Cloning, Identification And Application Of Fat Body Specific Promoter BmLSP, BmLP3 In Silkworm, Bombyx Mori

Slikworm,Bombyx. mori is an important economic, cultural, model insect and candidate for bioreactor of insect. With finish of the whole genome sequencing of silkworm,Bombyx. mori in 2003, most research in the world about silkworm have shifted to the gene identification and function analysis related to the important economic and biological characteristics of silkworm, such as the synthesis of silk proteins, metamorphosis and molecular immunology. Untill now significant progresses especially relevant to the growth, development and metamorphosis of silkwom have been achieved.Fat body is the center of nutrition, energy storage and supply of silkworm, which is the target tissue of intra hormones such as ecdysone and juvenile, and plays a critical role in the processes of the growth and metamorphosis of silkworm, Bombyx mori. The currently studies showed that the ecdysis and metamorphosis of insects are regulated by the ecdysone hormone and the arrows of the growth of insects are regulated by the juvenile hormone. It has been found that the transcriptional levels of a large numbers of fat body specific genes of silkworm are precisely controlled by the hormones, and their expressions change regularly with the growth of silkworm. Hence, to deeply understand the expressions, functions and regulations of fat body genes of silkworm have important meaning to elucidate the molecular mechanisms of the growth and metamorphosis of silkworm controlled by hormones. Furthermore, the fat body of silkworm contains a mass of proteins, especially in the span from the late of 5th instar larvae to pupation can reach up to 30% of the total body weight of silkworm, which means the proteins contained in the fat body of silkworm can reach up the 1.5g while the average body weight of silkworm is 5g. Thus the fat body is hopeful to be another late-model candidate for the bioreactors of insects besides the silk gland based the bioreactors of silkworm.Although a rich accumulation of research relevant to the fat body genes of silkworm and their expressions have been achieved, many issues especially related to the analysis of promoter and function research of fat body genes are still devoid. In order to overcome this issue, in this article, we set the BmLSP and BmLP3 genes which specifically expressed in fat body of silkworm as targets, on the basis of the promoter clones of these two genes, we focuse on the BmLP3 promoter. First, we analyze and identify the core elements of BmLP3 promoter in vitro by transient expression. Second, we analyze the activities and specificities of BmLSP and BmLP3 promoters in vivo by trangenic silkworm. Third, we investigate the ability of BmLP3 to be a candidate promoter for the bioreactor of silkworm by its property of high expression level. The main results are described as followed:1 Clone and bioinformatics identification of promoter BmLSP and BmLP3Based on the data of silkworm genome and reported information,we clone two promoter:BmLSP and BmLP3. The analysis of promoter BmLSP sequence indicate①The length of promoter BmLSP is 1 660bp. The sequence contains exon 1, intron 1, the core promoter region and 5'flanking region of BmLSP gene. Compared with promoter BmLSP of silkworm SujuxMinghu, a mariner-like transposon of 263bp is inserted in location+341 in the intron 1.②There are ecdysone response element of HSP23 and homologous regulation sequence of fat body specific motif.The analysis of promoter BmLP3 sequence indicate①The cloned promoter BmLP3 is about 1.1kb. The sequence contains exon 1, intron 1, part of exon 2, the core promoter region and 5'flanking region of BmLP3 gene. This sequence have not been reported.②The analysis of bioinformatics show there are a typical TATA box, a binding common sequence of Spl protein, a Bml element in this promoter sequence,suggest these motif may play an important role in expression of BmLP3 gene.2 Expression analysis of DsRed gene driven by promoter of BmLSP gene in transgenic Silkworm, Bombyx moriThe storage protein, larval serum protein (BmLSP), is synthesized in the fat body of silkworm, Bombyx mori, and is considered one of the target molecules for studying the regulation of larval development. Based on the data of silkworm genome, a 1.6 kb promoter sequence is cloned from the 5'flanking region of BmLSP gene to construct a piggyBac expression vector by using DsRed as a reporter gene, which is further used to conduct transgenic microinjection for verification of the promoter's characters in individual silkworm larvae. The results show that the reading frame of BmLSP-DsRed is inserted into the genome of silkworm in a single copy form and is expressed in fat body-specific pattern in transgenic individuals. Moreover, its temporal expression pattern is similar to that of the endogenous BmLSP gene and exhibit a regular variation in different stages of development. To be specifically, high expression is observed during the larval stage, no expression is detected in molting larvae of the 1st and 2nd instars, weak expression is observed in molting larvae of the 3rd and 4th instars, the expressed product is reduced gradually after spinning and disappeared at the eclosion stage. These results suggest that the expression of BmLSP gene might be regulated accurately by some factors such as juvenile hormone that bind to specific elements of the promoter.3. The activity analysis of the core element of BmLP3 promoter in BmE cell.BmLP3 gene is one of the members with highest expression level from 30K genes family of silkworm, Bombyx mori. Based on the silkworm genome database, we clone-1.1Kb promoter region from the 5'flanking region of BmLP3 gene. Subsequently, we conduct a series of luciferase(LUC) report vectors drived by different partly deleting patterns of BmLP3 promoter and