Expression Analysis Of Krüppel-like Factor 4 In Chlamys Farreri During Early Development And Gonadal Development

KLF4, a member of K.LF (Kruppel-like factor) family, is a transcriptional element binding protein of eukaryotes. Previous studies showed that KLF plays an important role in proliferation, differentiation, embryonic development, induced pluripotent stem cells (iPS cells) and tumorigenesis in vertebrates. However, information about KLF4 in marine invertebrates is just limited in expression level of several species, which restricts the systematic understanding of KLF4 and potential applications to some extent.In this study, Zhikong scallop Chlamys farreri, a commercially important aquatic bivalve, was employed and a full-length cDNA sequence of Klf4 was cloned by RACE. Its expression characteristics in adult tissues and gonads during the reproductive cycle were determined by semi-quantitative RT-PCR and real-time RT-PCR, respectively. Furthermore, its spatio-temporal expression pattern in embryos and larvae were analyzed by real-time RT-PCR and in situ hybridization techniques. The results will provide some basic data on probing the KLF4 founction in C. farreri. The main results are as follows:The full-length cDNA of Klf4 in C. farreri (Cf-Klf4) is 2,289 bp with an open reading frame of 1,005 bp, encoding a protein of 334 amino acids. Three zinc fingers were found in the predicted protein of KLF4, which are conversed in KLF family and we also found transcriptional activation domain (AD), transcriptional inhibitory domain (ID) and nuclear localization signal (NLS) which are conversed in KLF4 protein.Results of semi-quantitative RT-PCR showed that the Cf-Klf4 mRNA was ubiquitously expressed in all tissues analyzed in C. farreri at growing stage with high levels in adductor muscle, kidney and mantle, and little lower levels in testis, ovary, digestive gland and lowest in gills. The expression level of Cf-Klf4 mRNA detected by qRT-PCR was significantly higher in testis than in ovary at proliferative stage, growing stage and mature stage, and it increased with spermatogenesis while no change occurred with oogenesis, which revealed the expression during gametogenesis is sexual dimorphic. Our findings suggest that Cf-Klf4 may play an important role in spermatogenesis of C. farreri.Quantitative RT-PCR indicted that Cf-Klf4 mRNA cannot be detected in embryos until trochophore, and the expression level was highest in D-shaped larva and then it reduced significantly in umbo larva (P<0.05), suggesting that it is a zyotic gene which participates in larval development in C. farreri. Whole mount in situ hybridization showed that positive hybridization signals was hardly visible in embryos before trochophore. In trochophore, the positive signals were located mainly around shell gland cells and gut. In D-shaped larva, the strong signals were restricted in adductor muscle and gut. In umbo larva, weak signals were located mainly around gut. This expression pattern suggested the Cf-Klf4 may play a potential role in initiation and development of organogenesis in C. farreri.