae family and one of the three major tubers in the world (cassava, sweet potato, and potato). my country's technology for processing cassava into starch and alcohol is mature, and the cost of processing cassava into starch and alcohol is relatively low. However, my country's cassava production still cannot meet my country's demand for cassava starch and alcohol. Therefore, it is necessary to import a large amount of cassava starch and alcohol from abroad every year (Huang Jie et al., 2008). Therefore, increasing my country's cassava planting area and production will help reduce its dependence on foreign cassava.

[0003] Cassava is resistant to high temperatures and poor soil conditions, and can survive well in lands where other crops fail and soil nutrients are scarce (Bull et al., 2011). It does not require high planting technology, so it is suitable for growing cassava in areas with relatively backward agricultural technology. Cassava is a crop that is "moderately sensitive to salt stress" (Gleadow et al., 2016). It is difficult to grow on saline soil, so the increase in salinization and the increase in the area of ​​saline-alkali soil will affect its expansion (Carretero et al., 2007). Cultivating salt-tolerant cassava varieties will be beneficial to the utilization of saline-alkali land and the expansion of cassava planting areas.

[0004] The process of plant improvement and domestication is often accompanied by gene mutation. However, the probability of natural plant mutation is low, and excellent mutations in the direction of human needs are even rarer. Therefore, the initial improvement of crops often requires a lot of time and materials; then, although physical mutagenesis and chemical reagent mutagenesis have been widely used, due to the randomness of physical and chemical mutagenesis, the probability of producing excellent variant traits through this method is still very low, and it is difficult to directly obtain the expected trait mutation, so this method still has the problem of time-consuming and labor-intensive; gene editing technology can accurately insert or cut specific sequences of target genes to silence target genes, and with the development of gene sequencing technology, many species have completed whole genome sequencing. The two together provide convenience for the creation of mutants. Compared with traditional breeding methods, gene editing technology is more operational, more efficient, more direct and more time-saving. At this stage, gene editing technology has been widely used in the field of plant gene function identification and crop trait genetic improvement (Samanta et al., 2016). Currently, there are three systems used in gene editing technology, one is the ZFNs zinc finger nuclease system, the second is the TALENs transcription activator effector system, and the third is 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 fast speed.

[0005] The present invention intends to create salt-tolerant cassava germplasm through transgenic technology and cultivate new salt-tolerant cassava varieties, which is of great significance to increasing my country's cassava planting area, improving cassava yield and expanding my country's arable land area. Summary of the invention

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

[0007] The above object of the present invention can be achieved by the following technical scheme: application of cassava MeCML42 gene and the protein encoded by it in regulating the salt tolerance of cassava, 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, high salt-tolerant cassava is obtained by knocking out the MeCML42 gene or down-regulating its expression.

[0009] Optionally, the MeCML42 gene is knocked out or its expression is downregulated by CRISPR-Cas9.

[0010] Furthermore, the MeCML42 gene was knocked out by CRISPR-Cas9, comprising the following steps: using the pCAMBIA1301-Cas9-sgRNA editing vector to construct the pCAMBIA1301-Cas9-sgRNA-MeCML42 knockout vector, introducing the pCAMBIA1301-Cas9-sgRNA-MeCML 42 knockout vector into cassava by Agrobacterium-mediated method, and screening to obtain the cassava salt stress-tolerant MeCML42 gene knockout mutant.

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

[0012] (1) The present invention creates a mutant mecml42 cassava by 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 state of the mutant mecml42 is significantly better than that of the control WT. This result shows that mecml42 negatively regulates the salt tolerance of cassava.

[0013] (2) Salt stress was applied to the cassava mutants mecml42 and WT at the seedling stage. The results showed that the Ca content of the mutant mecml42 and WT plants decreased significantly. 2+ The contents of Ca in the roots and stems of the mutants increased after salt stress. 2+ The accumulation of mecml42 was significantly higher than that of WT, indicating that mecml42 could inhibit the Ca 2+ The malondialdehyde (MDA) content of both mutant mecml42 and WT plants increased under salt stress conditions, but the MDA content in the roots and stems of the mutant mecml42 was significantly lower than that of the WT, indicating that the cassava mutant was less damaged by stress, further indicating that the salt tolerance of the mutant mecml42 was increased. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0015] Figure 2 The process of genetic transformation of cassava FEC with CRISPR / Cas9 gene editing vector in Example 1, wherein 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 regenerated cotyledons (MSN medium); F: culture of regenerated cotyledons (CEM medium); GH: rooting screening (MS medium);

