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Peppermint is a herbaceous plant of the genus Peppermint in the Lamiaceae family, which is widely cultivated as a medicinal and edible homologous plant, in terms of medicinal use, peppermint can be used for common clinical diseases such as wind, heat and cold, wind temperature, headache, eye redness, measles, chest and flank swelling and stuffiness.
In terms of consumption, peppermint can be used as tea, spice, fresh vegetables, etc., indicating that peppermint has great research and development value, and jasmonic acid is an important natural plant endogenous hormone, which is widely involved in plant growth and development and stress response.
JAZ proteins are key repressors in the JA signaling pathway, and as a member of the plant-specific TIFY superfamily, JAZs proteins contain a conserved TIFY domain at the N-terminus and a Jas, also known as the CCT_2 domain at the C-terminus.
At the same time, the TIFY domain can mediate the interaction between JAZs proteins to form homologous or heterodimers, and the functions of different JAZs genes are redundant or differentiated.
Materials and methods of the experiment
The peppermint used in the experiment was M. canadensis, and the tobacco used in the subcellular localization experiment was N. benthamiana, and all the materials were planted in a light incubator, 23 °C, 16 hours of light/8 hours of darkness, and grown under normal conditions for 3~5 weeks for later use.
Collect the young 2-week-old mint leaves, place them in liquid nitrogen, and then use a mortar to grind them thoroughly into a powder, and extract the total RNA from the leaves according to the instructions of the RNA extraction kit.
Follow the steps of the reverse transcription kit to synthesize the first-strand cDNA and identify the McJAZ8 gene sequence based on the mint transcriptome data.
The full-length cloning primer McJAZ8-F:5'-ATGGCTTCCTTTGAGAACGTC-3' was used to clonate the McJAZ8 gene. McJAZ8-R: 5'-CTACCTGTCTCGTCTCTCTTCTCC-3', followed by PCR amplification of the candidate McJAZ8 gene.
The PCR amplification system was as follows: 1 μL of 5× cDNA template, 2 μL of ErimeSTAR®DNA polymerase, 5 μL of 5× PrimeSTARDNA polymerase buffer, 0.5 μL of forward and reverse primers, 16 μL of sterile ddH2O, for a total volume of 25 μL.
The amplification program was as follows: pre-denaturation at 95°C for 10 min, denaturation at 95 °C for 15 s, annealing at the specified temperature for 20 s, extension at 72 °C for 45 s, 34 cycles, 72 °C for 10 min, 4 °C for 1 min.
Subsequently, DNA bands were identified and isolated using a 1% agarose gel, and the PCR product of the McJAZ8 gene was recovered using a DNA purification kit and stored at -20 °C for later use.
The purified PCR product was ligated to the pClone007 vector, and the ligation product was transformed into E. coli DH5α competency, monoclonal monoclonal was selected to expand the culture, and the correctness of the McJAZ8 gene sequence was confirmed by bacterial solution PCR.
Analysis of experimental results
According to the steps of the RNA extraction kit, the total RNA of peppermint leaves was extracted, and the RNA bands were clearly visible and no degradation was detected by agarose gel electrophoresis.
Moreover, the ratio of A260nm/A280nm and A260nm/A230nm was 1.9~2.0, and the nucleotide absorption curve showed a single peak, indicating that the total RNA quality was good and could be used for follow-up experiments.
The above RNA was reverse transcribed to obtain cDNA, and the full-length CDS sequence of McJAZ8 was amplified by RT-PCR, and the results of agarose gel electrophoresis showed that there was a clearly visible PCR band at 500~750 bp.
Subsequently, the target PCR product was constructed into the pClone007 vector, and the positive recombinant was sequenced to indicate that the sequence was McJAZ8 gene.
The coding sequence CDS of peppermint McJAZ8 gene was 543 bp, and its CDS sequence was converted into amino acid sequence to predict the physicochemical properties of McJAZ8 protein sequence.
The results showed that the McJAZ8 protein sequence contained 180 amino acids, its molecular weight was 18.96 kDa, the theoretical isoelectric point was 6.30, and the protein instability coefficient was 55.31, which was greater than the limit of 40, indicating that the protein was an unstable protein.
