The β2-tubulin gene from three tephritid fruit fly species and use of its promoter for sperm marking

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Abstract

To isolate testis-specific regulatory DNA that could be used in genetically transformed insect pest species to improve their biological control, β2-tubulin genes and their proximal genomic DNA were isolated from three economically important tephritid pest species, Anastrepha suspensa, Anastrepha ludens, and Bactrocera dorsalis. Gene isolation was first attempted by degenerate PCR on an A. suspensa adult male testes cDNA library, which fortuitously isolated the 2.85 kb β1-tubulin gene that encodes a 447 amino acid polypeptide. Subsequent PCR using 5′ and 3′ RACE generated the 1.4 kb Asβ2-tubulin gene that encodes a 446 amino acid polypeptide. Using primers to conserved sequences, the highly similar A. ludens and B. dorsalis β2-tubulin genes, encoding identical amino acid sequences, were then isolated. To verify Asβ2-tubulin gene identification based on gene expression, qRT-PCR showed that Asβ2-tubulin transcript was most abundant in pupal and adult males, and specific to the testes. This was further tested in transformants having the DsRed.T3 reporter gene regulated by the Asβ2-tubulin 1.3 kb promoter region. Fluorescent protein was specifically expressed in testes from third instar larvae to adults, and fluorescent sperm could be detected in the spermathecae of non-transgenic females mated to transgenic males.To confirm these matings, a PCR protocol was developed specific to the transgenic sperm DNA.

Introduction

The ability to create transgenic strains of economically and medically important insect species has the potential to greatly enhance our ability to improve existing biological control methods and develop more novel means of control. An important facet of this technology is the use of sex- and tissue-specific regulatory systems for directed gene expression (Handler, 2002). In particular, a testis or spermatocyte-specific promoter that directs gene expression specifically in male gonadal tissue has been recognized as a means to improve the existing sterile insect technique (SIT) by facilitating genetic sexing, male sterility, and sperm marking. A visible fluorescent marker expressed in spermatocytes can be used for larval male selection, identification of mated females in the field, and for sperm precedence studies. Lethal gene expression similarly directed by a testis-specific promoter could confer male sterility that would provide a major advance over radiation-induced sterility. A primary candidate for testis-specific promoter regulation comes from the β2-tubulin gene. This gene was first identified in Drosophila melanogaster as functioning solely during spermatogenesis during larval development and continuing throughout male adulthood. The β2-tubulin isoform is first observed in early spermatocytes, as a switch from β1-tubulin isoform production (Buttgereit and Renkawitz-Pohl, 1993), where it is specifically used in the axoneme for motile sperm development (Hoyle et al., 1995). This function has been elucidated, in part, by Drosophila β2-tubulin mutations that result in aberrant axonemal microtubules that disrupt sperm motility, with resultant sterility (Kemphues et al., 1982, Rudolph et al., 1987). Axoneme function is dependent, in particular, on a C-terminal tail amino acid motif specific to the β2-tubulin isoform whose variable nucleotide sequence distinguishes β2-tubulin from other members of the conserved tubulin family (Raff et al., 2008).

After Drosophila, the β2-tubulin gene was first isolated from a testes cDNA library from the moth Heliothis virescens (Davis and Miller, 1988) and more recently identified in the mosquitoes Anopheles stephensi (Catteruccia et al., 2005) and Aedes aegypti (Smith et al., 2007), and the tephritid fly, Ceratitis capitata (Scolari et al., 2008). In the latter three species the 5′ upstream promoter was linked to fluorescent protein genes that resulted in fluorescent sperm that could be detected in the testes, and for A. aegypti and C. capitata, in the spermathecae of females mated to transgenic males. The transparent nature of mosquito larvae allowed fluorescent testes to be identified in third instar males, with efficient sexing of A. stephensi demonstrated using an automated fluorescent-sorting system (Catteruccia et al., 2005).

Here we describe the isolation of the β2-tubulin gene from three tephritid fruit fly species, the Caribbean fruit fly, Anastrepha suspensa, the Mexican fruit fly, Anastrepha ludens, and the oriental fruit fly, Bactrocera dorsalis. Linking the promoter of the A. suspensa β2-tubulin (Asβ2tub) to the DsRed.T3 fluorescent protein (Bevis and Glick, 2002) allowed characterization of the gene by the tissue and developmental specificity of its expression. Initial efforts to isolate the β2-tubulin gene from A. suspensa resulted in the fortuitous isolation of the closely related β1-tubulin gene, allowing structural and developmental comparisons.

Section snippets

Fruit fly rearing and nucleic acid preparation

A wild-type laboratory colony of A. suspensa (Homestead, Florida) and transgenic strains created from this colony were maintained at 25 °C in a larval diet of wheat germ-yeast-glucose. Third instar larvae were transferred to vermiculite with pupae maintained under humid conditions until adult emergence, and adults maintained on a yeast-sucrose diet. Pupal samples were provided from wild type strains of A. ludens (Tapachula, Mexico), B. dorsalis (Kahuku, Hawaii), and Bactrocera papayae (Penang,

β1-tubulin and β2-tubulin gene isolation from A. suspensa

Isolation of the β2-tubulin gene from A. suspensa was first attempted by PCR on an adult male testes cDNA library using degenerate primers to conserved sequences from the β1-tubulin and β2-tubulin isoforms. It was thought that enrichment for the β2-tubulin transcript in male testes would result in this being the favored amplified product. PCR-generated genomic sequences, however, indicated that the β1-tubulin isoform was first isolated based on the presence and position of a 1.5 kb 5′ intron

Discussion

The testis-specific β2-tubulin gene and its 5′ upstream regulatory sequence region have been isolated and sequenced from three economically important tephritid fruit fly species, and the constitutive β1-tubulin gene has been isolated from A. suspensa. Consistent with other β2-tubulin genes, there is a high level of amino acid conservation among the tephritid species and with other insects, as well as some nucleotide sequence conservation in the promoter region among the tephritids. Conservation

Acknowledgements

This article is dedicated to the memory of the lead author, Grazyna J. Zimowska, who passed away during its preparation. Dr. Zimowska made significant contributions to insect physiology, biochemistry and molecular genetics as a scientist and mentor, and her insights, expertise and thoughtfullness will be greatly missed. Grateful appreciation is extended to Ryan Smith and Peter Atkinson for discussions and sharing data previous to publication, and to Jennifer Mestas and Rob Harrell for technical

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