Visetnan Suwattana, Supungul Premruethai, Tang Sureerat, Hirono Ikuo, Tassanakajon Anchalee, Rimphanitchayakit Vichien
Center of Excellence for Molecular Biology and Genomics of Shrimp, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Phyathai Road, Bangkok 10330, Thailand.
Center of Excellence for Molecular Biology and Genomics of Shrimp, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Phyathai Road, Bangkok 10330, Thailand; National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Pathum Thani 10120, Thailand.
Fish Shellfish Immunol. 2015 Nov;47(1):572-81. doi: 10.1016/j.fsi.2015.09.053. Epub 2015 Oct 3.
In animals, infection by Gram-negative bacteria and certain viruses activates the Imd signaling pathway wherein the a NF-κB transcription factor, Relish, is a key regulatory protein for the synthesis of antimicrobial proteins. Infection by yellow head virus (YHV) activates the Imd pathway. To investigate the expression of genes involved in YHV infection and under the influence of PmRelish regulation, RNA interference and suppression subtractive hybridization (SSH) are employed. The genes in forward library expressed in shrimp after YHV infection and under the activity of PmRelish were obtained by subtracting the cDNAs from YHV-infected and PmRelish-knockdown shrimp with cDNAs from YHV-infected shrimp. Opposite subtraction gave a reverse library whereby an alternative set of genes under YHV infection and no PmRelish expression were obtained. Nucleotide sequences of 252 and 99 cDNA clones from the forward and reverse libraries, respectively, were obtained and annotated through blast search against the GenBank sequences. Genes involved in defense and homeostasis were abundant in both libraries, 31% and 23% in the forward and reverse libraries, respectively. They were predominantly antimicrobial proteins, proteinases and proteinase inhibitors. The expression of antimicrobial protein genes, ALFPm3, crustinPm1, penaeidin3 and penaeidin5 were tested under PmRelish silencing and Gram-negative bacterium Vibrio harveyi infection. Together with the results using YHV infection previously reported, the expression of penaeidin5 and also penaeidin3 but not ALFPm3 and crustinPm1 were under the regulation of PmRelish in the Imd pathway.
在动物中，革兰氏阴性菌和某些病毒的感染会激活Imd信号通路，其中一种核因子κB转录因子Relish是抗菌蛋白合成的关键调节蛋白。感染黄头病毒（YHV）会激活Imd通路。为了研究YHV感染相关基因的表达以及在对虾Relish（PmRelish）调控的影响下，采用了RNA干扰和抑制性消减杂交（SSH）技术。通过用YHV感染的对虾和PmRelish基因敲低的对虾的cDNA减去YHV感染对虾的cDNA，获得了YHV感染且在PmRelish活性作用下对虾中表达的正向文库中的基因。反向消减得到一个反向文库，从而获得了YHV感染且无PmRelish表达情况下的另一组基因。分别从正向和反向文库中获得了252个和99个cDNA克隆的核苷酸序列，并通过与GenBank序列进行比对注释。两个文库中参与防御和体内平衡的基因都很丰富，正向文库和反向文库中分别占31%和23%。它们主要是抗菌蛋白、蛋白酶和蛋白酶抑制剂。在PmRelish沉默和革兰氏阴性菌哈维氏弧菌感染的情况下，测试了抗菌蛋白基因ALFPm3、对虾抗菌肽Pm1、对虾抗菌肽3和对虾抗菌肽5的表达。结合先前报道的使用YHV感染的结果，对虾抗菌肽5以及对虾抗菌肽3（而非ALFPm3和对虾抗菌肽Pm1）的表达受Imd通路中PmRelish的调控。
