Primer / Sequence (5'-3') / Description
ELIP5R / ATCCCGCCCACTTGCGACCTCCACT / Reverse primer for 5' RACE, outer
ELIP5N / GAGGAGCAGCCGATGGGATGTTTG / Reverse primer for 5' RACE, nested
ELIP3R / TCGTCTCCTCGTGAAGTGTATGGCAG / Forward primer for 3' RACE, outer
ELIP3N / AACAAACATCCCATCGGCTGCTCCT / Forward primer for 3' RACE, nested
ELIPU / TGACCATAAGCACCTTCAGTTT / Full-length primer, forward
ELIPD / CGAGAATACAATCCCTACTTT / Full-length primer, reverse
ELIP1 / CTTCCATACACTTCACGAGGAGACGA / Reverse primer for 5' promoter, outer
ELIP2 / ACTGCGGGCATCAAATATCACTCTAT / Reverse primer for 5' promoter, nested
ELIPTU / AGACTCTAAACGGAAGACTGG / Probe primer, forward
ELIPTD / GGACCTCCTTTCAAATACTCA / Probe primer, reverse
ELIPF / GGCAGATGAGGCAGAATCAGA / Primer for RQPCR, forward
ELIPR / GTGCAGGCCCACTGAAAGC / Primer for RQPCR, reverse
GAPDHF / TGTCACGGTTTTCGGTTGTAG / Primer for RQPCR, forward
GAPDHR / ACCTTTTTGGCACCTCCCTTA / Primer for RQPCR, reverse
HIS5F / GCAAGGCCAAGTCATCGAAA / Primer for RQPCR, forward
HIS5R / CCAATACCGCAGCTAAATACA / Primer for RQPCR, reverse
Table S2 Procedures for PCRs
Name of PCRs / PCRs procedures
cDNA sequence of ELIP PCR / 94 °C for 5 min; 94 °C for 1 min, 54 °C for 1 min, 72 °C for 1 min 50 s (30 cycles); 72 °C for 5 min
Genomic sequence of ELIP PCR / 94 °C for 5 min; 94 °C for 1 min, 52 °C for 1 min, 72 °C for 1 min 50 s (30 cycles); 72 °C for 5 min
Probe sequence of ELIP PCR / 94 °C for 5 min; 94 °C for 1 min, 51 °C for 1 min, 72 °C for 1 min 50 s (35cycles); 72 °C for 5 min
Real-time PCR / 50 °C for 2 min; 95 °C for 15 min; 95 °C for 15 s, 60 °C for 1 min (35 cycles)
Figure S1 Melt curves of ELIP and two reference gene (GAPDH and HIS 5). Yellow line (GAPDH), Red lines (HIS), Green lines (ELIP); Tm value of the three application fragments were 76.04 (GAPDH), 80.41 (HIS) and 79.54 (ELIP).
Figure S2 Standard curve of ELIP and the two reference genes (GAPDH and HIS 5). Above: GAPDH (Slope: -3.012, Y-inter: 32.518, R2 0.979, Eff%: 114.769); Middle: HIS5 (Slope: -2.73, Y-inter: 30.065, R2 0.974, Eff%: 132.463); Bottom: ELIP (Slope: -2.73, Y-inter: 30.065, R2 0.974, Eff%: 132.463)
Figure S3 Comparison of slopes of Standard curves of ELIP and the two reference genes (GAPDH and HIS 5). Above: HIS5 to GAPDH (Slope: 0.018); Middle: HIS5 to ELIP (Slope: 0.007); Bottom: GAPDH to ELIP (Slope: 0.088). All the three slopes were less than 0.1, so 2−ΔΔCt method can be used for relative quantification with there two reference genes.
Figure S4 Genomic blot analysis of GbELIP. Genomic DNA was digested with BamH I, EcoR I and Hind III. The DNA bolt with the insert in the GbELIP cDNA clone.
Figure S5 Prediction of the presence and location of signal peptide cleavage sites in GbELIP using SignalP 4.1 server
Figure S6 Hydropathy plot of GbELIP using ProtScale by the method of Kyte & Doolittle
Figure S7 Prediction of transmembrane helices in GbELIP using TMHMM Server v. 2.0
Figure S8 Protein sequence alignment of GbELIP with other ELIPs using Vector NTI Advance 11. Following the aligned sequences is the accession numbers and homology percentage. The species names and accession numbers was stated at results in the manuscript.
The nucleotide sequence of deduced histone H2A protein
>his2
CTGGGAGATCGTTGGGGTTGCTTTCGTGAATAGGGTTTTGTTCGACATTTGAGGGACAATATCACCATGAGTTCCGCAGGTGGTGGCGGCCGAGGCAAGGCCAAGTCATCGAAATCCGTTTCAAGGTCTCAGAAGGCTGGTCTCCAGTTCCCTGTGGGAAGGATTGCTCGTTTTCTTAAGGCCGGCAAGTACGCAGAACGTGTGGGTGCAGGGGCTCCTGTGTATTTAGCTGCGGTATTGGAATATCTAGCAGCAGAGGTGCTCGAGTTGGCAGGGAATGCGGCCCGGGATAACAAGAAGAACAGAATTGTACCTCGACATATCCAGTTGGCTGTGAGAAATGACGAAGAGCTTAGCAAGTTATTGGGTTCTGTTACAATTGCAAATGGTGGTGTGCTACCCAATATTCATGGTGTTT