›› 2018, Vol. 61 ›› Issue (6): 627-636.doi: 10.16380/j.kcxb.2018.06.001

• 研究论文 •    下一篇

家蚕油蚕oc突变体突变基因的精细定位

殷娅茹, 胡建, 胡文波, 杨成飞, 王坤, 刘春, 林英, 朱勇*, 王凌燕*   

  1. (西南大学生物技术学院, 家蚕基因组生物学国家重点实验室, 重庆 400716)
  • 出版日期:2018-06-20 发布日期:2018-06-20

Fine mapping of mutant gene in the chinese translucent (oc) mutant of the silkworm, Bombyx mori

YIN Ya-Ru, HU Jian, HU Wen-Bo, YANG Cheng-Fei, WANG Kun, LIU Chun, LIN Ying, ZHU Yong*, WANG Ling-Yan*   

  1.  (State Key Laboratory of Silkworm Genome Biology, College of Biotechnology, Southwest University, Chongqing 400716, China)
  • Online:2018-06-20 Published:2018-06-20

摘要: 【目的】油蚕oc突变体是家蚕Bombyx mori油蚕突变体的一种,经典遗传学连锁图谱已经将oc突变基因定位在5号染色体40.8 cM座位。本研究旨在对oc突变体候选基因进行精细定位并克隆,探究oc性状形成的分子机制。【方法】以油蚕oc突变品系和野生型家蚕品系大造(Dz)为亲本,其杂交产生的F1代雄性个体与oc突变的雌性个体进行回交得到的1 397头BC1代个体为定位材料,以家蚕已经报道的基因组序列为参考设计markers,通过亲本及F1代个体筛选多态性markers,并利用多态性markers和BC1代个体对oc突变基因进行精细定位。通过半定量RT-PCR和实时荧光定量PCR(qPCR)筛选oc紧密连锁区间内的候选基因,确定目标候选基因,继而克隆和测序该基因,分析油蚕oc突变的原因。【结果】利用1 397头BC1代个体和11对有效的多态性markers将oc突变基因定位在M10与M11两个markers之间,物理图谱距离大约为234 kb。通过家蚕基因组数据库对oc连锁区域内的基因进行检索发现该区域有5个预测基因。对这5个预测基因在10个家蚕品系中进行的表达分析发现,只有BGIBMGA003572基因在oc突变个体体壁的表达量明显比正常个体中的低。通过基因的同源分析发现该基因编码的蛋白和人类单羧酸转运蛋白9(可能的尿酸转运蛋白)是同源蛋白,推测其为oc突变的候选基因。对BGIBMGA003572进行的克隆和测序结果显示其编码序列有5个氨基酸在oc突变体中发生了突变。【结论】通过定位克隆,本研究将oc突变基因定位在了234 kb的紧密连锁区间,其中编码单羧酸转运蛋白9的BGIBMGA003572可能和oc突变体的表型有关。
 

关键词: 家蚕, 油蚕突变体, 尿酸; oc突变体, 精细定位

Abstract: 【Aim】 The chinese translucent (oc) mutant is one of the translucent mutants in the silkworm, Bombyx mori. Its responsible gene has been mapped at 40.8 cM on chromosome 5. The purpose of this study is to fine map and clone the candidate gene for oc mutation and to explore the molecular mechanism of the oc mutant. 【Methods】 A BC1 generation was got by hybridizing male F1 (the mutant strain oc×the wild-type strain Dz) with female oc. The markers were designed according to reference genomes, and those showing polymorphism between oc and Dz were used for the genetic analysis of 1 397 BC1 individuals. The candidate genes in the oc tightly linked region were analyzed by semi-quantitative RT-PCR and qPCR, and the target candidate gene was identified, cloned and sequenced. The cause of oc mutation was analyzed. 【Results】 The oc locus was mapped to the 234 kb region between markers M10 and M11 using 1 397 BC1 individuals and 11 polymorphic markers. There are five predicted protein-coding genes in this region. The expression analysis of the five predicted genes in the integument of ten strains of B. mori indicated that only BGIBMGA003572 was severely suppressed in the oc mutant as compared with in the wildtype strain. The sequence homology analysis indicated that the encoded protein of BGIBMGA003572 is homologous to the human monocarboxylate transporter 9, a possible uric acid transporter, which might be the candidate gene for oc mutation. The cloning and sequencing results of BGIBMGA003572 showed that five amino acids changed in the oc mutant as compared with the wild-type strain. 【Conclusion】 In this study, the oc locus was mapped to the 234 kb region by positional cloning technique, in which BGIBMGA003572 encoding a monocarboxylate transporter 9 might contribute to the phenotype of oc mutant.

Key words: Bombyx mori; translucent mutant, uric acid, oc mutant, fine mapping