Mol Biol Rep (2012) 39:10325–10330 DOI 10.1007/s11033-012-1909-0
A new gene, SRP16, differentially expressed in the spermathecae of honeybee queens (Apis mellifera) related with reproduction status Liming Wu • Danping Wuxiang • Huoqing Zheng Jilian Li • Gang Pan
•
Received: 2 December 2010 / Accepted: 1 October 2012 / Published online: 16 October 2012 Ó Springer Science+Business Media Dordrecht 2012
Abstract Honey bee queens have the ability to store sperm in spermathecae for fertilizing eggs throughout their life. To investigate mechanisms for sperm storage in Apis mellifera, we employed suppression subtractive hybridization (SSH) to find differentially expressed fragments in spermathecae between virgin queens and newly mated queens. A new gene, named SRP16, was obtained by joining the SSH products with 50 -RACE and 30 -RACE. SRP16 is predicted to encode a 41 kDa protein with 363 amino acid residues. Its expression was found in the spermathecae dominantly in honey bee queens but not in honey bee workers, with the highest expression found in spermathecae of virgin and newly mated queens. SRP16 expression was weak in other tissues of queens other than in the spermathecae and showed no obvious change with reproductive status of queens. The results suggest that SRP16 may play important roles in sperm storage and honey bee reproduction. Keywords Apis mellifera Sperm storage Spermatheca SRP16 protein
L. Wu J. Li Bee Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China D. Wuxiang G. Pan (&) Sericulture Research Institute, Jiangsu University of Science and Technology, Zhenjiang, China e-mail:
[email protected] H. Zheng College of Animal Sciences, Zhejiang University, Hangzhou, China
Introduction Honey bee queens normally have only one mating opportunity at the start of their adult life [1–3]. During their mating flight virgin honey bee queens will mate with drones for many times [4–7]. After mating about 0.78 ll of sperm (more than 1 million spermatozoa) is passively and actively transferred into spermatheca, where it is stored during a queen’s life until it is used for the fertilization of eggs [8], and normally, sperm can remain viable and retain respiration in the spermatheca of mated female honey bees for several years [9]. But in vitro, there is no satisfactory long-time preservation of Apis sperm at room temperature or at slightly cooler temperature [10, 11]. It seems possible that for the long term storage of many years, honey bee queens produce special proteins which may be necessary for the survival of spermatozoa [12–14], and antioxidative enzymes are also believed to play important roles in sperm storage [9]. Another speculation believed the significant protein was brought into spermatozoa by sperm of drone. Harbo suggested that a queen releases a specific volume of spermatozoa-containing fluid from its spermatheca which can be replenished by the fluid produced by queen itself, so that the concentration of the spermatozoa in the spermatheca would be reduced by redistribution [15]. There are a few functional proteins in spermatheca were reported up to the present in which two were isolated by one and two dimensional SDS-PAGE electrophoresis in sexually mature queens’ spermatheca and a 29 kDa protein was characterized [16]. A new gene was found in mated bee queen spermatheca by suppression subtractive hybridization technology in the process of mechanisms investigation of sperm storage in Apis mellifera. Differential gene expression analysis
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revealed that this gene may play an important role in sperm storage of spermatheca.
Materials and methods Samples collection Honey bee queens (A. mellifera) were reared in the apiary of the Bee Research Institute, Chinese Academy of Agricultural Sciences. Virgin queens were collected immediately after they emerged from queen cells. Newly mated queens were collected once young queens were confirmed laying eggs after naturally mated. Aged mated queens (1 year old) were collected after they were discarded by beekeepers due to their low fertility. Spermathecae and ovaries of virgin queens, newly mated queens and aged mated queens were collected by dissecting on ice respectively, then immediately frozen in liquid nitrogen. Trizol reagent (Invitrogen) was added after the tissue was ground into a smooth, fine powder. Total RNA concentration was determined using a biophotometer (Eppendorf) by measuring absorbance at 260 and 280 nm (A260:A280). Poly A? RNA was purified from the total RNA using OligotexTM–dT30 \Super[ mRNA Purification kit (Takara). 