[0016] Figure 3 The cassava tissue culture seedlings WT and the mutant mecml42 in Example 1;

[0017] Figure 4 They are the tissue culture seedlings of cassava WT and mutants under salt stress treatment in Example 1;

[0018] Figure 5 They are the phenotype analysis of tissue culture seedlings of cassava WT and cassava mutant mecml42 under salt stress treatment 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 They are the seedlings of cassava WT and mutant mecml42 under salt stress treatment in Example 1;

[0020] Figure 7 They are the leaves of cassava WT and mutant mecml42 under salt stress treatment 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 They are the potted seedlings of cassava WT and mutant mecml42 under salt stress treatment 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] Fig. 9 They are 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] Fig.10 They are 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. Detailed implementation methods

[0024] Example 1

[0025] 1. Materials and reagents

[0026] 1.1 Plant materials

[0027] The plant materials used in this study are cassava variety South China 8 (Manihot esculenta Crantz. cv. SC8).

[0028] 1.2 Strains and vectors

[0029] The competent Escherichia coli DH5α strain and the competent Agrobacterium tumefaciens LBA4404 strain were purchased from Shanghai Weidi Biological Company.

[0030] The cassava gene editing vector is pCAMBIA1301-Cas9-sgRNA vector from our laboratory, and can be constructed by referring to the method in application number 202111588443.3.

[0031] 1.3 Experimental reagents and instruments

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

[0033] 1.4 Primers and main culture medium

[0034] Except for the primers in the following table, the remaining primers were conventionally designed or used as reported in 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 commercial culture media or prepared using existing literature.

[0038] 2. Obtaining mecml42 mutants

[0039] 2.1 Construction of editing vector

[0040] The nucleotide sequence of the MeCML42 gene is shown in SEQ ID NO: 1, and the amino acid sequence encoded by it 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] The amino acid sequence encoded by it is as follows:

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

[0045] The target sequence of MeCML42 gene editing is CATCATGTGAGTTTGATAGGAGG, with a GC content of 40% and a PAM region of AGG. The target site primers are designed according to the vector restriction site, that is, the 5' end of the upper primer of the target site is added with the linker sequence GATT, and the 5' end of the lower primer is added with the linker sequence AAAC. The gene sequence is shown in Table 1.

[0046] Dilute the upper and lower primers of the target site to the working solution concentration and mix them in equal amounts. The specific system is shown in Table 2. After mixing evenly, place in a 98℃ water bath for 3 minutes, turn off the power to the water bath, and let it cool naturally to room temperature. Store at -20℃ for later use.

[0047] Table 2 Target primer annealing system

[0048]

[0049] The gene editing vector pCAMBIA1301-Cas9-sgRNA was digested with a single enzyme. The enzyme used was BsaI-HFv2 endonuclease from NEB. The enzyme digestion system was referred to the instructions of the enzyme. After the digestion was completed, electrophoresis was performed to detect whether the vector was completely cut, and the uncut vector was used as a control. After complete digestion, the digestion product was recovered using the gel recovery kit of Kangwei Century. For specific operation steps, refer to the instructions of the kit. The concentration of the recovered linear vector was measured, and the linear vector was connected with T4 ligase at a molar ratio of 5:1 to the target primer. The specific system is shown in the instructions.

[0050] The ligation product was transformed into E. coli DH5α, screened with a plate containing Kana resistance, and the grown plaques were picked for PCR detection. The positive plaques were sent for testing, and the plaques that were successfully sent for testing were saved. The bacteria that were successfully sent for testing were expanded and cultured, and the recombinant plasmid pCAMBIA1301-Cas9-sgRNA-MeCML42 was extracted. The schematic 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 used for cassava transgenic after testing.

[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 FEC. The genetically transformed FEC was replaced with MSN medium (containing 250 mg / L carbenicillin and 15 mg / L hygromycin), and the operation was repeated every 14 days until cotyledons were induced to form, and then the newly grown cotyledons were transferred to CEM solid medium (the carbenicillin concentration remained unchanged) and cultured under light conditions (28°C). The culture medium was replaced every 14 days, and excess tissue was removed each time the culture medium was changed to avoid affecting the growth of the cotyledons. When the cotyledons were induced into young shoots, they were replaced with MS solid medium (hygromycin concentration was 15 mg / L), cultured under light conditions (28°C), and rooting screening was performed. The process results of genetic transformation of cassava FEC with CRISPR / Cas9 gene editing vector are shown in the figure. Figure 2 shown.

