Additional file 1: Methods 1. of Triterpenoid profiling and functional characterization of the initial genes involved in isoprenoid biosynthesis in neem (Azadirachta indica)
Creators: name: Pandreka, Avinash
name: Devdutta Dandekar
name: Saikat Haldar
name: Vairagkar Uttara
name: Shinde Vijayshree
name: Fayaj Mulani
name: Thiagarayaselvam Aarthy
name: Hirekodathakallu Thulasiram
Publisher: name: Figshare
Year: 2015
Subjects: subject: Biochemistry
subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Genetics
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: FOS: Chemical sciences
subject: Immunology
subject: FOS: Clinical medicine
subject: FOS: Clinical medicine
subject: 69999 Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: 110309 Infectious Diseases
subject: FOS: Health sciences
subject: FOS: Health sciences
subject: Plant Biology

description: Isolation of Neem triterpenoids from seed kernel and pericarp. Methods 2. Characterization of purified Neem triterpenoids. Figure S1. TLC profile of crude extracts and purified triterpenoids (developed in 70 % ethyl acetate in n-hexane for twice). Figure S2. UPLC-ESI(+)-quadrupole/orbitrap-MS extracted ion chromatograms of the fifteen pure triterpenoids from Neem. Chromatograms have been arranged in the order of increasing retention time. Figure S3. ESI(+)-quadrupole/orbitrap-MS spectra of the fifteen pure triterpenoids from Neem. Figure S4. Standard graphs for the purified triterpenoids prepared in UPLC-ESI(+)-quadrupole/orbitrap-MS; concentration range 0.040-0.003 mg/mL, injection volume 5 μL. Figure S5. Representative UPLC-ESI(+)-quadrupole/orbitrap-MS chromatograms of various Neem tissue extracts (× denotes non-triterpenoids with molecular mass less than 350). Figure S6. Quantitative abundance of individual triterpenoids in different tissues of Neem. Figure S7. Multiple sequence alignment of A. indica geranyl diphosphate synthases (AiGDS). Figure S8. Multiple sequence alignment of A. indica farnesyl diphoshate synthase (AiFDS). Figure S9. Multiple sequence alignment of A. indica Squalene synthase (AiSQS); Amino acid sequence alignment of C. annuum (CaSQS, AAD20626), N. tabacum (NtSQS, AAB08578), A. indica (AiSQS, AFJ15526), L. japonicas (LjSQS, BAC56854), G. max (GmSQS, NP_001236365), P. vulgaris (PvSQS, AHA84150). The solid lines indicate four highly conserved regions 1, 2, 3 and 4 which are considered to be the catalytic sites of squalene synthases. Figure S10. Phylogenetic analysis of AiGDS, AiFDS and AiSQS. Figure S11. Purification of recombinant AiGDS, AiFDS and AiSQS. Table S1. Predicted genes for Triterpenoid back bone biosynthesis. Table S2. Present Identity Matrix of AiGDS with plant homomeric GDS and heteromeric GDS Larger subunits. Table S3. Primers and vectors used for cloning of AiGDS, AiFDS and AiSQS and RT-PCR primers of 18S rRNA, GAPDH, Neem_transcript_10001, AiGDS and AiSQS. Table S4. Buffers used for AiGDS, AiFDS and AiSQS protein purification. Table S5. TargetP analysis Neem_transcript_10912 (AiGDS) and Neem_Transcript_10001. (DOCX 3387 kb)