1 lg of PolyA RNA was used as the starting material for reverse transcription to get cDNA. RNA samples were stored at -80 °C before analysis. cDNA synthesis was performed with Reverse Transcriptase M-MLV(Takara). RT products were stored at -80 °C before analysis. Suppression subtractive hybridization Suppression subtractive hybridization (SSH) was performed using the PCR-select cDNA subtraction kit (Clontech). The subtractions were constructed according to the manufacturer’s direction by using the cDNA of the newly mated queens as the tester and the cDNA of the virgin queens as the driver [17, 18]. cDNA was synthesized with 1 lg purification mRNA and cut into fragments by Rsa I digestion. cDNA from the tester was separated into two portions and adapters 1 and 2R were added each to one of the two parts. Testers were then separately hybridized with excess drivers in a ratio of 1:30 (tester:driver) for the first hybridization and followed by a second hybridization Table 1 Primers used for prokaryotic expression
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by adding 1 ll driver mixture to the first hybridization solution. Finally, suppression and nested PCR amplification was performed to amplify differentially expressed genes. PCR products were purified and cloned into the pMDTM 18-T vector (Takara) then transformed into DH10B competent cells. The clones were screened on LB broth plates containing ampicillin/X-gal/IPTG. Positive clones were chosen for sequencing. 50 - and 30 -RACE 50 - and 30 -RACE were applied to obtain full-length cDNA of the fragments from SSH. The procedure was according to the manufacturer’s protocol of Smart Race cDNA Amplification Kit (Clontech) [19, 20]. The CDS Primer A was used for reverse transcription to get 30 -RACE-Ready-cDNA. The Gene-Specific Primer (GSP) for 30 -RACE was designed based on the sequencing result of SSH and primer for 50 RACE was designed basing on 30 -RACE sequencing result. 30 -RACE GSP: 50 -GGTCCTGGATTAGATTCCGATG-30 , 50 -RACE GSP: 50 -CGGAATCTAATCCAGGACC-30 . PCR products were purified, cloned and transformed. Positive clones were selected for sequencing. Prokaryotic expression and SDS-PAGE Primers SAEST SRP16F and SAEST SRP16R (Table 1) were designed for amplification of the full length open reading frame of spermatheca related protein 16 (SRP16) gene. Restriction endonuclease sites for vector ligation were also considered and included. PCR products amplified by SAEST SRP16 primer pairs were double-digested with BamHI and HindIII, purified and ligated into the plasmid of PET-28a digested with the same restriction enzymes [21–23]. The positive recombinant plasmids PET-28aSRP16 were extracted (Ez-10 Spin Column Plasmid MiniPreps Kit, Sangon) and verified by PCR and double restriction enzyme digestion. The recombinant plasmids were then transformed into competent E.coli BL21. Simultaneously, the non-inserted pET-28a vector was acted as the control. When OD600 reached 0.4–0.6, the cells were induced with 1 mM IPTG for 4 h, then harvested by centrifugation (5,000 rpm, 4 °C, 10 min) and boiled for 10 min. The supernatant was collected after centrifugation (12,000 rpm, 4 °C, 5 min) for SDS-PAGE electrophoresis.
Primer name
Sequence
Restriction enzyme
SAEST SRP16F
50 -CGATCGGATCCATGCGTCCCGAACTCGAGAT-30
BamHI
SAEST SRP16R
50 -TGACTAAGCTTCTACGCGCTAGCATAACATC-30
HindIII
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Electrophoresis in denaturing conditions [24] was carried out at 15 mA. Sodium dodecyl sulfate-polyacrylamide gel electrophoreses (SDS-PAGE) were carried out using conventional method in a minigel apparatus. Stacking gels contained 3 % acrylamide with pH 6.8. Separation gel contained 10 % acrylamide with pH 8.8. RT-PCR for tissue expression profile analysis Total RNA from worker bees’ tissues, the spermathecae and ovaries of virgin queens, newly mated queens and aged mated queens were extracted by Trizol reagent. A semi-quantitative RT-PCR assay was designed to measure SRP16 gene expression in worker bees, queen bees, the spermathecae and ovaries of virgin queens, newly mated queens and aged mated queens. A. mellifera actin related protein gene (ARP1) (GenBank accession number NM 001185146), a house-keeping gene, was used as a reference gene to normalize mRNA levels. The sequence of the primers are in Table 2. The thermal cycling parameters were: 95 °C 4 min, 26 cycles of 95 °C 30 s, 58 °C 30 s, 72 °C 30 s, 72 °C 7 min. The PCR products were loaded on to ethidium bromide-stained 1.5 % agarose gels in TAE.
