Cnmt2 gene of chrysothemis naevius and expression vector, protein and application thereof

Abstract

The application provides a wedgedleaf chrysanthemum lanthanum-resistant gene CnMT2, an expression vector thereof, a protein and application thereof, and belongs to the technical field of genetic engineering.The nucleotide sequence of the wedgedleaf chrysanthemum lanthanum-resistant gene CnMT2 is shown as SEQ ID NO.1.It is found by the application that the CnMT2 gene is induced to express under LaCl3 stress treatment at different times, and the expression modes of the aboveground part and the underground part are different.Seed germination of transgenic Arabidopsis shows higher resistance to LaCl3 and H2O2, and the transgenic seedlings show better growth potential as the LaCl3 stress is prolonged, and the CAT and POD enzyme activities of the transgenic Arabidopsis are significantly increased.Therefore, the wedgedleaf chrysanthemum CnMT2 gene can improve the lanthanum resistance and the oxidation resistance of plants, and has the potential as an important gene resource for plant remediation.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and particularly relates to a Cnicus lanceolatus lanthanum-resistant gene CnMT2, an expression vector thereof, a protein and application thereof. BACKGROUND

[0002] Rare earths belong to one of the main chemical pollutants, and in recent years have been considered as a new type of pollutant in the international community, which can enter the environment through various ways. In the process of rare earth collection, the rare earth elements continuously accumulated in the soil around the rare earth tailings pond are polluted to the underground water through wind erosion, rainwater flushing and leaching, etc., which poses a serious threat to human health, and the closer to the tailings pond, the higher the content of La and Ce. The phytoremediation technology has the advantages of economy and ecological coordination, and is an important demonstration technology for ecological restoration. As the core of phytoremediation, hyperaccumulators have not been used on a large scale, because the currently discovered rare earth element hyperaccumulators are limited by regional ecological environment and cannot be applied in the local area, such as American pokeweed and Dicranopteris. Therefore, the local advantage plants with strong resistance and absorption and enrichment capacity of light rare earth elements are selected for the repair of light rare earth element over-standard soil, the key resistance genes are separated, the enrichment and accumulation of La element are improved, and the "ecological restoration" type of high resistance new varieties are cultivated, which provides a theoretical basis for in-depth understanding of the mechanism of plant rare earth enrichment and detoxification.

[0003] Metallothioneins (MTs) are proteins widely existing in animals and plants, which have the characteristics of low molecular weight, high Cys content and chelating metal ions, and are important gene resources for phytoremediation. Since the first discovery of plant MTs in wheat, multiple types of MTs have been isolated and identified in various plants, and show resistance to heavy metals. For example, the CeMT2b gene of the mountain yam makes transgenic tobacco exhibit better growth potential, and the level of H2O2 is significantly reduced, and the zinc tolerance is significantly improved; overexpression of the OsMT1a gene in rice significantly improves the drought resistance of rice, and exhibits ROS detoxification characteristics by increasing the activity of antioxidant enzyme mechanism; SsMT2 and PdMT2A of the alkali-tolerant Suaeda salsa and jujube tree enhance the activity of reactive oxygen species in transgenic Arabidopsis, and the tolerance to salt, drought and oxidative stress; the SbMT-2 gene of Hypneva musciformis can enhance the resistance of tobacco to zinc stress by transporting Zn 2+ and maintaining photosynthesis. It can be seen that MTs can improve the heavy metal resistance of plants, and are effective first-line defense agents for heavy metal stress. However, there are few reports on the tolerance of MTs to rare earth elements. SUMMARY

[0004] Therefore, the present application aims to provide a Cnicus oshimianus lanthanum-resistant gene CnMT2, an expression vector thereof, a protein thereof and application thereof, wherein the CnMT2 can significantly improve the lanthanum resistance of plants after overexpression in the plants, and has no influence on normal growth.

[0005] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions.

