Influence of clock genes knock-down on the transcriptional expression of clock genes in Blattella germanica

• Main Authors: Chun-Chiang Yang, 楊均強
• Other Authors: How-Jing Lee
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/33123270075925837813

碩士 === 國立臺灣大學 === 昆蟲學研究所 === 102 === Since the first clock gene, period (per), was found in Drosophila melanogaster, the circadian rhythm is known to be regulated by genes. A negative transcription-translation feedback loop is considered to be the core of clockwork. I have obtained 4 genes per, timeless (tim), cryptochrome1 (cry1), and cryptochrome2 (cry2) which are related to clockwork in Blattella germanica and investigated their interactions to unveil the core clockwork. By using RNA interference (RNAi), we could explore and compare the variation of clock genes'' expressions to determine their relationships. In addition, I discovered that clock genes also expressed in antennae. By comparing the clock genes'' expression after RNAi between head and antennae, I intended to find the difference in clockwork between central and peripheral clock. I found that after injecting dsTim and dsCry2, the relative amount of cry2, per and tim were influenced. Interestingly, for dsCry1 treatment, the relative mRNA expression level of per, tim and cry2 showed totally reverse pattern compared with control group in the head and antennae. To verify the interactions among the 4 genes at protein level, yeast two-hybrid (Y2H) technique was used. Although cry1 could be inserted into yeast plasmid, the other 3 genes were too large to be inserted successfully. Even without protein interaction results, I unveiled the molecular clockwork of the German cockroach may be different to the monarch butterfly model which PER and CRY2 formed heterodimer, then entering nucleus to act as a CLK/CYC repressor. Instead, TIM and CRY2 probably function as transcriptional repressors that regulate the expression of per, tim and cry2. For CRY1''s function, it served as a role to reset the clock phase as the CRY1 in the monarch butterfly model.