【作者】 杨玲；

【导师】 代方银；

【作者基本信息】 西南大学 ， 遗传学， 2021， 硕士

【摘要】 自然界昆虫卵色多样性与其环境适应性密切相关。探究昆虫卵色形成的生化与遗传基础,将为揭示昆虫外观色的演化提供重要线索。生物体外观颜色分为色素色和结构色。色素物质是昆虫色素色的物质基础,其种类、含量以及空间定位的变化都可能产生外观色多样性。因此,了解参与卵色形成的色素物质,其在体内的合成和代谢过程,以及这些物质如何被转运和沉积到特定位置,是揭示昆虫色素色及其多样性形成的重要基础研究。家蚕作为著名经济昆虫和鳞翅目的重要模式,不仅有深厚的遗传学研究基础和高质量的参考基因组,而且其突变体库中还保存有丰富的卵色突变体,这为研究昆虫卵色形成机制提供了便利。家蚕第4褐卵（b-4）是一种自然突变体,卵呈紫红色,成虫复眼为深红色,经典遗传学研究表明b-4位点位于第20号染色体。本研究以家蚕b-4突变体为研究材料,通过定位克隆方法鉴定其控制基因,并初步探究了卵色形成的分子机制,取得的研究结果如下:1.将家蚕b-4位点锁定在20号染色体约310kb的基因组区域根据家蚕雌完全连锁的特点,配制BC₁M代杂交分离群体,对b-4进行了分子定位。用筛选得到的13个位于20号染色体的多态分子标记对BC₁M代985个红色复眼个体的DNA进行基因分型实验,将b-4锁定到20号染色体约310kb的基因组范围,该区间内包含14个编码基因。2.通过表达分析、序列分析及功能注释,将b-4的候选基因锁定为Bm-tmem68和Bm-mah对定位区间内14个基因在大造和b-4突变体卵色形成过程中的表达模式进行调查,发现在卵色形成关键时期即产卵后24h-48h时间段内,KWMTBOMO12114KWMTBOMO12115、KWMTBOMO12116、KWMTBOMO12118和KWMTBOMO12119这5个基因在两品系的表达量存在显著差异;同时,对14个基因的编码序列进行克隆分析,发现KWMTBOMO12116和KWMTBOMO12119两个基因在大造和b-4中存在不同剪接形式的转录本,异常的转录本均会导致编码蛋白结构形成缺陷。基于表达分析及序列分析,将b-4的候选基因锁定为KWMTBOMO12116和KWMTBOMO12119。对两个基因进行功能注释:KWMTBOMO12116同源于大鼠Tmem68基因,编码跨膜蛋白;KWMTBOMO12119同源于果蝇mah基因,编码氨基酸转运蛋白,在果蝇中,该基因功能缺失导致复眼表型形成缺陷。3.Bm-mah基因的上游在b-4突变体中存在特异序列差异在大造和b-4突变体中调查了Bm-mah基因的调控序列,发现在b-4中该基因转录起始位点上游500bp处存在4个单碱基替换及4个碱基的缺失。随后在22个不同卵色的家蚕品系中调查了该位点序列,结果显示Bm-mah基因上游调控序列发生的碱基替换及缺失是b-4特有的。4.在大造中干涉Bm-mah基因后卵色变成红褐色为确定b-4突变体的控制基因,对Bm-tmem68和Bm-mah进行RNAi实验。结果显示,Bm-tmem68被干涉后卵色无变化,而Bm-mah在大造中被干涉后,卵的固有色呈红褐色,与b-4卵的颜色相似。因此,我们确定Bm-mah基因是b-4突变体的控制基因。5.Bm-mah基因与Bm-re基因可能共同参与调控家蚕卵色的形成在Bm-mah RNAi个体中调查了眼色素合成通路中Bm-KMO、Bm-white、Bm-scarlet、Bm-cardinal和Bm-re的表达情况。结果显示Bm-mah干涉后,红卵（re）的控制基因Bm-re表达量显著降低。进一步调查了Bm-mah和Bm-re在b-4突变体、re突变体与野生型大造中的表达情况,发现Bm-mah和Bm-re基因具有相似的表达模式,且在卵色形成的关键期,它们在b-4、re突变体中的表达量都显著低于大造。综上所述,本研究通过分子定位、基因表达模式及表达差异分析、上游序列分析,锁定了b-4的候选基因,并通过RNAi对候选基因进行了功能研究,证明Bm-mah为b-4的控制基因,编码具有11个跨膜结构域的氨基酸转运蛋白,是昆虫眼色素代谢通路的新基因。本研究为进一步探明昆虫眼色素合成通路下游关键步骤提供了重要线索。更多还原