[0006] A Cnicus oshimianus lanthanum-resistant gene CnMT2, wherein the nucleotide sequence of the CnMT2 is shown as SEQ ID NO. 1.

[0007] The present application also provides a primer pair for amplifying the Cnicus oshimianus lanthanum-resistant gene CnMT2, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 2, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3.

[0008] The present application also provides an expression vector pEarlyGate101-CnMT2 of the Cnicus oshimianus lanthanum-resistant gene CnMT2, wherein the expression vector comprises the nucleotide sequence shown as SEQ ID NO. 1.

[0009] The present application also provides a lanthanum-resistant regulatory protein encoded by the Cnicus oshimianus lanthanum-resistant gene CnMT2, wherein the amino acid sequence of the lanthanum-resistant regulatory protein is shown as SEQ ID NO. 10.

[0010] The present application also provides application of the Cnicus oshimianus lanthanum-resistant gene CnMT2 in cultivating transgenic plants.

[0011] Preferably, the Cnicus oshimianus lanthanum-resistant gene CnMT2 is overexpressed in plants.

[0012] Preferably, the expression vector pEarlyGate101-CnMT2 is transformed into plants by an Agrobacterium-mediated method to obtain a plant overexpressing the Cnicus oshimianus lanthanum-resistant gene CnMT2.

[0013] More preferably, the Agrobacterium-mediated method is a floral dip method.

[0014] More preferably, the plants are Arabidopsis thaliana.

[0015] More preferably, the plants are lanthanum-resistant plants.

[0016] Compared with the prior art, the present application has the following beneficial effects:

[0017] This invention provides a lanthanum-tolerant gene, CnMT2, from *Arabidopsis thaliana*. Transgenic *Arabidopsis thaliana* seeds showing increased resistance to LaCl3 and H2O2 during germination, and the transgenic seedlings exhibited better growth vigor with prolonged LaCl3 stress. Simultaneously, the activities of CAT and POD enzymes in the transgenic *Arabidopsis thaliana* were significantly increased. Therefore, the *Arabidopsis thaliana* CnMT2 gene can enhance plant resistance to lanthanum and antioxidant capacity, and has the potential to serve as an important gene resource for plant repair.

[0018] This invention utilizes genetic engineering to improve plants, adjusting their ability to absorb, transport, and accumulate La element, creating rapidly growing, high-biomass transgenic plants. This provides a new approach to improving phytoremediation efficiency and has broad application prospects. Attached Figure Description

[0019] Figure 1 Homologous sequences of CnMT2 and CnMT3 from *Chrysanthemum septemlobum* and MTs proteins from different plants were compared.

[0020] Figure 2 Phylogenetic tree of amino acid sequences of CnMT2 and CnMT3 of *Chrysanthemum septemlobum* and MT proteins of other species;

[0021] Figure 3 The expression level of CnMT2 in different tissues and organs of *Chrysanthemum indicum* is shown.

[0022] Figure 4 The expression level of CnMT2 in different tissues and organs of *Chrysanthemum indicum* seedlings treated with 800 μM LaCl3 for different time periods;

[0023] Figure 5 The expression levels of CnMT2 in different transgenic Arabidopsis thaliana species;

[0024] Figure 6 The effects of LaCl3, NaCl, and H2O2 on the germination of transgenic Arabidopsis seeds are shown in the figure. From left to right, the concentrations are MS, 200 μM LaCl3, 150 mM NaCl, and 3 mM H2O2.

[0025] Figure 7 The effects of LaCl3, NaCl, and H2O2 on the rooting of transgenic Arabidopsis seeds are shown in the figure. From left to right, the concentrations are MS, 200 μM LaCl3, 150 mM NaCl, and 3 mM H2O2.

[0026] Figure 8 To show the resistance of transgenic Arabidopsis seedlings to LaCl3, the top four groups in the figure represent the 200 μM LaCl3 treatment, and the bottom four groups represent the 400 μM LaCl3 treatment.

