| In this study,CRISPR/Cas9 gene editing technique was used to further explore the molecular regulation mechanism of the gene CPS2 for the synthesis of cis-abienol by means of gene knockout.Using high aroma strain 8306 as material,8306 CPS2 gene exon 2 region sequence was selected to design gRNA sequence.The recombinant plasmid of gRNA sequence and vector was constructed and transformed into tobacco by agrobacterium tumefaciens-mediated method.Meanwhile,the leaf secretions of the transformed plants were detected and the expression of related genes was analyzed.The transcriptome sequencing technique was used to compare the expression profiles of T0 transformed plants and unedited 8306 genes,and the differentially expressed genes-related metabolic pathways were analyzed and verified by q-PCR.The main findings are as follows:1.The CPS2 gene in high aroma flue-cured tobacco line 8306 was edited by CRISPR/Cas9 system.Two target sites of CPS2 were designed and inserted into CRISPR expression vector,and 31 positive transformed plants were successfully obtained.PCR amplification and sequencing of the plants were performed,and 7 tobacco plants with mutations in the target site sequence were obtained.Mutations occur between the 3rd base and the 4th base upstream of the PAM,and the mutation type is a single base A/T/C insertion,resulting in early termination of the encoded peptide chain.The expression of CPS2 and ABS was significantly decreased in the T0 generation transformed plants,and the content of cis-abienol and labdenediol was significantly decreased.It is indicated that knockout CPS2 inhibits labdane diterpenoid synthesis pathway.The expression of DXR was significantly increased,the content of GGPP is also greatly increased.indicating that knockout of CPS2 promotes the expression of scorpion genes in the upstream pathway and contributes to the accumulation of the first generation precursor GPP in the upstream pathway.The expression levels of CYC-1 and CYP71D16 were significantly different,and some were increased and decreased,and some of them were not significantly different.The content of cembratrienol was increased or some were not significantly different.The expression of cembratriendiol decreased slightly except for one strain.There was no obvious change in the content of sucrose esters and alkanes.The plant height,stem circumference,leaf spacing,maximum leaf length and maximum leaf width of the T0 generation transformed plants increased significantly,indicating that knocking out CPS2 would change the phenotypic traits of the plants.2.The RNA-seq technique was used to analyze the difference in expression patterns between T0 transformed plants and 8306 8306.A total of 255 differentially expressed genes were obtained.Through GO analysis,220 differentially expressed genes were used to obtain GO annotations.Many genes are significantly enriched in the reductive pentose-phosphate cycle(GO:0019253),photosynthesis,dark reaction(GO:0019685),photorespiration(GO:0009853),carbon fixation(GO:0015977),cellular metabolic compound sal vage(GO:004309),extracellular region(GO:0005576),ribulose bisphosphate carboxylase activity(GO:0016984),carboxy lyase activity(GO:0016831)and other GO items.In addition,by KOG analysis,190 differentially expressed genes were divided into 5 categories and 18 subclasses.The KEGG analysis showed that differential genes were mainly enriched in the environmental adaptation,metabolism of other amino acids,transport and catabolism,energy metabolism,metabolism of terpenoids and polyketides,lipid metabolism,carbohydrate metabolism.These include plant-pathogen interaction,taurine and hypotaurine metabolism,endocytosis,carbon fixation in photosynthetic organisms,limonene and pinene degradation,fatty acid degradation,alpha-linolenic acid metabolism,cutin,suberine and wax biosynthesis,glyoxylate and dicarboxylate metabolism,arachidonic acid metabolism.3.255 differentially expressed genes in the KEGG pathway analysis show that,knock out CPS2 can inhibit labdane diterpenoid synthesis,promote the expression of CPS1,GA2ox and GA20ox genes,and then promote gibberellin anabolism.Knock out CPS2 can promote auxin signal transduction,and inhibit abscisic acid and cytokinin signal transduction.In addition,knockout of CPS2 has a certain promoting effect on the anabolism of caffeine,flavonoids,vitamin B6,wax esters,glycerophospholipids,and glycerolipids,and on ubiquinone and other terpenoids,folic acid carbon pools,alpha-linolenic acid,and peanuts.The olefinic acid anabolism has a certain inhibitory effect.Knockout of CPS2 has little effect on the anabolic metabolism of sibutane.Knockout of CPS2 also plays a role in the plant-pathogen interaction-related genes,The up-regulated expression of calmodulin,disease resistance protein RPM1,chitin elicitor receptor kinase 1 and brassinosteroid insensitive 1-associated receptor kinase 1 gene could improve the defense ability of tobacco plants against diseases and insect pests.The down-regulated expression of respiratory burst oxidase and LRR receptor-like serine/threonine-protein kinase FLS2 was unfavorable to the defense of tobacco pathogen. |