The results showed that the secondary structure of McJAZ8 protein was analyzed, and the results showed that the α helix accounted for 24.44%, the β fold accounted for 6.11%, the extended chain accounted for 10.5% and the irregular coil accounted for 58.89%, and the tertiary structure of McJAZ8 protein was analyzed.
The identity of the protein with the template sequence was 83.43%, and the quality of the model was estimated to be 0.8, which was consistent with the prediction of secondary structure, and contained four secondary structures: α helix, β fold, extended chain and random coil, among which random coil was the main structure.
In order to identify the similarity between the amino acid sequences of McJAZ8 protein and other JAZs proteins of other species, 12 JAZs proteins of Arabidopsis thaliana, 15 rice, 9 Artemisia annua and 13 spruce were obtained, and the amino acid sequences were compared.
All JAZs proteins contain highly conserved TIFY and Jas domains, and the amino acid sequences within the conserved domains are highly conserved, suggesting that McJAZ8 may be involved in JA signaling.
In addition, in order to understand the evolutionary relationship between McJAZ8 and other known species of JAZ proteins, the rootless phylogenetic tree of McJAZ8 protein and the above-mentioned different species JAZs proteins was constructed using MEGA software.
结果显示，McJAZ8与拟南芥AtJAZ1、AtJAZ2、黄花蒿AaJAZ4和AaJAZ5蛋白聚为一支，同源关系较近。
In order to determine the subcellular localization of McJAZ8 protein, the WoLFPSORT online tool was used to predict the subcellular localization of McJAZ8 protein, and the prediction results showed that McJAZ8 protein may be localized in the nucleus, cytoplasm and mitochondria.
构建McJAZ8基因,融合绿色荧光蛋白GFP的重组表达载体McJAZ8-GFP,将GFP空、McJAZ8-GFP、核定位载体SV40-mCherry分别转化农杆菌GV3101。
Equal volumes of Agrobacterium resuspension containing different expression vectors were mixed and injected into the lower epidermis of tobacco leaves for transient co-expression.
Tobacco leaf cells containing control GFP empty vector or McJAZ8-GFP fusion vector were observed to have green fluorescence signals in both the nucleus and cytoplasm, and the green fluorescence in the nucleus overlapped with the red fluorescence.
In conclusion, the results showed that McJAZ8 protein was localized in the nucleus and cytoplasm.
In order to explore whether McJAZ8 can form homodimers with itself or other JAZs proteins in peppermint, different plasmids of BD-McJAZs and AD-McJAZ8, or BD-McJAZ8 and AD-McJAZs, were transformed into yeast AH109 by yeast two-hybrid experiments, and the interaction between them was analyzed.
所有转化的酵母组合均能在对照培养基DDO上正常生长,然而,只有AD-McJAZ8分别与BD-McJAZ1、BD-McJAZ3、BD-McJAZ4、BD-McJAZ5、BD-McJAZ8共转化的酵母。
或者BD-McJAZ8分别与AD-McJAZ1、AD-McJAZ4、AD-McJAZ5、AD-McJAZ8共转化的酵母,能在选择培养基QDO上正常生长。
表明McJAZ8能与自身,或McJAZ1、McJAZ3、McJAZ4、McJAZ5互作。
Discussion of experimental results
The results showed that jasmonic acid signal suppressor JAZ gene was widely present in plants, and JAZ protein inhibited the conduction of jasmonic acid signaling by binding to downstream transcription factors, thereby regulating plant growth and development, disease resistance response and stress response.
At present, JAZs genes have been cloned in Arabidopsis thaliana, rice, Fertia, wheat, Artemisia annua, Salvia miltiorrhiza and other species, however, the research on JAZs genes in peppermint is still in its infancy.
Based on the previous mint transcriptome data, a complete JAZ gene named McJAZ8 was cloned from peppermint, and the gene sequence analysis showed that the coding sequence length of McJAZ8 gene was 543 bp, which was similar to the coding sequence length of Arabidopsis AtJAZs gene.
In addition, the pI value of McJAZ8 protein is 6.30, which is due to the high proportion of acidic amino acids in the amino acid sequence of McJAZ8 protein, and this phenomenon is prevalent in JAZ protein of other plants.
