3 and 5 full race core set

For cloning the mango MiSOC1 gene, we first isolated partial mango MiSOC1 cDNA fragment with degenerate PCR (polymerase chain action) method, then obtain the full length mango MiSOC1 cDNA sequence with 3′and 5′ RACE. The primer sets are listed in Table 1 designed based on the 5′ and 3′-terminal sequence of partial mango MiSOC1 sequence obtained in our laboratory. The reaction was performed in an S1000TM thermal cycler (Bio-Rad, USA). The sequencing results of the 3′ and 5′ RACE PCR products were searched against the NCBI database, then determined the splice and clone the full length of MiSOC1gene. The 3′ and 5′ RACE PCR were performed with the protocol of the 3′ and 5′ full RACE Core Set (TaKaRa, Japan). The PCR production was purified using a PCR purification kit (Qiagen), inserted into the pGEM-T Easy Vector (Promega) and transformed into competent Escherichia coli DH5a cell. The recombinant plasmids were identified and the positive clones were picked and sequenced in Shanghai Sangon Biological Engineering Technology and Services Co., Ltd (Shanghai China). The confirmed full-length cDNA of MiSOC1was was deposited at GenBank (accession number KP404094) and used for molecular characterization and bioinformatics analysis later.

Total RNA extracted from frozen P. australis leaf tissue using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) formed the template for the M-MLV reverse transcriptase driven synthesis of cDNA (TaKaRa Bio Group, Otsu, Japan). The resulting cDNA was amplified using the primer pair 5′-CTTCCAG(A/T)CTCA(G/A)TCGGAGC and 5′-ATTGC(G/C)ACTCCT(T/A)GACAGCA to obtain an initial PaPCS sequence. After sequencing this amplicon, further primers were derived to perform 3′-RACE and 5′-RACE (TaKaRa 3′- and 5′-Full RACE Core set), according to the manufacturer's protocols.

A single female spider was used as a sample to extract the total RNA, and using 5′ and 3′ full RACE Core Set (TaKaRa, Dalian, China) to amplify the full cDNA ends according to the manufacturer’s instructions. Based on the P. pseudoannulata transcriptome annotation and sequence blast using NCBI online services, one putative ace gene was selected and acquired full sequence using RACE technology with its specific primers (Table 5). The complete amino acid sequence of the new putative AChE was aligned with other species AChEs using VectorNTI 11.5 (Invitrogen, Carlsbad, CA, USA) and GeneDoc 2.7 software (Softpedia, Romania). The phylogenetic relationships among different species AChEs were analyzed through MEGA 7.0.21 software (http://www.megasoftware.net/) using the neighbour-joining method and evaluated via bootstrapping with 1000 iterations.