By this we mean that large and thermodynamically stable proteins may be arisen by the simple expedient of intragenic duplications, rather than the more complex processes of de novo a-helix and Rosiridin bsheet creation. Unfortunately, a co-immunoprecipitation assay could not verify these interactions in BmN4 cells, suggesting that silkworm Piwi interacts loosely or transiently with HP1 proteins. Thus, we carried out an insect two-hybrid analysis, a method with high sensitivity for the detection of this weak protein-protein interaction. Similarly to the results obtained with BiFC, the co-expression of DBD-Ago3 or Siwi with either of the two AD-HP1 proteins increased the reporter activity more than 10-fold in BmN4 cells. thus, there remains the possibility that the DBD-mediated nuclear import of silkworm Piwi proteins may lead to false-positive results for the interaction between silkworm Piwi and HP1 proteins. To exclude this possibility, we constructed another I2H system in which TetR without typical NLS and tetO sequences was used instead of the GAL4 DBD and UAS sequences, respectively. In agreement with the results obtained with the GAL4-based I2H, the reporter activity of the TetR-based I2H analysis indicated the interaction between silkworm HP1 and Piwi-subfamily proteins. Taken Ergosterol together, these results clearly indicate that silkworm Piwi proteins interact with two canonical HP1 proteins in the nucleus of BmN4 cultured cells. In Drosophila, dimeric dHP1a binds to a PxVxL-type motif in the N-terminal domain of dPiwi, which is found only in the Drosophila Piwi protein, through its C-terminal chromoshadow domain. To investigate which region of the Piwi protein interacts with HP1s, i.e., the N- or C-terminal domain, we performed I2H analyses between split silkworm Piwi and HP1 proteins. In all silkworm Piwi proteins and dPiwi undergoing the I2H assay with HP1 proteins, the N-terminal domain showed stronger luciferase activities than the C-terminal domain. Thus, similar to the case in Drosophila, these results suggest that the Nterminal domains of Piwi have a major contribution to the interaction with HP1a/b in silkworm BmN4 cells. The silkworm genome sequence database, however, revealed that Ago3 and Siwi do not contain PxVxL-type motifs in their amino acid sequence, suggesting that the silkworm Piwi-subfamily proteins interact with HP1s at regions distinct from the PxVxLtype motif, unlike other canonical HP1 interactors. Additionally, the interaction of the N-terminal domain of Piwi proteins with HP1s was stronger than that of full-length Piwi with HP1s, even in the case of dPiwi-dHP1a interaction. This result may indicate that the potential high affinities of Nterminal domains for HP1s are modulated by other regions of the Piwi proteins. These results thus suggest the possibility that Piwi proteins of other species lacking typical HP1 interaction motifs can interact with HP1 in the same way as silkworm Piwi proteins. Allene oxide cyclase is the key enzyme of jasmonate biosynthetic pathway, which catalyses the formation of OPDA and establishes the stereochemical configuration of naturally occurring JA10. Here, our results of GC-MS demonstrated that overexpression of AaAOC gene could significantly increase the content of endogenous JA and artemisinin in transgenic A. annua plants.