Results Differentially expressed gene in spermatheca of virgin queens and newly mated queens. Dozens of DNA fragments were obtained from the SHH experiment. A full length cDNA was obtained by joining of 50 -RACE and 30 -RACE among these fragments. This cDNA is predicted to encode a polypeptide of 363 amino acid residues (Fig. 1) with a theoretical molecular mass of 41 kDa. No similarities were found between this cDNA translated product and other amino acid sequences in the GenBank database. Hydrophobicity plot analysis showed that the amino acid sequence was high hydrophilic except two predicted transmembrane domains with 5 amino acid residues length situated in 126–130 position and 15 amino acid residues situated in 186–200 position (Fig. 2). Glycosylation analysis revealed there were two N-linked glycosylation sites located in 224–226 and 297–299 respectively. Because this cDNA was derived from No.16 EST fragment Table 2 Specific primers used for semi-quantitative real-time Primer name
Sequence
Apis actinF
50 -GACGGTGTTTCACACACTGTACC-30
Apis actinR
50 -CCATTCCTAAGAAGGATGGTTTGG-30
SRP16F
50 -GAGAATGCTAAACAAGCGCTAG-30
SRP16R
50 -AGGTCTTGGGGGGTGGTACTT-30
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and found in A. mellifera spermatheca tissue firstly, we named this new gene as SRP16 Gene. GenBank accession number is HQ214486. Prokaryotic expression and SDS-PAGE To confirm the SRP16 translated product, SRP16-PET28a prokaryotic expression was constructed and the positive clones were expressed in BL21 cells. SDS-PAGE electrophoresis results indicated that the expression product located between 35 kDa and 45 kDa, which were consistent with the gene coding prediction. (Fig. 3). Tissue transcription profile In order to know whether the gene is induced expression by mating or specifically expressed in spermatheca tissue, the queen reproductive tissues were analyzed. Between spermatheca and ovary, SRP16 expression was more abundant in spermatheca than in ovary. In spermatheca, SRP16 expression was higher in virgin queens and newly mated queens than in aged mated queens. In ovaries, SRP16 exhibited no obvious difference in virgin queens, newly mated queens and aged mated queens (Fig. 4). To further understanding this gene, we compared the gene expression among worker bee tissues (Fig. 5) and among queen bee tissues (Fig. 6). No SRP16 expression was detected in worker bee tissues including head, hypopharyngeal gland, thorax, abdomen, midgut and legs. SRP16 expressed in all queen bee tissues we examined. But compare to spermatheca, SRP16 just expressed weakly in other tissues.
Discussion The spermatozoa storage in honey bee queens is a complex trait that is still not well understood. Very few proteins have been shown to be required for honey bee spermatozoa storage. To understand how the honey bee queens store spermatozoa throughout their life, it is necessary to find proteins or clone genes in honey bee queen spermatheca. Klenk found a 29 kDa protein in spermatheca by twodimensional SDS-PAGE method [16]. This study employed a suppression subtractive hybridization method to try identifying genes specific to spermatheca or mated queen spermatheca at the mRNA level. A new gene was found which we named A. mellifera spermatheca related protein gene 16 (SRP16). SRP16 is predicted to have hydrophilic structure in most of its sequence with two potential N-linked glycosylation sites. No gene was found homologous to SRP16 in the GenBank database. SRP16 expressed in queen bees not in worker bees and the tissue transcription profile revealed
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Fig. 1 Full length cDNA sequence of SRP16 gene N-linked glycosylation sites were underlined in amino acid sequence and ‘‘aaata’’ signal was underlined in nucleotide sequence
Fig. 3 Prokaryotic expression of A. mellifera SRP16 gene M, Protein molecular weight Marker. Lane1, pET-28a/BL21 induced by IPTG. Lane2, pET-28a-SRP16/BL21 induced by IPTG Fig. 2 Hydrophobicity plot analysis of A. mellifera SRP16
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important to sperm storage. More knowledge of the function of proteins in the spermatheca may provide a step towards the understanding of the sperm storage mechanisms in females.
Fig. 4 The expression of SRP16 gene in honey bee tissues 1–3, ovary from virgin queens, newly mated queens and aged mated queens. 4–6, spermathecae from virgin queens, newly mated queens and aged mated queens. ARP1, A. mellifera actin related protein gene, GenBank accession number NM 001185146
Fig. 5 The expression of SRP16 in worker bee tissues. 1 head without hypopharyngeal gland. 2 hypopharyngeal gland. 3 thorax. 4 abdomen without midgut. 5 midgut. 6 legs
SRP16
ARP1 Fig. 6 The expression of SRP16 in queen bee tissues. 1 head. 2 thorax. 3 ovary. 4 spermathecae. 5 midgut. 6 legs
that SRP16 expressed in spermatheca dominantly in queen bees. These results suggest that SRP16 may be associated with honey bee reproduction. SRP16 expression was found in spermatheca of all three kinds of queens tested, not only in spermatheca of mated queens. This fact suggests that SRP16 expression is not induced by fertilization. SRP16 is expressed at a higher level in spermatheca of virgin queens and newly mated queens than in spermatheca of aged mated queens. Suggesting that SRP16 may pre-exist in spermatheca for spermatozoa storage and SRP16 expression will decrease with decreasing amounts of spermatozoa stored in spermatheca. Studies on Drosophila melanogaster have suggested that the male seminal fluid proteins introduced into females by sperm ejaculation were essential for efficient sperm storage by females [25, 26]. Also there are some other factors which contribute to germ storage by honey bee queens such as pH value, Zn2? concentration, antioxidase activity etc. [9, 27, 28]. In this study, we believe that proteins produced by honey bee queens themselves in spermatheca are
Acknowledgments We thank Tim Bryant and Dan Ward for preparation our manuscript. This work was supported by grants of national natural science foundation of China (No.30771636) and the earmarked fund for Modern Agro-industry Technology Research System of China (CARS-45-KXJ8).
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