perform the transient expression in spli-221 and BmE cell lines to investigate the correlative regulation elements.Firstly,7 variants of BmLP3 promoter by PCR-based sequential segments deletion from the 5'flanking of total length of BmLP'3 promoter are obtained, and ligated into pGL3-basic plasmid to achieve 7 transient expression vectors named LUC-F1～LUC-F7. The results of transient expression of these vectors in BmE cell line indicate that the-250 to-151 region of BmLP3 promoter enhances the transcription level of luciferase gene. Further, two synthesized specific cy3 labeled probes of this region named probe 1, probe 2 and nuclear extracts from fat body at stage of five star 7th of silkworm are used to perform the EMSA, the results show that probe 1 bind to the nuclear extracts specifically. In order to figure out which parts in probe 1 participate in binding the nuclear extracts, we design Probe 1-1, Probe 1-2, Probe 1-3 in which some sequence miss at three positions, the results of EMSA that Probe 1-1 binds to the nuclear extracts significantly whereas Probe 1-2 and Probe 1-3 bind to the nuclear extracts weakly indicats that the core binding domain of BmLP3 promoter to nuclear extracts locates into the 21bp of Probe 1-1.To further understanding the function of this 21bp sequence, a transient expression vector in which the 21bp sequence is ligated to the upstream of the core BmLP3 promoter is created and tranfect into the BmE cell line, the analysis of LUC activity show this 21bp sequence enhance the activity of LUC up to about three folds, compared with the control vector LUC-F6, this indicate that the 21bp sequence can enhance the transcription activity of BmLP3 promoter in vitro.4 Expression analysis of DsRed gene driven by promoter of BmLP3 gene in transgenic Silkworm, Bombyx moriTo detect the activity of BmLP3 promoter in individual, we construct a piggyBac expression vector by using DsRed as a reporter gene, which is further used to conduct transgenic microinjection for verification of the promoter's characters in individual silkworm larvae. The results show as follow.1)The(?)eading frame of BmLP3-DsRed is inserted into the genome of silkworm in a single copy form and expressed in fat body-specific pattern in transgenic individuals. Insertion site is located on chromosome 20. DsRed expressed from day 4 of 5th instar larvae, reach a higher level in day 5 of 5th instar larvae, then continue to rise,achieve the highest expression in pupation. The expression levels gradually decrease after pupation, completely disappear in day 7 of pupae, in agreement with the expression pattern of endogenous gene BmLP3. Western blot results show that only a clear hybridization signal is in transgenic silkworm fat body, the position of hybridization signals is about 31K, consistent with the molecular weight of red fluorescent protein. A His-tag DsRed standard hybridization signal locate in about 35KD, no hybridization signal in other organizations. The results suggest red fluorescent protein express specifically in fat body, consistent with the result of fluorescence observation. The western blot hybridization signal of fat body sample is carried out with the standard gray scale to estimate the expression level of Red,the results show that body fat per 1 Oug contains 0.54μg of DsRed.2) The continuous observation of positive individuals find that rear end of positive individuals begin to send out red fluorescence in day 4 of 5th instar larvae.With the development of transgenic silkworm, the whole body can be observed to emit red fluorescence. The observation of different organizations of silkworm, such as silk gland, midgut, fat body and gonads, suggest red fluorescence is only in fat body, not in other tissues. After pupation, the entire pupa send a bright red fluorescence signal, no red fluorescence signal in control individuals. Observation of many transgenic individuals indicate the expression of red fluorescent protein is no difference in gender, express in both male and female individuals. Expression levels have some difference in individuals, such difference appear in different individuals of transgenic lines, as well as in the backcross progeny of the single copy individual.5 Activity determination of phytase driven by BmLP3 promoter in transgenic silkworm.As a feed additive, phytase play an important role in the feed industry. We explore the research using promoter BmLP3 to drive phytase expression in fat body of the silkworm, which lay the foundation for fat body-based bioreactor of silkworm. The phytase gene is synthesized and the recombinant pBac[BmLP3-phy A-SV40,3xP3EGFP] vectors for injection is obtained in two-steps using the cloning shuttle vector pSL[BmLP3-DsRed-SV40]. Transgene vector is microinjected into the preblastodermic eggs, transgenic silkworm is obtained by screening specific fluorescence. Finally, we establish transgenic silkworm that express recombinant phytase under the control of BmLP3 promoter. PCR is performed to identify the insertion and the result shows only the transgenic silkworm have positive signal while the control show none, which demonstrate the insertion of phytase gene. Total RNA is extracted from the second day of pupa, then RT-PCR is used to detect the expression of phytase gene. The result show phytase gene is transcripted only in transgenic silkworm. Total protein of transgenic silkworm is extracted from the second day of pupa, then we determine phytase activity under the temperature 0℃,37℃and 55℃using measures GBT 18634-200, phytase standard as positive control. The results show that protein from transgenic silkworm have enzyme activity. It has the same curve with phytase standard from 0℃to 55℃and show the highest activity up to 5.1U/g at 55℃. The research primarily confirmed that using fat body of silkworm as bioreactor to produce value-added exogenous protein is feasible.