[0027] Figure 9Antioxidant enzyme activity determination for different transgenic Arabidopsis. DETAILED DESCRIPTION

[0028] The application provides a Chrysanthemum naktongense lanthanum-resistant gene CnMT2, and a nucleotide sequence of the Chrysanthemum naktongense lanthanum-resistant gene CnMT2 is shown as SEQ ID NO. 1.

[0029] Chrysanthemum naktongense is a perennial mesophyte, is widely distributed in the grassland belt of China, has huge biomass, is cold and drought resistant, and has strong sprouting, and meets the requirements of plant remediation technology on plants. Previous studies show that Chrysanthemum naktongense has strong tolerance to light rare earth elements La and Ce. In order to further study the La tolerance mechanism of Chrysanthemum naktongense and screen the dominant gene, the application clones an MT homologous gene CnMT2 from Chrysanthemum naktongense, selects the differentially expressed CnMT2 gene, and performs function verification in Arabidopsis.

[0030] The Chrysanthemum naktongense lanthanum-resistant gene CnMT2 provided by the application can improve the lanthanum tolerance of plants, and after overexpression of the lanthanum-resistant gene CnMT2 in plants, the transgenic plants can obtain significant lanthanum tolerance, the survival ability of the plants in the soil environment with excessive lanthanum is improved, and the adverse effects of lanthanum stress on the growth of the plants are reduced.

[0031] The Chrysanthemum naktongense lanthanum-resistant gene CnMT2 is obtained by amplification from Chrysanthemum naktongense cDNA, the coding region cDNA of the Chrysanthemum naktongense lanthanum-resistant gene CnMT2 is 234 bp in length, and the nucleotide sequence is as follows:

[0032] ATGTCTTGCTGCAATGGAAAGTGTGGTTGTGGATCAAGCTGCTCTTGTGGCAGCTCTTGCAAGGGATGTGGGATGTATCCTGACATCGAGGCATCTACCACTGCCACCATGATCGTTGATGGTGTTGCACCAAAGAAGATGTATGATGATGGAAGTGAGGGAAGCTTTGTAGCTGAGGGTGGACATGCATGCAAGTGTGGAGCAAACTGCAAGTGTGATCCTTGTAATTGTTAA (as shown in SEQ ID NO. 1).

[0033] The application provides a primer pair for amplifying the Chrysanthemum naktongense lanthanum-resistant gene CnMT2, and the nucleotide sequence of the upstream primer of the primer pair is shown as SEQ ID NO. 2, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3.

[0034] In the present application, the upstream and downstream primer sequences for amplifying the CnMT2 lanthanum tolerance gene of Chorispora tenella are as follows:

[0035] The upstream primer is 5'-CAAAAAAGCAGGCTCAGGGGATATCATGTCTTGCTGCA ATGGAAAG-3' (as shown in SEQ ID NO. 2);

[0036] The downstream primer is 5'-AAAGCTGGGTGCAGGGCGATATCACAATTACAAGGATC ACAC-3' (as shown in SEQ ID NO. 3).

[0037] The primer pair provided by the present application can be used to amplify the CnMT2 lanthanum tolerance gene of Chorispora tenella from Chorispora tenella, and can be used in the construction of an expression vector of the CnMT2 lanthanum tolerance gene of Chorispora tenella and the construction of a transgenic lanthanum-tolerant plant.

[0038] The present application provides an expression vector pEarlyGate101-CnMT2 of the CnMT2 lanthanum tolerance gene of Chorispora tenella, which comprises the nucleotide sequence shown in SEQ ID NO. 1.

[0039] Specifically, the construction of the expression vector pEarlyGate101-CnMT2 of the present application comprises the following steps:

[0040] (1) Taking Chorispora tenella cDNA as a template, the CnMT2 lanthanum tolerance gene of Chorispora tenella is amplified by PCR using the primer pair described in the above technical solution;

[0041] (2) The amplified CnMT2 lanthanum tolerance gene of Chorispora tenella is connected to the QBV3 vector.

