Mining,identification And Functional Characteristics Of CaSLS-Like Homologous Genes In Camptothecin Biosynthesis Pathway

Camptotheca acuminata is a plant of the Nyssaceae,belonging to the Camptotheca Decne.Camptotheca acuminata is a specific Chinese medicinal plant,and its main physiological substance is camptothecin.Camptothecin can be used to treat cancer and other diseases,so it has more invaluable medical value.However,the content of camptothecin that can be extracted from Camptotheca acuminata is very low and cannot meet the needs of the market.Therefore,it is necessary to researchudy the biosynthetic pathway of camptothecin and increase the production of camptothecin.The biosynthetic pathway of camptothecin is more complicated,which involves many key enzyme genes.Secologanin synthase(SLS)is a very important regulatory enzyme in the biosynthetic pathway of camptothecin.Secologanin synthase belongs to the cytochrome P450 family,and this family can be used to catalyze the oxidative cleavage of the cyclopentane ring of the Loganin to form Secologanin.The catalytic activity of most P450 enzymes is determined by cytochrome P450 reductase(CPR).Therefore,the coding gene of Secologanin synthase and its partner gene CPR are of great significance for the study of camptothecin biosynthetic pathways.Therefore,this topic mainly conducts the following research:1.The four SLS gene sequences in Camptotheca acuminata were obtained through screening(CaSLS1、CaSLS2、CaSLS3、CaSLS4).Design primers according to the sequence of the SLS gene(CaSLS1、CaSLS2、CaSLS3、CaSLS4)to clone these four CaSLSs.Bioinformatics analysis and multiple alignments of protein sequences were performed on CaSLSs.The analysis results show that the characteristic domains of CaSLS enzyme protein include: N-terminal hydrophobic membrane anchoring domain,conserved oxygen binding and activation I-helix motif(AGQETT,yellow box),heme-binding domain PFS(G)WGPRI(V)CL(V/I)G.2.The open reading frame of the Secologanin synthase gene of Camptotheca acuminata was constructed into the Bam H I/Xho I site of the yeast expression vector p ESC-Leu2d-CaCPR1 to obtain a recombinant plasmid.The vector was transferred into yeast strain MKP-0 for inducing expression and finally analyzed by HPLC-MS.The results showed that both CaSLS1 and CaSLS4 can complete the catalytic function in yeast,and the substrate conversion rate is 29.47%and 19.78%.3.This paper tested the specific expression of CaSLSs in different tissues of wild-type Camptotheca acuminata.The expression level of CaSLS1 in young leaves was the highest,which was 68.21 times that of old stems;followed by roots,which was 9.91 times that of old stems.The expression level of CaSLS4 was the highest in old stems,which was 23.29 times that of tender leaves.Therefore,CaSLSs may be generally involved in the biosynthesis of schiscyclanin/acid in leaves at the same time,which is consistent with the tissue site where MIA synthesis is the highest;CaSLS4 may be the main enzyme protein isomers that catalyze SLA biosynthesis in the stems of the aerial parts of plants.4.In this paper,CaSLS1,CaSLS4 and its companion genes CaCPR1 and CaCPR2 were silenced.The results showed that the accumulation of camptothecin in leaves that silenced CaSLS1 decreased by about 49% compared with the blank control group,the accumulation of camptothecin in leaves that silenced CaSLS4 decreased by about 46%,and that in leaves that silenced CaCPR1.The accumulation of CaCPR2 decreased by about 10%,while the accumulation of camptothecin in the leaves of silent CaCPR2 decreased by about 60%.Therefore,the accumulation level of camptothecin in the leaves after CaCPR1 and CaCPR2 gene silencing is different,and the CPT accumulation result of CaCPR2 is consistent with the gene silencing level,indicating that CaCPR2 participates in the biosynthesis of CPT.