[0053] 2.3 Identification of transgenic effects of cassava resistant seedlings

[0054] The transgenic cassava obtained by screening was preliminarily tested for molecular detection to determine whether it was transgenic cassava. Leaves of cassava seedlings that could normally root were selected for leaf PCR detection. The enzyme used was M5 Superluminal Enzyme MIX from Polymer, and the template was DNA obtained by cleaving the leaves with the lysis solution matched with M5 Superluminal Enzyme. The primers were target detection-F / R. The specific sequences of the primers are shown in Table 1. Electrophoresis detection was performed. Sanger sequencing was selected as the identification method for gene editing targets, and the PCR products obtained by successful electrophoresis detection were sent to Shanghai Biotech for sequencing.

[0055] The obtained regenerated cotyledons were replaced on CEM medium for culture. After 30 days, the regenerated cotyledons were induced to sprout and regenerated seedling lines were obtained, among which the cassava tissue culture seedlings WT and the mutant mecml42 were as follows Figure 3 shown.

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

[0057] According to the Sanger sequencing results, the corresponding editing gene of the transgenic cassava was determined. Hi-TOM primers (including Hi-TOM adapter sequences, Table 1) were designed according to the editing gene sequence. The target gene fragment was amplified using M5 enzyme, and the PCR product was verified by gel electrophoresis. The successfully verified PCR was used as the second-round template of Hi-TOM. The second-round primers and operation procedures were referred to the Hi-TOM system (Liu et al., 2019). Then, the obtained Hi-TOM second-round products were subjected to gel electrophoresis detection and the gel was cut to recover the correct target fragment. The recovered products were sent to the sequencing company (Tianjin Novogene Company) for high-throughput sequencing. The sequencing results were analyzed using the Hi-TOM website (http: / / www.hi-tom.net / hi-tom / ).

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

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

[0060] strain Edited gene sequence Edit Type WT CATCATGTGAGTTTGATAGGAGG mecml42 CATCATGTGAGTTTGATTAGGAGG Missing T

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

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

[0063] The obtained gene-edited cassava of different strains were propagated and cultured. After a certain number of gene-edited cassava of different strains with the same growth status were obtained, salt stress was applied. Three kinds of MS culture medium were prepared, namely MS culture medium without NaCl, and MS culture medium with NaCl concentrations of 20mM and 40mM respectively. The transgenic cassava tissue culture seedlings of different strains and transgenic unedited cassava tissue culture seedlings were cultured in tissue culture bottles containing these three kinds of MS culture medium, and the cassava tissue culture seedlings of different strains were propagated in 3 bottles of MS culture medium containing different NaCl concentrations, and 3 cassava stem segments were cut in each bottle, each stem segment contained only 1 bud point, and the status was similar, and cultured at room temperature of 28℃ under light. After 2 months, the growth status of transgenic culture seedlings of different strains was observed, and the transgenic unedited cassava tissue culture seedlings were used as controls to conduct preliminary statistical analysis of the morphology of cassava tissue culture seedlings, and the strain with the best growth status was selected for subsequent experiments.

[0064] The results are as follows Figure 4 As shown in the figure, the cassava mutants and the control WT that were not subjected to salt stress can grow normally; the mutant mecml42 grew significantly better than the control WT under 20mM NaCl stress; and the mecml42 grew better than the control WT under 40mM NaCl stress. This shows that the cassava mutant mecml42 has good tolerance to salt stress.

[0065] In order to further analyze the salt tolerance of the mutant mecml42 and the effect of gene mutation on plant growth, this study conducted statistics on various growth indicators (A: aboveground weight; B: number of roots; C: root length; D: root weight; E: stem length; F: number of leaves) of cassava WT and mutants after salt stress treatment, and used data analysis software to perform significant analysis on the 9 sets of repeated data obtained ( Figure 5 ). When no stress treatment was applied, the cassava mutant mecml42 showed no significant difference in other parameters except root length which was significantly lower than WT.

[0066] When the salt stress concentration was 20mM, the number and weight of fibrous roots of the cassava mutant mecml42 were similar to and slightly higher than the control WT, while the other indicators were significantly or extremely significantly higher than the control WT. The negative impact of salt stress on the mutant mecml42 was not obvious overall, and the root growth was even better than that of the untreated mutant, and the overall biomass was also increased compared to the untreated mutant.

