番石榴实蝇性别决定基因Bcotra和Bcotra-2的克隆、序列特征及表达分析

Cloning, sequence characterization and expression analysis of sex determining genes Bcotra and Bcotra-2 in the guava fruit fly,Bactrocera correcta( Bezzi)( Diptera: Tephritidae)

【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。

【Aim】This study aims to isolate two sex determining genes（ transformer and transformer 2）,analyze their genomic DNA and c DNA structures and determine their expression profiles during different developmental stages and in adult tissues of the guava fruit fly,Bactrocera correcta. The findings will provide basic knowledge for further functional study of these two sex-determinating genes and the construction of the genetic sexing strain of the guava fruit fly. 【Methods 】 The full-length c DNA sequences and their intronic seqences were isolated by PCR and RACE technique. The structure prediction,sequence alignment and phylogenetic analysis of the coding products of these two sexdetermining genes were performed by using different bioinformatics softwares. Based on the c DNA sequences of these two genes,specific primers were designed to investigate their developmental and tissue expression profiles by semi-quantitative RT-PCR. 【Results 】 Two full-length c DNA sequences of transformer and transformer 2 were isolated from B. correcta and named as Bcotra and Bcotra-2,respectively. Bcotra is transcribed sex-specifically： female transtranscript is 1 673 bp in length,which encodes a polypeptide of 413 amino acids（ Gen Bank accession number KP712876）, while male transcript is 2 025 bp in length,which encodes a truncated and non-functional polypeptide with in-frame stop codons on the two addtional exons that do not exist in female transcript（ Gen Bank accession number KP712877）. No sex-specific transtript was detected in Bcotra-2. Bcotra-2 is 1 458 bp in length,which encodes a polypeptide of 251 amino acids with the classic characteristics of RNA binding proteins（ Gen Bank accession number KM658207）. Bcotra-2 harbors 8 exons and 7 introns. Sequence alignment and phylogenetic analysis of the products coded by these two sex determining genes respectively with the known Tra and Tra-2 from other Dipteran insects revealed that Bcotra-2 shows higher homology than that of Bcotra with their respective homologues. The semi-quantitative RT-PCR result showed that Bcotra and Bcotra-2 was expressed in different developmental stages and various adult tissues of B. correcta.【Conclusion】 In this study,the genomic and c DNA stuctures of two sex determining genes were identified in B. correcta. Both Bcotra and Bcotra-2 were detected during different developmental stages and in various adult tissues. The presence of Tra / Tra-2 binding sites,intronic splicing silencer sequence and RNA binding protein binding sites suggests that the regulation of sex development in B. correcta may rely on the interation between the post-translation of Bcotra and Bcotra-2. The fragment of 963 bp femalespecific intron of Bcotra can be used for the vector construction to develop genetic sexing strains.