[0042] According to the QBV3 vector information, a suitable enzyme cutting site EcoRV is selected, a gene F / R primer (MT2-F: CAAAAAAGCAGGCTCAGGGGATATCATGTCTTGCTGCAATGGAAAG; MT2-R: AAAGCTGGGTGCAGGGCGATATCACAATTACAAGGATCACAC) containing the EcoRV enzyme cutting site is designed, the vector resistance is Chl+, and the following method is used to obtain the QBV3-MT2 fragment: 2xGflxBuffer (25 μl), TKS Gflx DNA Polymerase (1 μl), Forward Primer (2 μl), Reverse Primer (2 μl), cDNA (1 μl), ddH2O (19.4 μl), DMSO (0.6 μl).

[0043] Restriction enzyme digestion was performed on QBV3 vector, and the enzyme digestion site was EcoRV. The enzyme digestion reaction system was 10x Cutsmart Buffer (5 μl), QBV3 (3000 ng / c), EcoRV (3 μl), RNase Free ddH2O (added to 50 μl). The enzyme digestion was performed in a 37℃ constant temperature incubator for 2 h. Agarose gel electrophoresis (125 v for 25 min) was performed, and the gel was recovered.

[0044] The target gene fragment was homologously recombined with the enzyme-digested vector and transformed into E. coli competent cells. The recombinant system was 5x CEII Buffer (2 μl), Exnase II (1 μl), QBV3 (50 ng / c), target gene fragment (35 ng / c), and ddH2O (added to 10 μl). Finally, Exnase II enzyme was added, and the mixture was gently mixed and centrifuged at low speed. The mixture was incubated at 37℃ for 30 min, and then placed on ice for 5 min. The DH5α competent cells were taken out from the -80℃ refrigerator and placed on ice. When the competent cells were semi-melted, the ligation product was added to the competent cells, and the mixture was gently mixed. The mixture was immediately placed on ice for 30 min, heated in a 42℃ water bath for 70 s, and then immediately placed on ice for 2 min. 600 μL of LB liquid medium without resistance was added, and the mixture was transferred to a shaker. The mixture was activated and recovered at 37℃ and 200 rpm for 1 h. 200 μL of bacterial solution was uniformly spread on LB solid medium containing Chl+ using a sterile spreader. The mixture was cultured in a 37℃ incubator overnight.

[0045] PCR was used to detect positive monoclonal strains. In a clean bench, single colony E. coli was randomly streaked on another new LB solid medium containing Chl+. The medium was cultured at 37℃. The number of streaked colonies was increased by 1-2 to prepare PCR mix. The PCR mix system for 1 bacterial solution was as follows: 2x PCR mix (10 μl), forward primer (0.25 μl), reverse primer (0.25 μl), ddH2O (9.5 μl), and DMSO (0.7 μl). The PCR reaction program was as follows: 94℃ for 5 min, 94℃ for 40 s, 54℃ for 30 s, 72℃ for 1 min, 35 cycles, 72℃ for 10 min, and then 10℃ storage. The PCR product was subjected to agarose gel electrophoresis, and 3 positive monoclonal colonies with target bands were randomly selected for sequencing. The plasmid with correct sequencing was transformed into E. coli DH5α, which was cultured in LB medium containing Chl+. Single colonies were selected and cultured, and the recombinant QBV3-CnMT2 plasmid DNA was extracted and stored at -20℃.

[0046] (3) Construction of pEarlyGate101-CnMT2 vector

[0047] GatewayTMLR reaction was used to construct pEarlyGate101-CnMT2 final vector, and the vector resistance was Kan, and the LR reaction system was: QBV3-CnMT2 (75 ng / c), pEarlyGate101 vector (90 ng / c), LRmix (0.5 μl), TE Buffer (supplemented to 4.5 μl), and the reaction was placed at room temperature for 1 h, 0.5 μL Proteinase K was added, mixed, 37℃ water bath for 10 min, placed on ice for 5 min, then transferred into E. coli competent cells according to the above method, and sent to the company for sequencing, and the recombinant vector pEarlyGate101-CnMT2 was obtained.

