Cloning, Expression And Functional Analysis Of LeTCP4Gene In Lithospermum Erythrorhizon

Shikonin and its derivatives, important secondary metabolites with anti-tumor, anti-bacterial, anti-inflammatory activities and other important medicinal value, accumulate in the roots of Boraginaceae plants. Many attentions have been focused on the regulatory mechanism of shikonin and its derivatives biosynthesis. Currently, plant tissue culture technology has been applied to the commercial production of shikonin and its derivatives by a two-phase culture system. Our previous studies showed that the biosynthesis of JAs was rapidly induced after the Lithospermum cells were transferred from B5solid medium to M9liquid medium. These hormones play important roles in regulating stress signaling, plant stress tolerance and secondary metabolism. In plants, miRNAs mostly regulate other regulators, the DNA-binding transcription factors. The downstream events regulated by five TCP (TEOSINTEBRANCHED/CYCLOIDEA/PCF) transcription factors that are controlled by the microRNA miR319have been studied in Arabidopsis thaliana. The miR319-regulated TCPs were previously known to be important for limiting the growth of leaves. By applying a combination of genome-wide, biochemical, and genetic studies, new TCP targets that included enzymes responsible for the synthesis of the hormone jasmonic acid were identified. Analysis of leaf extracts from plants with increased activity of miR319confirmed that altered expression of the biosynthetic genes led to changed jasmonic acid levels. These plants also showed that an altered senescence behavior became normal again when the plants were treated with jasmonate. In Jaw mutant, many TCP genes were repressed by the overexpression of miR319.To elucidate the function of TCP transcription factor in the regulation of shikonin biosynthesis, part of the LeTCP4gene was amplified using primers designed according to the conserved sequence in TCP gene family and the full length of LeTCP4was then obtained by RACE method. Bioinformatics analysis results showed that it belonged to the CIN subgroup of Class II TCP family. The transgenic plants of LeTCP4in jaw mutant exhibited expanding leaves and smooth siliques surface, which showed that LeTCP4gene can restore the mutant phenotype nearly to wild type. Overexpression of LeTCP2promoted the JAs synthesis and signal transduction-related gene expression, and also regulated the expression of hormones such as IAA, ABA and GA. These results implied that LeTCP2gene may play a vital role in the processes of MJ synthesis and the regulation of shikonin production.