The Induction Of Hairy Roots With Overexpression Of LeEIL-1 And Regulatory Effect On Biosynthesis Of Shikonin By LeEIL-1 In Lithospermum Erythrorhizon

Lithospermum erythrorhizons are used as medicinal plants in our country. Shikonin and its derivatives, important secondary metabolites synthesized in the roots of Lithospermum erythrorhizon, attract much attention for their anti-bacterial, anti-inflammatory, anti-tumor, anti-HIV and other important medicinal activities. In few past decades, the pathway of shikonin biosynthesis has been clarified, and key enzymes and genes related to shikonin biosynthesis were isolated, cloned and characterized. It has been reported that the pathway of shikonin biosynthesis was been regulated by many kinds of impact factors, such as plant hormones (ethylene and jasmonic acid, etc), light signals, inorganic ions, and fungal elicitors and so on. However, the regulatory mechanism of these factors remains unclear.Ethylene, an important plant hormone, plays a key role in regulating shikonin biosynthesis. The key transcription factor EIN3/EIL-1 (Ethylene insensitive 3/ Ethylene insensitive like-1) of ethylene signal transduction pathway also possibly participates in the regulation of shikonin biosynthesis by light signal. Therefore, we guess that EIN3/EIL-1 may play an important regulatory role in shikonin biosynthesis pathway.In this study, we induced the transgenic hairy roots of L. erythrorhizon by overexpressing LeEIL-1 gene and investigated the regulatory effect on shikonin formation. Firstly, we constructed the plant overexpression vector LeEIL-1-PBU21-eGFP. Then the vector was transformed into Agrobacterium rhizogenes strain ATCC15834 (American type culture collection 15834) and the transgenic hairy roots were induced by infecting the seedling of L. erythrorhizon. Finally, we measured the shikonin content and analyzed the expression pattern of genes related to shikonin biosynthesis. The results showed that shikonin content increased with time when the hairy roots cultured in M9 liquid medium in the dark, and a large amount of shikonin was accumulated in the 6-day cultured hairy roots. Shikonin content of the LeEIL-1 transgenic hairy roots was significantly higher than that of the wild type hairy roots (WT, without LeEIL-1 gene), suggesting that overexpressing LeEIL-1 gene could enhance the formation of shikonin. Gene expression result showed that the expression level of LeEIL-1 gene of the LeEIL-1 transgenic hairy roots was higher than that of WT hairy roots, that can tell us improving LeEIL-1 gene expression may be the important reason of enhancing the formation of shikonin.These results lay the foundation for further investigation of molecular mechanism of regulation of shikonin biosynthesis by ethylene, and provide a new idea for increasing shikonin content.