[0048] The expression vector pEarlyGate101-CnMT2 constructed in the application can be used for expressing the lanthanide tolerance regulatory protein coded by CnMT2, and can also be used for transduction into plants to construct transgenic lanthanide tolerance plants.

[0049] The application provides a lanthanide tolerance regulatory protein expressed by the lanthanide tolerance gene CnMT2 of Wedelia trilobata, and the amino acid sequence of the lanthanide tolerance regulatory protein is shown as SEQ ID NO. 10.

[0050] MSCCNGKCGCGSSCSCGSSCKGCGMYPDIEASTTATMIVDGVAPKKMYDDGSEGSFVAEGGHACKCGANCKCDPCNC

[0051] The lanthanide tolerance regulatory protein has 77 amino acid sequences. Through multi-sequence comparison with other plant MT2 proteins, it is shown that the CnMT2 protein has high homology with MTs of other plants. The N-terminal and C-terminal domains of the CnMT2 protein respectively contain 8 and 6 Cys residues, and are separated by an intermediate Cys-free interval.

[0052] The application also provides application of the lanthanide tolerance gene CnMT2 of Wedelia trilobata in cultivating transgenic plants. Preferably, the lanthanide tolerance gene CnMT2 of Wedelia trilobata is overexpressed in a plant body, and further preferably, the overexpression is achieved by transferring the expression vector pEarlyGate101-CnMT2 into the plant body by an agrobacterium-mediated method, so that a plant overexpressing the lanthanide tolerance gene CnMT2 of Wedelia trilobata is obtained.

[0053] Preferably, the agrobacterium-mediated method is a flower inflorescence dip method, which comprises the following steps:

[0054] (1) Take sequencing identified as positive Agrobacterium pEarlyGate101-CnMT2, if stored at -80℃, need to be streaked on LB solid medium (50 mg / L Kan + 50 mg / L Rif) activation. After the single colony, inoculated in 3 ml of the same containing double liquid LB medium with the small shake overnight. The turbid bacteria liquid according to 1:100 ratio into two containing LB continue to expand culture, 28℃, 180rmp avoid light shock culture to OD 600 = 0.8-1.5 between

[0055] (2) The bacteria liquid is put into 50 ml centrifuge tube, 5500r / min, centrifugal 15 min at room temperature, collect bacteria, discard the supernatant, and suspend the bacteria to OD 600 = 0.8-1.0 with infiltration medium (1 / 2MS + 50 g / L sucrose + 0.03% Silwet L-77). The infiltration medium is prepared and used immediately, without sterilization.

[0056] (3) Select the flowering period of Arabidopsis, the day before the inflorescence dip, the flower pot is completely immersed to keep enough humidity, which helps to improve the transformation efficiency. The flowers and silks that have completely opened are cut off, and the unopened flower buds are left for transformation. The inflorescence of Arabidopsis is completely immersed in the resuspended bacteria solution and shaken gently, and the excess bacteria solution on the leaves and stems is removed after 1 min with a clean absorbent paper. The infected Arabidopsis is wrapped with plastic wrap to keep the humidity, inverted on the tray, and cultured in the dark for 24 h before returning to normal growth conditions. In order to improve the transformation efficiency, the Arabidopsis can be infected again according to the above steps after 7 days. The infected Arabidopsis is prone to leaf yellowing due to the residual Agrobacterium, and the vitality is weak, so special attention should be paid to the diseases and pests, and the nutrient solution can be appropriately irrigated to promote growth. About 40 days or so, the yellow mature Arabidopsis silks can be collected, dried at room temperature for 5-7 days, and stored at 4℃.

[0057] In the present application, the plant body is preferably Arabidopsis or lanthanide-resistant plant.

[0058] The technical solutions provided by the present application are described in detail below in conjunction with examples, but they should not be understood as limiting the scope of protection of the present application.

