| The Pacific white shrimp, Litopenaeus vannamei, is an important economic aquaculture species found worldwide that accounted for over 79% of total shrimp aquaculture production and generated the highest commercial value(USD 16.5 billion) in 2013. However, over the past 20 years, shrimp farming has suffered enormous losses with an estimated amount of 1 billion USD per year due to infectious diseases. In present, most research has focused on the disease control, genetic breeding and aquaculture. Among these fields, current development of shrimp functional gene overexpression research has been hampered due to the lack of the suitable expression system. An expression system is consisted of expression vector and host system, and an effective expression vector should possess an efficient promoter.Till now, few research are focused on the development of shrimp homologous promoter. β-actin promoters have been reported to be efficient ubiquitous regulatory elements and are widely used in transgenic mammalians and fish. In this thesis, in continuation of our previous description of the isolation and identification of genomic sequence actin T1 from the pacific white shrimp, we obtained the 5’-flanking sequence of actin T1, and further analyze its activity, application scope and cis-regulatory elements, meanwhile, we conducted a trial for the application of Sba P(ENX) in a baculovirus delivery system in vitro and in vivo. The objectives were to develop an efficient shrimp homologous promoter and effective expression system.Three major research results are as follows: 1. Molecular cloning, functional and application scope analysis of 5’upstream sequence of L. vannamei β-actin geneA 1,642 bp sequence, containing 5’-flanking sequence, exon 1, intron 1 and partial exon 2, which are responsible for transcriptional initiation of shrimp β-actin(actin T1), was isolated from L. vannamei by inverse PCR method, and the sequence was VIII designated as Sba P(Shrimp beta-actin Promoter).To determine Sba P’s function, the structure domains were examined by constitutive expression of the luciferase reporter gene. We identified 5’ flanking region that played a central role in the expression of the β-actin gene, which contained two highly conserved transcriptional sites, CCAAT box and CAr G motif. However, Sba P exhibited stronger promoter activity than 5’ flanking sequences. In order to evaluate and characterize the promoting function of Sba P, a systematical test was performed to compare this putative shrimp promoter with Cytomegalovirus(CMV), Simian vacuolating virus 40(SV40), Polyhedrin(Polh) and white spot syndrome virus immediate early gene 1(WSSV ie1) four constitutive promoters commonly used and one β-actin promoter(Tba P) isolated from tilapia fish, using eight cell lines derived from different animal species. Transfection of these promoter-luciferase constructs followed by luciferase quantitation assays revealed that Sba P was able to drive luciferase gene expression in all eight cell lines including sf21(insect), PAC2(zebrafish), EPC(carp), CHSE-214(chinook salmon), GSTEF(green turtle), MS-1(monk seal), 293T(human) and He La(human), but at different levels. Compared to other promoters tested, the promoting activity of Sba P was 10-fold lower than CMV promoter but much higher than Polh promoter in most of these cell lines(except for CHSE-214 cells). On the contrary, Sba P mediated luciferase expression in sf21 cells was over one order of magnitude higher than that of CMV, SV40, Polh, and Tba P promoters. 2. The structural and functional analysis of Sba P cis-regulatory elements, and the promoting activity examination of compact Sba PStructural domain functional assay indicated that there might be cis-regulatory elements in 1st intron of Sba P. To characterize the function of regulatory element in 1st intron, a serial deletion promoter-luciferase constructs were generated on the basis of p GL-Sba P, and two negative(-1140/-925,-222/-21) and one positive(-810/-223) regulatory elements were identified in 1st intron according to dual-luciferase assay. Transient transfection assay with a construct containing proximal promoter and enhancer regions of the shrimp β-actin(Sba PΔ-222/+1Δ-1325/-925 which were named as Sba P(ENX)) coupled with luciferase and egfp(enhanced green fluorescent protein) showed its promoter activity was more than 8-fold higher than a viral-origin promoter(WSSV ie1) in sf21 cells. Particularly, Sba P(ENX) also drove a successful expression of luciferase in vivo and also showed higher promoter activity than the ie1 promoter. In addition, compared to the Sba P, Sba P(ENX) exhibited a relatively stronger promoter activity in EPC and PAC2 cells, but a 5% and 16% lower promoting effect in 293 T and He La, respectively. 3. The establishment of recombinant baculovirus system under the control ofSba P(ENX) and its potential applicationFor further application of Sba P(ENX) and establishment of an effective foreign gene expression system, we constructed a recombinant baculovirus system under the control of Sba P(ENX). Bac-Sba P(ENX)-RFP was confirmed that could successfully drive RFP gene expression in sf21 cells, and the stability tested indicated that there was no significantly decreased in titre in seawater and seawater mixture environment within 12 hours. Moreover, Bac-Sba P(ENX)-RFP was tested in both in vitro of transduced primary shrimp cells and in vivo of injected and immersed indicator shrimp. The results suggested that the expression of RFP could be detected in muscle and hepatopancreas by injected method.In this study, we obtained the 5’ flanking sequence of L. vannamei β-actin gene, and further analyzed the function of its structural domain and cis-regulatory elements, which will benefit the understanding of shrimp homologous promoter; furthermore, these results warrant the potential value of the newly isolated shrimp promoter Sba P, particularly its derivative(Sba P(ENX)) in ectopic gene expression, which will facilitate the development of foreign gene expression system in shrimp functional gene overexpression research in future. |
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