Effects Of Overexpression Of Squalene Synthase Gene (TaSS) On Taraxasterol In Taraxacum Antungense Kitag.

Taraxasterol has anti-mastitis and anti-vascular activity.The content of taraxasterol in taraxacum plant is extremely low,so the enhancement of taraxasterol in taraxacum plant is research emphasis.In this study,the variation on triterpenoids and key enzyme genes expression which related to triterpenoids biosynthesis pathway under the Me JA treatment were analyzed.The squalene synthase(TaSS),which catalyzes the first key enzyme gene on triterpenoids biosynthesis pathway was cloned and transgenic dandelion strains which over-expressed the TaSS gene plants were obtained using Agrobecteria mediated transformation.Preliminarily explore of the influence of TaSS gene on taraxasterol content in Taraxacum antungense Kitag.Here,the promoter of TaSS was cloned by Genome Walking PCR and the cis-elements was predicted to investigate the expression patterns of TaSS.In this study,Ta MYC2 was isolated from c DNA which is a key regulator in jasmonate signal transduction and transgenic hairy roots which over-expressed the Ta MYC2 were obtained.Preliminarily explore of the influence of Ta MYC2 gene on taraxasterol content and TaSS expression.Main results as follows:1.The expression of the key genes of taraxasterol biosynthesis pathway was induced by Me JA was analyzed by real-time quantitative PCR.The results showed that Ta FPS1,Ta FPS2,Ta HMGR3,Ta MYC2,Ta MVD,Ta HDR and TaSS were induced by Me JA in the early stage of the treatment,and was inhibited in the later stage.The content of total triterpenoids was significantly higher than that of the control,and reached the highest yield after treatment at 3 h,indicating that Me JA can elevate total triterpenoids content by up-regulate the expression level of triterpenoid biosynthesis-related genes.2.Based on the T.antungense Kitag.transcriptome data,the full coding region of the TaSS gene was successfully cloned,and the TaSS overexpression vector was constructed by double enzyme digestion method.The TaSS gene was overexpressed in T.antungense.by Agrobacterium tumefaciens-mediated method,and two transgenic lines OE-1 and OE-2 were obtained.The expression of TaSS gene in transgenic lines was up-regulated by 12.86 and 7.04 folds,respectively.The content of taraxasterol was also up-regulated in the transgenic lines.The results showed that overexpression of TaSS gene could significantly increase the taraxasterol content,indicating that TaSS is a key gene in the biosynthesis pathway.3.The promoter of TaSS gene was successfully cloned by chromosome walking method.The cis-acting element analysis of TaSS gene promoter was carried out by PLANTCARE website.The results showed that there were mechanical damage response elements and jasmonate acid response element,auxin response element(,abscisic acid response element,photoresponsive element,salicylic acid response element,MYB recognition site,CAAT-box and TATA-box,etc.These results indicate that the TaSS gene can be induced by a variety of hormone and mechanical damage.4.The full coding region of the Ta MYC2 gene was cloned,and the Ta MYC2 overexpression vector was constructed by Infusion method.The expression of Ta MYC2 and TaSS genes were up-regulated in transgenic hairy roots which overpressed Ta MYC2,and the content of taraxastetol was up-regulated in transgenic hairy roots.The results indicated that the Ta MYC2 gene may increase the taraxasterol content by regulating the expression of TaSS gene.