[0059] Example 1

[0060] Cloning and sequence analysis of CnMT2 gene

[0061] The materials for expression analysis of CnMT2 gene in different tissues and organs of Chamaemelum nobile include: root, stem, leaf, and stem tip. The Chamaemelum nobile seedlings were treated with 800 μM La 3+The expression patterns of CnMT2 gene were analyzed after collecting the aboveground and underground parts of the plants at different times (0 h, 1 h, 3 h, 6 h, 12 h, 24 h, 48 h, 72 h). All samples were rapidly frozen in liquid nitrogen after collection and stored at -80°C for standby.

[0062] The Arabidopsis thaliana Columbia type (Col-0) wild type was preserved in the laboratory, and the culture conditions were 21°C, 16 h light / 8 h darkness, and 70% humidity.

[0063] The specific primers of CnMT2 gene were designed according to the previous obtained transcriptome data, as shown in Table 1. PCR amplification was performed according to the following system: 1 μl of cDNA template, 1 μl of upstream primer CnMT2-F and downstream primer CnMT2-R, 12.5 μl of PCR MIX (high fidelity), and ddH2O to 25 μl. The reaction program was 94°C for 5 min, 94°C for 30 s, 58°C for 1 min, 72°C for 30 s, 35 cycles, 72°C for 10 min, and then 4°C storage.

[0064] The characteristics of CnMT2 protein were analyzed by ProtParam. Similar protein sequences were searched and downloaded in NCBI, and multiple sequence alignment was performed by ClustalX, and the phylogenetic tree was constructed by the neighbor-joining method in MEGA7.0 software (bootstrap was set to 1000 times).

[0065] Table 1 Primer sequences used in the application

[0066]

[0067] The full length of CnMT2 gene is 234 bp, encoding 77 amino acids (Cenbank: OP785087). The theoretical isoelectric point of CnMT2 protein is 5.04, the molecular weight is 7.68 kDa, and the instability index is 62.8, which is an unstable protein. Multiple sequence comparison of MT2 proteins of different plants showed that CnMT2 has high homology with MTs of other plants. The N-terminal and C-terminal domains of CnMT2 protein contain 8 and 6 Cys residues, respectively, which are separated by an intermediate Cys-free interval, as shown in Figure 1 The phylogenetic tree of MTs of Cynara cardunculus and other 14 plants was constructed by the neighbor-joining method of MEGA6 software, as shown in Figure 2As shown in the diagram, the CnMT2 protein and MT proteins from Artemisia annua, Helianthus annuus, Tripoliumpannonicum, Lactuca sativa, and Ergieron canadensis cluster in one group, including both MT1 and MT2 proteins, indicating that MT1 and MT2 proteins are closely homologous.

[0068] Example 2

[0069] CnMT2 gene expression pattern analysis

[0070] Real-time quantitative PCR was used to analyze the CnMT2 gene at different tissues and time points. 3+ Expression patterns under treatment. The reaction was performed according to the instructions of the SYBR Premix Ex Taq™ (TAKARA) kit. The primers for the CnMT2 gene were CnMT2-qF and CnMT2-qR, and the primers for the internal control gene were TY-DN13135-C0-F and TY-DN13135-C0-R, as shown in Table 1.

[0071] PCR program: 50℃ for 2 min, 95℃ for 10 min, 95℃ for 15 s, 60℃ for 50 s (35 cycles), 72℃ for 2 min, with 3 replicates per sample. After the reaction, the reliability of the data was determined by analyzing the amplification curve and melting curve. Based on the obtained Ct values ​​of each sample, the expression levels of each tissue and organ were calculated using a 2-1... -△Ct Methods, different time-stress treatment expression patterns were adopted using 2 -△Ct△Ct The CnMT2 gene was relatively quantified using a method.