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

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

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

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

[0071] In order to further clarify that the enhanced salt tolerance of mecml42 mutants is related to mecml42 mutations, rather than off-target editing of other genes. Based on the target sites of the best salt-tolerant cassava mutant plants obtained in the above experiment, the possible off-target sites of gene mecml42 were compared and analyzed on the analysis website (CRISPR-P v2.0 (hzau.edu.cn)), and the target fragment (including potential off-target sites) was amplified using transgenic cassava DNA as a template. The obtained PCR products were sent to a sequencing company for Sanger sequencing, and the gene sequences near the PAM region of the potential target sites were observed and analyzed. The corresponding sequences of SC8 were referred to determine whether there was an off-target phenomenon.

[0072] This study analyzed potential off-target sites. The results showed that there were 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, were located in the CDS region. The off-target scores of these five sites were not high, with the highest not exceeding 0.4. Moreover, when there was a difference in bases with the target site in the 0-8bp core region upstream of PAM, there would be no off-target phenomenon in theory. Other potential off-target sites were all located in non-coding regions, and the off-target in these regions would not affect other functional genes in theory.

[0073] Five potential off-target site detection primers located in the CDS region were designed, and the DNA of mutant mecml42 and SC8 were used as templates to perform PCR amplification of the potential off-target site region. Sanger sequencing analysis results showed that the five potential off-target sites of mutant mecml42 overlapped with the peak of SC8. Figure 1 This indicates that the DNA at these potential sites has not been edited and there is no off-target problem.

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

[0075] The cassava transgenic unedited plants were obtained by infecting cassava FEC with Agrobacterium and transferring them into empty vector cassava transgenic unedited seedlings, which were then hardened and transplanted.

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

[0077] In order to further determine whether the selected cassava mutant quemecml42 is salt-tolerant, a preliminary experiment was conducted on cassava mutant quemecml42 seedlings with the same growth status and hardened cassava seedlings 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, respectively, for stress treatment of cassava seedlings. The control was treated with clean water. Three plants of each line were treated at each concentration. Phenotypic observations were carried out every 7 days after salt stress treatment, and the salt stress treatment lasted for a total of 7 days.

[0078] The results are as follows Figure 6 As shown in the figure, the growth of the cassava mutant mecml42 and WT before and after the treatment of clear water was the same; when the salt treatment concentration was 130mM, the growth of the cassava mutant mecml42 and WT was not significantly negatively affected at 7d and 14d of treatment, and only a small number of leaves at the bottom of the plant turned yellow; when the treatment concentration reached 160mM, some leaves of cassava WT began to turn yellow after 7d of salt stress treatment; at 14d of salt stress treatment, a large number of leaves of the control WT began to wilt and fall off, and for the cassava mutant mecml42, only a small number of leaves wilt and fall off at 14d of salt stress treatment. This shows that the cassava mutant mecml42 is more tolerant to salt stress than the control WT.

[0079] Select the mutant mecml42 and the control WT (transgenic unedited cassava) with the same and mature leaves for punching, avoiding the main veins on the leaves, and then soak the mutant mecml42 and WT leaves obtained by punching in H 2 Ten leaves were placed in each treatment solution of 100 mM 0 and 150 mM NaCl solution, and phenotypic analysis was performed 5 days after treatment.

[0080] The punched leaves were immersed in clean water and 150 mM NaCl solution for 5 days. There was no significant difference between the leaves of the cassava mutant mecml42 and WT immersed in clean water ( Figure 7 (A and B in the middle); the leaves of cassava WT soaked in NaCl solution became thinner, lighter in color, and more easily broken ( Figure 7 C), while the leaves of the cassava mutant mecml42, which was also immersed in NaCl solution, did not show any significant changes ( Figure 7 (D in the middle). In summary, the cassava mutant mecml42 has a stronger salt tolerance.

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

[0082] Cassava mutant mecml42 potted seedlings with consistent and good growth status and transgenic unedited cassava (WT) potted seedlings were selected for salt stress treatment. The NaCl concentration of the stress treatment was 200 mM. Cassava seedlings treated with clean water were used as controls. Five cassava seedlings were treated with each strain. After 7 days of treatment, root, stem and leaf tissues were sampled, and the malondialdehyde content in fresh samples of various plant tissues was determined using the malondialdehyde content determination kit of Suzhou Keming Biological Company; the calcium ion content in various plant tissues was determined by flame photometry using the company's calcium content determination kit. For specific steps, please refer to its instruction manual.

