ate for the inner primer, and the thermocycler conditions were as follows: 30 cycles at 95uC for 30 sec, 58uC for 30 sec, and 72uC for 1 min. To clone the H. armigera Vg gene, degenerate primers were designed. PCR amplifications were performed in 25 ml volumes containing 1 ml of primers, 2.5 ml of 106 buffer, 2 ml of each dNTP, 0.15 ml of Ex Taq and 1 ml of cDNA template. The following thermocycler program was used: denaturation at 95uC for 30 sec, annealing at 55uC for 30 sec and extension at 72uC for 5 min for 35 cycles. To obtain the complete cDNA sequence of the Vg gene, a new set of gene-specific primers matching the primers in the 39- and 59-Full RACE kit were designed. The 39-RACE outer and inner PCR reactions were carried out with 20 cycles at 95uC for 30 sec, 55uC for 30 sec, and 72uC for 1 min followed by 
30 cycles at 95uC for 30 sec, 60uC for 30 sec, and 72uC for 1 min. The 59-RACE outer and inner PCR amplification conditions were the same as the 39- RACE outer and inner protocols. All clones were sequenced by Invitrogen. Sequence Alignments and Comparisons To compare the identified HaHMGR sequence to other insect species, we used the previously published HMGR amino acid sequences of the following species: A. aegypti, A. ipsilon, A. grandis, B. mori, C. quinquefasciatus, D. jeffreyi, D. melanogaster, I. pini, I. paraconfusus, Apis mellifera, Bombus impatiens, I. confuses, Chrysomela populi, P. cochleariae, Gastrophysa viridula, S. cynthia ricini, and B. germanica. Sequence alignments were carried out with MEGA4 Molecular Evolutionary Genetics Analysis Software Version 4.0. HaHMGR and Vg Quantitative real-time PCR To determine the appropriate age for initiating RNAi EW-7197 price silencing of the target gene, we used qPCR to investigate the relative expression of HaHMGR in female pupae from day 1 to day 9. The same method was also used to assess the expression levels of Vg in female pupae from day 1 to day 9. 3 RNAi of the HMGR Gene in the Helicoverpa armigera Total RNA was isolated using Trizol following the manufacturer’s instructions, and it was suspended in 30 ml of RNase-free water. DNase I was used to remove the residual genomic DNA. PrimeScript RT Master Mix Perfect Real Time was used to obtain the first-strand cDNA. qPCR was used for the analysis of the relative expression by iCycler iQ5. Three sets of gene-specific primers for qPCR were designed for the b-actin, HaHMGR and Vg gene fragments. The PCR protocol consisted of 95uC for 30 sec followed by 40 cycles of 95uC for 5 sec and 60uC for 30 sec using SsoFastTM EvaGreenH Supermix. The total reaction volume was 20 ml. All reactions were performed in triplicate. The relative expression of HaHMGR mRNA was compared using ANOVA and Tukey’s post-hoc test for multiple comparisons. 4 RNAi of the HMGR Gene in the Helicoverpa armigera Preparation, Quantification and Purification of 18039391 dsRNA The dsRNAs prepared from different sections of the coding region showed 11078888 the same activity as the full-length dsRNA. A randomly chosen section of the coding region of HaHMGR was amplified from moth cDNA. A control fragment from EGFP was amplified from the pBCMiLR-3xP3 EGFP-PL2 vector. A T7 RNA polymerase promoter was added to the EGFP and HaHMGR sequences using PCR by adding the T7 promoter sequence at the 59 end of the amplification primers . The PCR products were excised from the ethidium bromide-stained gel and purified using a DNA purification kit. The dsRNA was synthesized using the T7 RiboMAXTM Express