[0072] The expression level of the CnMT2 gene in various tissues and organs of *Chrysanthemum indicum* was analyzed using qRT-PCR. The transcriptional level of CnMT2 in roots and stems was significantly higher than that in leaves and stem tips, such as... Figure 3 As shown in the figure. Using *Chrysanthemum indicum* seedlings treated with 800 μM LaCl3 at different times as material, the transcriptional level of CnMT2 was detected to assess its induced expression. Time-series analysis showed that under 800 μM LaCl3 stress, CnMT2 expression in the aboveground parts (leaves and stems) continuously increased with increasing treatment time, decreased after 6 h of treatment, and peaked at 24 h, reaching 7.49 times the pre-treatment level. Unlike the expression pattern in stems and leaves, CnMT2 expression in roots showed a decreasing-increasing-decreasing-increasing trend with increasing treatment time, reaching its lowest value at 9 h of treatment. Furthermore, CnMT2 expression in aboveground tissues was strongly induced, and expression showed significant differences under different treatment times, such as... Figure 4As shown in the figure, this indicates that CnMT2 is highly expressed in roots, but the roots are sensitive to LaCl3 stress, and the expression level decreases significantly with prolonged stress. Meanwhile, the CnMT2 gene shows a significant increasing trend in the aboveground parts, suggesting its involvement in LaCl3 stress. 3+ Upward transport.

[0073] Example 3

[0074] CnMT2 gene transformation Arabidopsis thaliana

[0075] The plant expression vector pEarlyGate101-CnMT2 was constructed using the Gateway method and transformed into Agrobacterium EH105. Genetic transformation of Arabidopsis thaliana was then performed using the inflorescence immersion method. Transgenic lines that tested positive by PCR and RT-PCR in the T1 generation were harvested as single plants and sown in a solution containing 50 mg·L⁻¹ of [unspecified substance]. -1 The resistant seedlings obtained were placed on 1 / 2 MS medium plates containing kanamycin. These were the T2 generation transgenic lines, and selection continued until the T3 generation. Using the Arabidopsis thaliana Actin gene as an internal control, quantitative qPCR was used to detect the expression levels of CnMT2 in the roots and stems of the transgenic plants.

[0076] Three transgenic lines carrying the CnMT2 gene were obtained: L4, L7, and L10. The expression level of the CnMT2 gene in all transgenic lines was significantly higher than that in wild-type Arabidopsis thaliana, and the expression level of the CnMT2 gene in the roots was higher than that in the stems and leaves. Figure 5 As shown in the figure, the CnMT2 gene expression level was highest in the roots of the L7 line, which was 5.3 times that of the control. The CnMT2 gene expression level was highest in the stems and leaves of the L10 line, which was 5.1 times that of the control. Compared to other lines, the CnMT2 gene was highly expressed in both the roots and stems and leaves of the L10 line.

[0077] Example 4

[0078] Stress resistance analysis of transgenic Arabidopsis seeds

[0079] Wild-type and T3 transgenic Arabidopsis seeds were sterilized with 70% ethanol for 1 min, followed by sterilization with 1% NaClO solution for 3 min, and rinsed three times in sterile water. Seeds were sown on agar plates containing MS basal medium, 1% (w / v) sucrose, and 0.8% (w / v) agar, with filtered and sterilized 200 μm LaCl3, 5 mM H2O2, and 150 mM NaCl added. Lines sown on MS medium served as controls, with three replicates. Photographs were taken on day 14 after stress treatment, and the germination rates of transgenic and wild-type Arabidopsis were calculated.

[0080] Under 200 μm LaCl3 stress, germination of WT seeds was restricted, with a germination rate of 50%. Even after true leaves emerged, rooting was inhibited, while the germination rates of seeds from different transgenic lines ranged from 67% to 76.66%. Under stress of 150 mM NaCl and 3 mM H2O2, the germination rates of wild-type seeds were 32% and 64%, respectively, as shown in Table 2.

[0081] Table 2. Effects of LaCl3, H2O2, and NaCl on seed germination of wild-type and different transgenic Arabidopsis thaliana.

