Study Of Efficient Transformation System And NtLTP1 Effect On The Artemisinin Content In Artemisia Annua L.

Malaria is a disease of serious damage to human,especially in developing countries.In the world,about one million people die from malariaeach every year.Artemisinin,a sesquiterpene lactone endoperoxide,is currently the most important antimalarial agent,it extracted from A.annua L.The content of artemisinin in Artemisia annua is very low,only about 0.1%-0.5%of the dry weight of A.annua.At present,the production of artemisinin is still based on the cultivation of A.annua.And this is far from enough to meet the need of artemisinin in medicine.So,improving the content of artemisinin has become an important research topic in A.annua.As the short of breeding resources,it is difficult to get the varieties with high artemisinin in A.annua in a short period of time using routine breeding.However,transgenic technology provides an efficient breeding technology to get it in short time.NtLTP1 gene code the membrane transport protein--NtLTP1.It was from tobacco.NtLTP1 has the function of transporting sesquiterpene lactone endoperoxide.We have established the high efficient regeneration system and efficient genetic transformation system in A.annua,and we used the transgenic technology transferred NtLTP1 gene into the plant,obtained the transgenic plants with high artemisinin content,increased 1.5 times than wild type.The results are as follows:1.Efficient Agrobacterium-mediated transformation of A.annua.I used leaves as explant materials,using the method of orthogonal experiments and single factor experiments,explore 6-BA,NAA,AgN03 influence of artemisinin explant differentiation;NAA and sugar to the influence of the explant rooting;Infection time,bacteria liquid concentration,incubation time,the influence of the transformation system;Explant types(leaves and young inflorescence)the influence of the transformation system.The best genetic transformation system is that:immature inflorescence as explant,Agrobacterium strain EHA105,OD600 is 0.2,infected time is 15 minutes,co-culture time is 2 days.Co-culture medium is MS + 0.5 mg/L 6-BA + 0.3 mg/LNAA +30 g/Lsugar +6.5 g/L agar;resistance screening medium:MS + 0.5 mg/L 6-BA + 0.3 mg/LNAA + 300 mg/L carboxy benzyl penicillin + 20 mg/L kanamycin + 30 g/L sugar + 6.5 g/L agar,successive transfer culture once three weeks,until the explant differentiation form bud seedling;screening of rooting medium:MS + 0.2 mg/LNA + 300 mg/L carboxy benzyl penicillin + 10 mg/L kanamycin 30 g/L A + sugar + 6.5 g/L agar.This experiment to obtain the highest conversion efficiency is 17.2%,With the same conditions compared with leaf as explant,the transformation efficiency improve 2 times.2.NtLTP1 enhanceed the artemisinin content in A.annua L.Seven transgenic plants with NtLTP1 gene were obtained by Agrobacterium-mediated transformation using leaves and identificated by PCR,RT-qPCR,HPLC was used for analysising of artemisinin content of transgenic plants and wild type.Six out of seven transgenic plants with increased artemisinin content compared with wild type.It suggested the expression of NtLTP1 gene can enhanced the artemisinin content in A.annua.The highest Artemisinin content in transgenic plants was 21.2089�0.57 mg/g,it is 2.47 times higher than the wild type.