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【目的】对番石榴实蝇Bactrocera correcta(Bezzi)性别决定基因transformer和transformer 2的c DNA和基因组DNA序列进行克隆和分析,明确这2个基因的结构特征及其在不同发育阶段和雌、雄成虫不同组织中的表达模式,为进一步的功能研究和番石榴实蝇遗传性别品系(genetic sexing strain,GSS)的建立奠定基础。【方法】利用PCR结合RACE技术克隆番石榴实蝇2个性别决定基因的c DNA全长和内含子序列,利用不同的生物信息学软件对序列进行结构预测、序列比对和进化树分析;利用半定量RT-PCR检测这2个基因在番石榴实蝇的不同发育阶段及雌、雄成虫不同组织(精巢、卵巢、中肠和脂肪体)中的表达分布。【结果】克隆得到番石榴实蝇transformer和transformer 2的c DNA全长序列,分别命名为Bcotra和Bcotra-2。Bcotra存在性别特异剪接,雌虫Bcotra的c DNA全长1 673 bp,其开放读码框(ORF)为1 242 bp,编码413个氨基酸(Gen Bank登录号为KP712876);雄虫Bcotra c DNA全长2 025 bp,比雌虫多2个外显子,但由于外显子上有多个终止密码子,因此,不能编码完整的有功能的Tra蛋白(Gen Bank登录号为KP712877)。Bcotra-2不存在性别特异剪接,c DNA全长1 458 bp,其开放读码框(ORF)为756bp,编码251个氨基酸,具有RNA结合蛋白的典型特征(Gen Bank登录号为KM658207)。Bcotra-2有8个外显子,7个内含子。氨基酸序列比对和系统进化关系表明,两个基因的系统发育关系一致,Tra-2与目前已报道的双翅目Tra-2具有很高的同源性,而Tra的保守性较Tra-2要低。半定量RT-PCR结果显示,Bcotra和Bcotra-2在番石榴实蝇的不同发育阶段及雌、雄成虫不同组织中都有表达。【结论】本研究明确了Bcotra和Bcotra-2的基因组DNA和c DNA结构特征,Bcotra和Bcotra-2在番石榴实蝇的不同发育阶段和成虫不同组织中均有表达,序列分析发现这两个性别决定基因均具有Tra/Tra-2结合位点、内含子剪接抑制序列位点,其中Bcotra具有RNA结合蛋白的结合位点,暗示了这2个基因可能通过翻译后相互作用调控雌、雄体性发育。Bcotra存在性别特异剪接,雌虫特有的一段963 bp的内含子序列可以用于番石榴实蝇遗传定性品系的载体构建。
【Objective】 The objective of this study was to clone and analyze the c DNA and genomic DNA sequences of Bactrocera correcta (Bezzi) sex-determining genes transformers and transformer 2, to clarify the structural characteristics of these two genes and their roles in female and male at different developmental stages The expression patterns of different adult tissues of adult worms lay the foundation for further functional studies and establishment of genetic sexing strain (GSS). 【Method】 The full-length c and intron sequences of two sex-determining genes of guava fruit fly were cloned by PCR and RACE technology. The structure prediction, sequence alignment and phylogenetic tree analysis were carried out using different bioinformatics softwares. Semi-quantitative RT-PCR was used to detect the expression profiles of these two genes in different developmental stages of guava fruit fly and in different tissues of female and male adults (testis, ovary, midgut and fat body). 【Result】 The full-length cDNA of c DNA of transformer and transformer 2 was cloned and named as Bcotra and Bcotra-2 respectively. Bcotra had sex-specific splicing. The c DNA of female Bcotra was 1 673 bp in length and 1 242 bp in open reading frame (ORF), encoding 413 amino acids (Gen Bank Accession No. KP712876) It is 2 025 bp long and 2 exons more than the female. However, due to multiple stop codons on exons, it can not encode a complete functional Tra protein (GenBank Accession No. KP712877). Bcotra-2 does not have sex-specific splicing. The c DNA is 1 458 bp in length. Its open reading frame (ORF) is 756 bp and encodes a polypeptide of 251 amino acids with typical characteristics of RNA-binding protein (GenBank accession number is KM658207). Bcotra-2 has eight exons and seven introns. The amino acid sequence alignment and phylogenetic relationship indicated that the phylogenetic relationship between Tra-2 and Trap 2 was highly homologous to that of Tra-2, while Tra was more conservative than Tra-2 To be low. Semi-quantitative RT-PCR results showed that Bcotra and Bcotra-2 expressed in different tissues of female and male adults at different development stages of guava fruit fly. 【Conclusion】 The study identified the structural characteristics of genomic DNA and c DNA of Bcotra and Bcotra-2. Bcotra and Bcotra-2 were expressed in different developmental stages and different adult tissues of guava fruit fly. The sequence analysis showed that the two Sex-determining genes all have Tra / Tra-2 binding sites and intron splice-inhibiting sites, of which Bcotra has a binding site for RNA-binding proteins, suggesting that these two genes may regulate post-translational interactions between female and male Physical development. Sex-specific splicing exists in Bcotra, and a 963-bp intron sequence specific to females can be used to construct vectors for the qualitative genetic line of guava fruit fly.