Amino acid sequence alignment analysis showed that although the amino acid similarity between McJAZ8 and other plant JAZs proteins was low, McJAZ8 and other plant JAZ proteins contained a conserved N-terminal TIFY domain and a C-terminal Jas domain, indicating the conserved evolutionary evolution of TIFY and Jas.
It was also found that the TIFY domain played a dual role in regulating the interaction between homologous or heterologous JAZs proteins, as well as the interaction between JAZs proteins and NINJA proteins to recruit TPL/TPRs.
The Jas domain plays an important role in the interaction between JAZs protein and the jasmonic acid receptor F-box protein Coronatineinsensitive1 and various transcription factors, and participates in the regulation of subcellular localization of JAZ protein.
The results of subcellular localization and yeast two-hybrid showed that McJAZ8 protein was localized to the nucleus and could interact with other McJAZs proteins to form homologous or heterodimers.
Suggesting that similar to JAZ protein in other plants, the McJAZ8 protein in peppermint may play an important role in JA signaling response and regulation of JA-mediated downstream gene expression, and the tissue expression pattern of the target gene is closely related to gene function.
The expression of different JAZs genes in different tissues of different plants has certain constitutiveness and differences, and the expression of JAZs genes in different tissues of plants is closely related to plant growth and development.
McJAZ8 gene was expressed in different tissues of peppermint, but its expression level was different in different tissues, suggesting that McJAZ8 gene may play an important role in regulating the growth and development of different tissues of peppermint.
The JAZs genes of Arabidopsis thaliana and rice play an important regulatory role in the development of flowers and stamens, and the expression of McJAZ8 gene in leaves was higher than that in all other tissues, suggesting that McJAZ8 gene may have important regulatory functions in leaf development.
In different plants, JA induced fluctuations in the transcriptional abundance of JAZs genes, and the changes in the transcription level of JAZs genes were closely related to the regulatory functions of JAZ genes in JA-mediated plant development, defense, and special metabolite synthesis.
Compared with the control group, the McJAZ8 gene was significantly up-regulated in the leaves and roots of peppermint after MeJA treatment, further suggesting that the gene may be involved in JA signaling.
For example, in wheat, rice, tea and cabbage, drought, salt and low temperature induced the expression of JAZs genes to varying degrees, and the up-regulated expression of these JAZs genes was related to the salt tolerance and drought tolerance of plants.
In this study, it was found that the expression of McJAZ8 gene was up-regulated in the leaves and roots of peppermint treated with drought and NaCl stress, and it was speculated that the McJAZ8 gene may play an important role in regulating the response of peppermint to drought and salt stress.
Previous studies have shown that JA treatment improved plant resistance to exogenous cadmium and copper ion stresses, and found that CdCl2, CuCl2 and AlCl3 treatments induced the expression of McJAZ8 gene in leaves and roots of peppermint, suggesting that McJAZ8 may regulate the response of peppermint to different metal ion stresses.
In addition, JA treatment up-regulated the expression of the abiotic stress response gene McJAZ8, suggesting that peppermint McJAZ8 may regulate plant responses to different stresses through the JA signaling pathway.
The McJAZ8 gene was cloned from peppermint, which encoded 180 amino acids with a length of 543 bp, a molecular weight of 18.96 kDa, a theoretical isoelectric point of 6.30, a TIFY and Jas conserved domains, and high homology with Arabidopsis AtJAZ1 and AtJAZ2.
McJAZ8蛋白定位于细胞核、细胞质,能与自身或McJAZ1、McJAZ3、McJAZ4、McJAZ5互作形成同源或异源二聚体。
It was widely expressed in different tissues of peppermint, with the highest expression level in young leaves, and the expression of McJAZ8 gene in leaf order fluctuated, with the highest expression level in the fourth leaf and the lowest expression level in the second leaf.
However, the responses to MeJA, drought, salt, CdCl2, CuCl2 and AlCl3 treatments in the leaves and roots of peppermint suggested, suggesting that McJAZ8 may play an important role in regulating JA signaling in peppermint and regulating menthol response to abiotic stress.