| Dryopteris fragrans(L.) Schott, a deciduous perennial fern belonging to the family Dryopteridaceae, is widely distributed throughout the Heilongjiang Province of China, especially the Wudalianchi Region. The unique environment and growth characteristics of D. fragrans contribute to its unique medicinal value. Infusions and decoctions prepared from the aerial parts of D. fragrans have been used in traditional folk medicine to treat arthritis and various dermatoses such as psoriasis, acne, erythra, and dermatitis. International research has demonstrated that D.fragrans contains effective anthelmintic, antitumor, antibacterial, antioxidant, and antiviral compounds. Researchers have previously focused on morphological and anatomical observations,extraction and separation of chemical compounds, and the pharmacological activity of D. fragrans,whereas there have been few studies that have focused on the key enzyme genes involved in secondary metabolic pathways. Accordingly, it is important to determine appropriate temperature and light stress conditions that match the special growth environment of D. fragrans. D. fragrans produces many of its active secondary compounds in reaction to such stresses, and thus this step is necessary to elucidate molecular regulation mechanisms and to effectively protect wild D. fragrans populations while sustainably harvesting plants for their active ingredients.In the plant, including secondary metabolites lignin and flavonoids are from phenylpropanoid metabolic pathways generated by these secondary metabolites not only on the plant itself has an important role, but also on human health has important role, therefore, benzene, propane metabolic pathways and pathway key enzyme has become a hot research. The key point in this metabolic pathway, 4-coumaric acid coenzyme A ligase(4CL) located on the main route to the branch transition pathways, control goes to a different branch of the compound is metabolized Phenylpropanes, produce different secondary metabolites and found genes belong 4CL gene family form, 4CL gene can control different metabolic pathways in different directions, the researchers4 CL gene family members are important to the regulation of the accumulation of secondarymetabolites and secondary metabolites, but also for a wide range of research and application of secondary metabolites fragrant Dryopteris lay a solid foundation.One study examining the effect of temperature and light on D. fragrans transcription determined that the phenylpropanoid metabolism pathway is an important metabolic pathway in the species, as it is in angiosperms. The 4CL enzymes have been studied extensively in many species of angiosperms, gymnosperms, and mosses; however, those of ferns remain poorly characterized. This study combined RNA-Seq analysis results in D. fragrans, specifically focusing on 4CL genes. The main results obtained are as follows:1. Four genes(Unigene15437_All, Unigene18514_All, Unigene21777_All, and Unigene32945_All) were obtained from a library constructed from D. fragrans exposed to different temperatures and light stresses. Sequence comparison revealed that these four genes were highly consistent with known plant 4CL genes and were hence used as core fragments for subsequent experiments.2. RACE was used to obtain and amplify four full-length fragments, from which four full-length genes, Unigene15437_All, Unigene18514_All, Unigene21777_All, and Unigene32945_All were identified, which were named Df4CL1, Df4CL2, Df4CL3, and Df4CL4,respectively. Further analysis of their predicted amino acid sequences revealed that the four genes contain two highly conserved peptide motifs, Box I(SSGTTGLPKGV) and Box II(GEICIRG), as well as substrate binding regions Sbd I and Sbd II, which also existed in other 4CL genes. Nucleic acid sequence analyses determined a sequence identity of 66.00% among these four genes, which contain 539, 565, 552, and 573 amino acids, respectively. Further sequence analysis revealed a56.59% shared identity among these four predicted amino acid sequences, indicating that the four genes belong to the same family.3. Cloned 4CL gene(Df4CL) sequences from D. fragrans were analyzed online with the programs CHIPS, CUSP, and Codon W and then compared with sequences from E. coli, yeast,Arabidopsis thaliana, and Nicotiana tobacum. The codon usage bias of Df4 CL was characterized to help select appropriate gene expression assay systems. Df4 CL exhibited no bias among synonymous codons at the third codon position, and Physcomitrella patens exhibited consistent codon-use characteristics. The differences in codon usage frequency between Df4 CL and Arabidopsis thaliana were less than those between Df4 CL and 4CL sequences from Nicotiana tobacum, E. coli, or yeast. Gene codon usage bias cluster analysis of 4CL sequences placed D.fragrans, Physcomitrella patens, and Arabidopsis thaliana into one group. Therefore, the Arabidopsis thaliana expression system was inferred to be more suitable for assaying 4CL expression in D. fragrans.4. Characteristics of 4CL proteins in D. fragrans. Four 4CL proteins in D. fragrans were inferred to be amphoteric proteins. DF4CL2 was found to lack a transmembrane region, whileDF4CL1, DF4CL3, and DF4CL4 each had two transmembrane domains. Four 4CL proteins had no signal peptides. Additionally, the three-dimensional structure was inferred for each 4CL protein. In future studies on the structure and function of 4CL proteins, these findings will provide a theoretical reference basis as well as help indicate the mechanism of action of these proteins in plants, thus suggesting gene expression regulation mechanisms.5. There was a positive correlation between Df4 CL transcript expression levels and both total flavonoids and total lignin content across tissues. Overall, 4CL genes were highly expressed in the tissues in which flavonoids and lignin were most concentrated. This demonstrates that the 4CL genes cloned from D. fragrans are involved in the biosynthesis of flavonoids and lignin.6. Environmental stress, such as temperature, induced 4CL expression. Under both low temperature(4°C) and high temperature(35°C) conditions, the expression of four genes decreased after initially increasing. The content of both flavonoids and lignin reflected relative expression of4 CL, but the peak values were not the same. In general, temperature-stress-induced expression changes in Df4 CL could change flavonoid and lignin content, but increases or decreases in total flavonoids and total lignin lagged behind 4CL expression changes. In addition to affecting gene expression, the temperature may also affect enzyme activity in the synthesis of substances,resulting in the observed changes in content.7. UV radiation can affect 4CL expression and hence the content of flavonoids and lignin.Under UV irradiation, Df4CL3 and Df4CL4 transcript levels increased significantly, especially that of Df4CL3. Flavonoids increased significantly, while lignin increased gently. In general, under UV irradiation, plants protect their tissues with increased flavonoid levels. This is consistent with the experimental results obtained in this study.8. Of the Df4 CL genes identified, only Df4CL2 was expressed in prokaryotes, though at a low level; furthermore, the codons of the prokaryotic Df4 CL gene needed to be optimized.9. The plant expression vector p BI121-Df4 CL was transformed into wild-type Arabidopsis via the floral dip method. PCR was used to detect Df4 CL transgenic plants and obtain homozygous transgenic plants. Subsequently, the flavonoid and lignin contents were assayed. The transgenic plants contained significantly higher levels of flavonoids and lignin than those of the wild type. |
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