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Functional Analysis of the Shrimp Putative Molt Inhibiting Hormone Cdnas (LIV-Mih1 and Pem-Mih1) by RNA Interference

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This dissertation, "Functional Analysis of the Shrimp Putative Molt Inhibiting Hormone CDNAs (Liv-MIH1 and Pem-MIH1) by RNA Interference" by Chun-yin, Mak, 麥俊然, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any wa This dissertation, "Functional Analysis of the Shrimp Putative Molt Inhibiting Hormone CDNAs (Liv-MIH1 and Pem-MIH1) by RNA Interference" by Chun-yin, Mak, 麥俊然, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled FUNCTIONAL ANALYSIS OF THE SHRIMP PUTATIVE MOLT INHIBITING HORMONE cDNAs (Liv-MIH1 AND Pem-MIH1) BY RNA INTERFERENCE Submitted by Mak Chun Yin For the degree of Master of Philosophy at The University of Hong Kong in July 2007 The CHH/MIH/GIH family is an important group of neuropeptides regulating a wide range of physiological processes in crustaceans. Molting in crustaceans is negatively regulated by the molt inhibiting hormone (MIH). It is hypothesized that MIH could inhibit ecdysteroids (molting hormone) synthesized from and released by the Y-organ. Liv-MIH1 and Pem-MIH1, collectively known as MIH1, are two recently characterized putative MIH cDNAs in Litopenaeus vannamei and Penaeus monodon, respectively. As demonstrated by Northern blot and RT-PCR, Liv-MIH1 and Pem-MIH1 were expressed mainly in the eyestalks in a molt stage-related pattern with a higher expression level in the postmolt and intermolt stages. To develop a technique for specific and efficient functional analysis on the two MIH1 cDNAs, long double-stranded RNA (dsRNA) was used for an in vivo gene knockdown study. The results showed that injection of 3 μg of MIH1 dsRNA caused an effective and specific knockdown of MIH1 homologous transcripts in 48 hours. Injection of dsRNA for Liv-MIH1 and Pem-MIH1 resulted in 70% and 67% decrease in MIH1 transcript level respectively. In contrast, expressions of the molt-related cysteine protease, cathepsin L, were up-regulated to 3 and 4 folds in hepatopancreas. In addition, molting was stimulated in shrimps injected with MIH1 dsRNA as shrimp showed an advancement of molt stages from D to D . The result suggested that 0 1 MIH1 regulated molting in a negative manner. The advancement of molt stage was represented by an average shortening of 1.8 days (or 20%) as compared to the control. To investigate the regulatory mechanism of shrimp molting, the interaction between MIH1 and the molt-related gene farnesoic acid O-methyltransferase (FAMeT) was studied. The results indicated that gene knockdown of MIH1 has no effect on FAMeT gene expression in the eyestalk, nerve cord and hepatopancreas. The result suggested that other CHH neuropeptide family member(s) other than MIH1 was involved in FAMeT regulation. In conclusion, by dsRNA-mediated RNAi approach, results from functional analyses of Liv-MIH1 and Pem-MIH1 indicated that MIH1 regulate shrimp molting in a negative manner. The specificity and effectiveness of gene knockdown by RNAi technology will provide further application to study the functions of the CHH/MIH/GIH genes. DOI: 10.5353/th_b3890075 Subjects: Molting Ecdysteroids Neuropeptides RNA Gene silencing Shrimps - Physiology


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This dissertation, "Functional Analysis of the Shrimp Putative Molt Inhibiting Hormone CDNAs (Liv-MIH1 and Pem-MIH1) by RNA Interference" by Chun-yin, Mak, 麥俊然, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any wa This dissertation, "Functional Analysis of the Shrimp Putative Molt Inhibiting Hormone CDNAs (Liv-MIH1 and Pem-MIH1) by RNA Interference" by Chun-yin, Mak, 麥俊然, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled FUNCTIONAL ANALYSIS OF THE SHRIMP PUTATIVE MOLT INHIBITING HORMONE cDNAs (Liv-MIH1 AND Pem-MIH1) BY RNA INTERFERENCE Submitted by Mak Chun Yin For the degree of Master of Philosophy at The University of Hong Kong in July 2007 The CHH/MIH/GIH family is an important group of neuropeptides regulating a wide range of physiological processes in crustaceans. Molting in crustaceans is negatively regulated by the molt inhibiting hormone (MIH). It is hypothesized that MIH could inhibit ecdysteroids (molting hormone) synthesized from and released by the Y-organ. Liv-MIH1 and Pem-MIH1, collectively known as MIH1, are two recently characterized putative MIH cDNAs in Litopenaeus vannamei and Penaeus monodon, respectively. As demonstrated by Northern blot and RT-PCR, Liv-MIH1 and Pem-MIH1 were expressed mainly in the eyestalks in a molt stage-related pattern with a higher expression level in the postmolt and intermolt stages. To develop a technique for specific and efficient functional analysis on the two MIH1 cDNAs, long double-stranded RNA (dsRNA) was used for an in vivo gene knockdown study. The results showed that injection of 3 μg of MIH1 dsRNA caused an effective and specific knockdown of MIH1 homologous transcripts in 48 hours. Injection of dsRNA for Liv-MIH1 and Pem-MIH1 resulted in 70% and 67% decrease in MIH1 transcript level respectively. In contrast, expressions of the molt-related cysteine protease, cathepsin L, were up-regulated to 3 and 4 folds in hepatopancreas. In addition, molting was stimulated in shrimps injected with MIH1 dsRNA as shrimp showed an advancement of molt stages from D to D . The result suggested that 0 1 MIH1 regulated molting in a negative manner. The advancement of molt stage was represented by an average shortening of 1.8 days (or 20%) as compared to the control. To investigate the regulatory mechanism of shrimp molting, the interaction between MIH1 and the molt-related gene farnesoic acid O-methyltransferase (FAMeT) was studied. The results indicated that gene knockdown of MIH1 has no effect on FAMeT gene expression in the eyestalk, nerve cord and hepatopancreas. The result suggested that other CHH neuropeptide family member(s) other than MIH1 was involved in FAMeT regulation. In conclusion, by dsRNA-mediated RNAi approach, results from functional analyses of Liv-MIH1 and Pem-MIH1 indicated that MIH1 regulate shrimp molting in a negative manner. The specificity and effectiveness of gene knockdown by RNAi technology will provide further application to study the functions of the CHH/MIH/GIH genes. DOI: 10.5353/th_b3890075 Subjects: Molting Ecdysteroids Neuropeptides RNA Gene silencing Shrimps - Physiology

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