The Cloning Of AaGL2in Artemisia Annua L. And Inheritance Analysis Of Transgenic A. Annua

In order to study the molecular mechanism of trichome development inArtemisia annua L. and the inheritance of transgenic A. annua, for thepurpose of obtaining higher artemisinin content transgenic A. annua. AnHD-ZIP IV transcription factor was cloned from A. annua leaf cDNA byhomologous cloning strategy and RACE. It was named AaGL2for its highamino acids sequence similarity to GL2in Arabidopsis thaliana. Thededuced protein structure of AaGL2and phylogenetic tree were analyzedusing bioinformatics softwares. The expression of AaGL2in different tissuesof A.annua and that under the treatment of methyl jasmonate (MeJA) weredetected by Quantitative Real-Time PCR. The subcellar localization ofAaGL2protein was detected by fluorescent fusion protein and tobaccotransient expression system. Its capacity to activate transcription was detectedby yeast hybrid system. The copy number of AaGL2was detected by Southern blotting. The results showed that AaGL2was the closest to HAHR1from Helianthus annuus in the evolutionary relationship, and the secondclosest to GL2from Arabidopsis thaliana. AaGL2was mainly expressed instem and ten days-old bud. AaGL2was also very sensitive to MeJA. TheAaGL2protein was localized to nucleus and did not have the capacity toactivate transcription itself. AaGL2had at least two copies in the A. annuagenome.The inheritance of CYP71AV1and CPR genes in the progenies ofprevious generated transgenic A. annua plants (GYR) in which CYP71AV1and CPR were over-expressed was also studied. Southern blotting andQuantitative Real-Time PCR methods were used to identify the copy numberof transgenes and expression stability of the introduced genes in the T2generation plants respectively. Segregation ratio of the introduced genes inT2generation plants was analyzed by detecting the CYP71AV1gene by PCR.Artemisinin content and key agronomic trait (biomass) were also analyzed.The results showed that although the CYP71AV1and CPR genes can beinherited into the progenies and were over-expressed in the whole, thesegregation ratio of the introduced genes were lower than the expectedtheoretical ratio. There exists some copy number variation in the transgenicprogenies. The highest artemisinin content in transgenic progenies was46.9% higher than that of the control. No significant difference was observedregarding the plant fresh weight and leaf dry weight between transgenic linesand wild type control lines.In sum, this study showed that AaGL2was a transcription factor whichbelongs to the HD-ZIP IV family. It was possibly involved in the A. annuatrichome development regulation which needs further investigation. Theintroduced genes can be inherited into following generations of transgenic A.annua lines. This research lays the foundation to explore the regulatorymechanism of trichome development and the pratical application oftransgenic A. annua.