Transcriptome Analysis Of Artemisia Annua From Different Habitats And Application Of CRISPR/Cas9 In Artemisia Annua

Artemisia annua?scientific name:Artemisia annua Linn.?,compositae,Artemisia,annual herbs,widely distributed in all parts of China,mainly in guangxi,sichuan,hunan and other southern regions of China a large number of cultivation.Artemisia annua is the base plant of the traditional Chinese medicine Artemisia annua and the only natural plant source of artemisinin.Artemisinin,a new sesquiterpene lactone with oxygen bridge structure,is currently the best therapeutic agent against plasmodium falciparum resistant and pathogenic strains of cerebral malaria,saving millions of lives.China has an absolute resource advantage in the supply of artemisinin raw materials and is a supplier of artemisinin raw materials in the world market.However,the content of artemisinin in natural Artemisia annua is very low?0.01%¹%,DW?,which makes it difficult to meet the demand of the international market due to the limitation of resources and environment.Therefore,how to improve the yield of artemisinin in Artemisia annua has become the focus of Artemisia annua research.Firstly,although the biosynthesis pathway of artemisinin has been clearly resolved,there is still a bottleneck in the synthesis from artemisinin to Artemisinin,and the regulatory mechanism of artemisinin in Artemisia annua is still unclear.Therefore,this study collected Artemisia annua resources from different habitats,analyzed the changes in gene expression levels of Artemisia annua germplasm with different artemisinin contents through detection of artemisinin and other terpenoids and combined transcriptomic analysis,and thus explained the possible molecular mechanism of artemisinin synthesis regulation.Secondly,in order to cultivate artemisinin-rich Artemisia annua varieties,the CRISPR?Clustered interspaced shortpalindromic repeats?/Cas9 technology was applied to Artemisia annua in this study.CRISPR/Cas9 is an effective gene knockout tool,which uses a small piece of RNA as a guide to specifically identify the targeted sequence,and then uses the endonuclease Cas9 to perform enzyme digestion of the targeted sequence to destroy its DNA structure,so as to achieve gene editing successfully.Compared with ZFNs and TALEN,CRISPR/Cas9 is simple to construct and highly specific to target.In this study,five key enzymes of competitive branches with FPP as the substrate in artemisinin synthesis pathway were screened,and the multi-target CRISPR/Cas9 expression vector was constructed and transformed into Artemisia annua,laying a foundation for the application of CRISPR/Cas9 technology in Artemisia annua and the cultivation of Artemisia annua germplasm with high artemisinin content.Specific research results are as follows:1.Chemical and transcriptome analysis of Artemisia annua from different habitatsThis study collected from China's jilin?JL?and Beijing?BJ?,guangxi?GX?,hubei?HUB?and hainan?HAN?and other places of A.annua provenance,through the ultra high performance liquid chromatography-tandem test level 4 pole mass spectrometry?UPLC-ESI-QQQ-MS?method of artemisinin and artemisinin B content analysis,the results found in jilin,Beijing,guangxi,hubei,hainan and YQ2the artemisinin content increases,in turn,Beijing,jilin,guangxi,hubei,YQ2 and hainan assumes the declining trend in turn.In order to investigate the differences in terpene metabolism of artemisinin with different artemisinin contents in Artemisia annua.,the germplasm resources of Artemisia annua l.with representative artemisinin contents in jilin,hubei and hainan regions were selected from the collected samples,and their RNA was extracted and three biological replicates were set in each group for transcriptomic analysis.In this study,the sequencing results of three samples from three regions were compared with 31,938-32,612 genome sequences,and pare-to-pare comparison of the three regions revealed that there were 12,343 differential genes in HAN and HUB,12,912 differential genes in HUB and JL,and 11,697 differential genes in JL and HAN,with a total of 2,410 differential genes annotated between the three groups.Through GO functional annotation analysis,the differential expression of HUB and JL Artemisia annua was analyzed,and 30,847 genes were annotated,and39,726 genes were annotated in HAN and JL Artemisia annua.Based on the analysis of differentially expressed genes in artemisinin biosynthesis pathway,it was found that the gene expression on the main route of artemisinin synthesis?CYP71AV1,DBR2,DXR,HMGS,ADS?was generally high?SQS,BFS,ECS,GAS?,while that on the branch route was low?SQS,BFS,ECS,GAS?.In addition,KEGG analysis found that the differentially expressed genes not only enriched in terpenoids such as artemisinin synthesis,but also significantly enriched in plant-pathogen interaction pathways,suggesting that the regulation of artemisinin synthesis may be related to the interaction between pathogens.This study laid a foundation for further elucidating the mechanism of artemisinin and its derivatives accumulation through chemical and transcriptome analysis of artemisia annua from different habitats.2.Application of CRISPR/Cas9 technology in Artemisia annua.In this study,by adjusting the key steps of genetic transformation,such as concentration of agrobacterium solution,infection time,co-culture time and hormone concentration,the existing genetic transformation methods of Artemisia annul l.were optimized,and an efficient genetic transformation system of Artemisia annul l.was established.It was found that the best explants in Artemisia annua l.were the two true leaves which had been growing for 10 days.The optimal infection concentration OD₆₀₀00 was 0.3 and the infection time was 10 min.The most suitable co-culture time is3 days;The optimal kanamycin selective pressure was 50 mg/L.The rooting rate of explants was highest?>90%?on 1/2ms+0.1 mg/L NAA?naphthylacetic acid?+0.5mg/L IBA?indolebutyric acid?+200 mg/L Cef medium.On this basis,genome sequences and CDS sequences of 5 known FPP competitive terpene synthase genes SQS?squalene synthase?,BFS?beta-farnesene synthase?,GAS?germacrene A synthase?,CPS?beta-caryophyllene synthase?and ECS?8-epicedrol synthase?were downloaded and collated from GenBanK in this study.The exons of BFS,ECS,GAS and CPS were 7,introns were 6,SQS exons were12,introns were 11.Through the CRISPR-P v2.0?http://crispr.hzau.edu.cn/CRISPR2/?website,designed five competitive terpenoids FPP synthase gene sgRNA each 2,build respectively based on the carrier of the CRISPR/Cas9 targets editor PTG/Cas9-SQS,PTG/Cas9-BFS,PTG/Cas9-ECS,PTG/Cas9-GAS,PTG/Cas9-CPS;Then,33regenerated plants of Artemisia annua were obtained after PCR identification by transforming Artemisia annua seeds preserved in the laboratory.Among them,25strains were PTG/Cas9-SQS and 5 strains were PTG/cas9-ECS and 3 strains were PTG/cas9-GAS.