[0082]

[0083] Although the seeds of the transgenic plants were also affected, their germination rate was significantly higher than that of the wild type. Furthermore, the germination phenotypes of the three transgenic lines were very similar under different stresses, such as... Figure 6 As shown in the figure. This indicates that the CnMT2 gene improves the germination rate of transgenic Arabidopsis seeds under different stresses. Further investigation was conducted on the effects of LaCl3, NaCl, and H2O2 on the rooting of transgenic lines. On the control medium, there were no significant phenotypic differences between transgenic lines and WT plants. When the medium contained 200 μm LaCl3, 150 mM NaCl, and 3 mM H2O2, rooting growth in both WT and transgenic lines was inhibited. However, transgenic plants grew better than WT plants. Under 200 μm LaCl3 stress, WT plants almost failed to root, while the roots of the CnMT2 gene-transgenic lines were less affected by stress. On 150 mM NaCl medium, the roots of both WT and transgenic lines were inhibited. On 3 mM H2O2 medium, the root length of the CnMT2 transgenic lines was significantly longer than that of the wild type. Figure 7 As shown in the figure. The results indicate that expression of the CnMT2 gene in transgenic Arabidopsis thaliana enhances the early root system tolerance of seedlings to rare earth element lanthanum and oxidative stress.

[0084] Example 5

[0085] Analysis of stress resistance and physiological and biochemical indicators of transgenic Arabidopsis seedlings

[0086] To verify the stress resistance of mature plants, wild-type and different transgenic Arabidopsis seeds were sown on MS plants, germinated, and then transplanted into soil. Two-week-old wild-type and transgenic Arabidopsis seedlings were irrigated with 200 μM LaCl3 and 400 μM LaCl3 solutions, respectively, every 4 days for 12 days, with three replicates. On day 12 after treatment, the survival rate was calculated, and the plant roots were rinsed clean. The fresh weight of the aboveground stems and leaves and the underground roots were measured.

[0087] The results show that LaCl3 has an inhibitory effect on the growth of Arabidopsis thaliana after 12 days of treatment, and shows obvious stress characteristics such as leaf yellowing, purple, dwarf plants, and early flowering. However, with the extension of stress time, the transgenic Arabidopsis thaliana with CnMT2 gene shows better growth potential, with more new green true leaves, as shown in Fig. 1. Figure 8

[0088] At the same time, the activities of four kinds of antioxidant enzymes such as malondialdehyde (MAD), catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) were determined. The results show that after 12 days of treatment with 200 μM LaCl3, the activities of CAT and POD enzymes of the transgenic Arabidopsis thaliana are significantly higher than those of the wild type Arabidopsis thaliana, the MAD enzyme activity of L10 strain is significantly increased, and the activities of other strains have no obvious change, and the activity of SOD enzyme is significantly reduced, as shown in Fig. 2. Figure 9

[0089] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.​​

Claims

1. The use of overexpression of CnMT2, a lanthanide-tolerant gene from Chamaemelum nobile, in breeding transgenic plants, characterized in that, The nucleotide sequence of the lanthanum-tolerant gene CnMT2 of the Wedelia trilobata is shown as SEQ ID NO.

1. The lanthanum-tolerant gene CnMT2 of the Wedelia trilobata is used to improve the resistance of plants to lanthanum and the antioxidant capacity. The plant is Arabidopsis thaliana.

2. Use according to claim 1, characterized in that, The vector for overexpressing the lanthanum-tolerant gene CnMT2 of the Wedelia trilobata is pEarlyGate101-CnMT2.

3. Use according to claim 2, characterized in that, The pEarlyGate101-CnMT2 is introduced into a plant body by an Agrobacterium-mediated method to obtain a plant overexpressing the lanthanum-tolerant gene CnMT2 of the Wedelia trilobata.

4. Use according to claim 3, characterized in that, The Agrobacterium-mediated method is specifically a floral dip method.

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