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

›› 2015, Vol. 58 ›› Issue (5): 516-525.doi:

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

刘桂清^1,2, 武强¹, 和丹阳¹, 张桂芬¹, 李建伟¹, 万方浩^1,3,*

(1. 中国农业科学院植物保护研究所, 植物病虫害生物学国家重点实验室, 北京100193;2. 广东省昆虫研究所, 广东省野生动物保护和利用公共实验室, 广东省农业害虫综合治理重点实验室, 广州 510260; 3. 青岛农业大学农学与植物保护学院, 山东青岛 266109)

出版日期:2015-05-20 发布日期:2015-05-20
作者简介:刘桂清, 女, 1981年生, 江西新余人, 博士研究生, 助理研究员, 研究方向为昆虫分子生物学与外来生物入侵, E-mail: pepsiliu81@163.com

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

LIU Gui-Qing^1,2, WU Qiang¹, HE Dan-Yang¹, ZHANG Gui-Fen¹, LI Jian-Wei¹, WAN Fang-Hao^1,3,*

(1. State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China; 2. Guangdong Key Laboratory of Integrated Pest Management in Agriculture, Guangdong Public Laboratory of Wild Animal Conservation and Utilization, Guangdong Entomological Institute, Guangzhou 510260, China; 3. College of Agronomy and Plant Protection, Qingdao Agricultural University, Qingdao, Shandong 266109, China)

Online:2015-05-20 Published:2015-05-20

摘要/Abstract

摘要： 【目的】对番石榴实蝇 Bactrocera correcta (Bezzi)性别决定基因 transformer 和 transformer 2 的cDNA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cDNA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cDNA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cDNA全长1 673 bp，其开放读码框(ORF)为1 242 bp，编码413个氨基酸(GenBank登录号为KP712876)；雄虫Bcotra cDNA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(GenBank登录号为KP712877)。 Bcotra-2 不存在性别特异剪接，cDNA全长1 458 bp，其开放读码框(ORF)为756 bp，编码251个氨基酸，具有RNA结合蛋白的典型特征(GenBank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cDNA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。

关键词: 番石榴实蝇, 性别决定, transformer, transformer 2, 性别特异剪接, 表达谱

Abstract: 【Aim】 This study aims to isolate two sex determining genes (transformer and transformer 2), analyze their genomic DNA and cDNA 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 cDNA 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 sex determining genes were performed by using different bioinformatics softwares. Based on the cDNA 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 cDNA sequences of transformer and transformer 2 were isolated from B. correcta and named as Bcotra and Bcotra2, respectively. Bcotra is transcribed sex-specifically: female transtranscript is 1 673 bp in length, which encodes a polypeptide of 413 amino acids (GenBank 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 (GenBank 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 (GenBank 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 cDNA 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 female-specific intron of Bcotra can be used for the vector construction to develop genetic sexing strains.

Key words: Bactrocera correcta, sex determination, transformer, transformer 2, sex-specific splicing, expression profile

刘桂清, 武强, 和丹阳, 张桂芬, 李建伟, 万方浩. 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[J]. , 2015, 58(5): 516-525.

LIU Gui-Qing, WU Qiang, HE Dan-Yang, ZHANG Gui-Fen, LI Jian-Wei, WAN Fang-Hao. Cloning, sequence characterization and expression analysis of sex determining genes Bcotra and Bcotra-2 in the guava fruit fly, Bactrocera correcta (Bezzi) (Diptera: Tephritidae)[J]. , 2015, 58(5): 516-525.

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