[0083] The results showed that after 7 days of salt stress treatment, the phenotype of the cassava mutant mecml42 potted seedlings did not change significantly, but the lower leaves of the control cassava WT plants had turned yellow and curled ( Figure 8 ), and samples were taken from the roots, stems, leaves, and other parts of the treated and untreated cassava potted seedlings for subsequent determination of physiological and biochemical indices.

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

[0085] like Fig. 9 As shown in the figure, there was no significant difference in the MDA content in each tissue before salt stress treatment. After salt stress treatment, the MDA content in the roots and stems of 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 shows that the degree of salt stress damage to the roots and stems of the mutant mecml42 is significantly lower than that of the control WT, and the mutant mecml42 is more salt tolerant than the control WT.

[0086] When plants are subjected to salt stress, Ca 2+ The content of MECML42 will change, and MECML42 is a calcium sensor that can 2+ The mechanism of plant response to salt stress can be inferred by analyzing the changes in Ca content and distribution. 2+ The ion content is as follows Fig.10 After salt stress treatment, the Ca content in various tissues of the WT control and the mecml42 mutant was 2+ The contents of Ca 2+ Most of them are concentrated in the roots and leaves of cassava, especially in the roots. 2+The increase was the largest, and the Ca 2+ The increase was greater than that of the WT control. It is speculated that the cassava mutant mecml42 may accumulate more Ca 2+ To reduce Na + Therefore, the root Ca is the most directly and seriously affected by salt stress. 2+ The accumulation amount was greater than that of the control WT.

[0087] Therefore, the present invention has been found through experiments:

[0088] 1) In this study, CRISPR / Cas9 technology was used to transform cassava FEC through Agrobacterium-mediated transformation, and the cassava mecml42 gene was edited to obtain a mutant. Compared with WT, the mutant mecml42 was significantly salt-tolerant and the degree of inhibition by salt stress was significantly less than that of the control WT.

[0089] 2) In this study, under low salt stress (NaCl concentration of 20 mM), the aboveground biomass of the mutant mecml42 seedlings was not significantly different from that of the untreated seedlings, the length of the newly added stems exceeded that of the untreated seedlings, and the root biomass was significantly increased compared with that before treatment, indicating that the mecml42 mutant had good salt tolerance in the tissue culture bottle; as the salt stress concentration increased, the inhibitory effect on the plant also increased;

[0090] 3) In the present application, after the cassava mutant mecml42 potted seedlings were treated with a salt stress of 200 mM NaCl, the MDA content increased, and the MDA content in the control WT plants increased more, especially the MDA content in the control WT roots 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 of the WT, and the mecml42 mutant was more resistant to salt stress;

[0091] 4) Ca in the cassava mutant mecml42 and the control WT after salt stress 2+ The contents of Ca in the cassava mutant mecml42 were significantly increased. 2+ The accumulation was higher than that of the WT control, especially the Ca 2+ The accumulation of Ca in the untreated mutant mecml42 was significantly greater than that in the control WT. 2+ The concentration increased, and mecml42 is a calcium ion sensor. The plant salt tolerance SOS pathway requires the joint regulation of multiple calcium ion receptors. Therefore, it is speculated that the mecml42 gene may be involved in the SOS pathway.

[0092] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Application of cassava MeCML42 gene or protein encoded by MeCML42 gene in regulating salt tolerance of cassava, 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.

2. The use according to claim 1, characterized in that: Highly salt-tolerant cassava was obtained by knocking out or downregulating the expression of the MeCML42 gene.

3. The use according to claim 2, characterized in that: The MeCML42 gene was knocked out or its expression was downregulated by CRISPR-Cas9.

4. The use according to claim 3, characterized in that: The MeCML42 gene was knocked out by CRISPR-Cas9, comprising the following steps: using a pCAMBIA1301-Cas9-sgRNA editing vector to construct a pCAMBIA1301-Cas9-sgRNA-MeCML42 knockout vector, introducing the pCAMBIA1301-Cas9-sgRNA-MeCML42 knockout vector into cassava by using an Agrobacterium-mediated method, and screening to obtain a cassava salt stress-tolerant MeCML42 gene knockout mutant.

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