【Abstract】 The diversity of insect egg color in nature is closely related to its environmental adaptability.It is an important clue,provided by exploring the biochemical and genetic basis of insect egg color formation,to reveal the evolution of insect appearance color.The appearance color of organisms can be divided into pigment colors and structural colors.Pigment material is the substance basis of insect pigment color,and the change in its type,content,and spatial positioning may produce a variety of appearance colors.Therefore,understanding the pigment substances involved in egg color formation,their synthesis and metabolism processes in the body,and how these substances are transported and deposited to specific locations is an important basic research to reveal the formation of insect pigment colors and their diversity.As a famous economic insect and an important model organism of Lepidoptera,Bombyx mori not only has a deep genetic research foundation and high-quality reference genome,but also has a wealth of egg color mutants stored in its mutant library,which is provided convenience to study the mechanism of insect egg color formation.The fourth brown egg（b-4）is a mutant of silkworm which has reddish-brown serosa and dark red compound eyes in adult and this mutant of egg color formed naturally.The site of b-4 is located in the 20th chromosome by classical genetic studies.In this study,the molecular mechanism of insect egg color formation was initially explored and its controlling genes were identified through the positional cloning method by using the b-4 mutant of silkworm as research material.The research results obtained are as follows:1.Mapping the b-4 locus to the 310kb genomic region of silkworm chromosome 20According to the genetic law of complete linkage of silkworm females,the molecular positioning was performed by preparation segregation population of the BC₁M generation.The 13 polymorphic molecular markers located on chromosome 20were used to perform genotyping experiments on the DNA of 985 red compound eye individuals from the BC₁M generation,finally,the b-4 was locked to the 310kb genome range of chromosome 20,which contained 14 coding genes.2.The candidate genes of b-4 are locked into Bm-tmem68 and Bm-mah through expression analysis,sequence analysis and functional annotationThe expression patterns of 14 genes in the mapping interval during the egg color formation of Dazao and b-4 mutants were investigated,and it was found that the expression levels of KWMTBOMO12114,KWMTBOMO12115,KWMTBOMO12116,KWMTBOMO12118 and KWMTBOMO12119 in the two strains are significantly different during the key period of egg color formation within 24h-48h after spawning;At the same time,the coding sequence of 14 genes was cloned and analyzed,and it was found that the two genes of KWMTBOMO12116 and KWMTBOMO12119 had different splicing forms of transcripts in Dazao and b-4,and abnormal transcripts would lead to defects in the formation of the encoded protein structure.Based on expression analysis and sequence analysis,the candidate genes of b-4 were locked into KWMTBOMO12116 and KWMTBOMO12119.Annotate the functions of two genes,KWMTBOMO12116 is homologous to Tmem68 gene in the rat and encodes a transmembrane protein;KWMTBOMO12119 is homologous to mah gene in the Drosophila and encodes an amino acid transporter.In Drosophila,the loss of this gene function leads to the formation of compound eye phenotypes defect.3.The upstream of the Bm-mah gene has specific sequence differences in the b-4 mutantThe four single-base substitutions and four-base deletions at 500 bp upstream of the transcription start site of the gene in b-4 was found by investigating regulatory sequence of the Bm-mah genein Dazao and b-4 mutants,then,the sequence of this locus was investigated among 22 silkworm strains with different egg colors,and the results showed that the base substitutions and deletions in the upstream regulatory sequence of the Bm-mah gene are unique in b-4 mutant.4.The red-brown egg appeared in Dazao by RNAi with Bm-mah geneIn order to identify the control genes of the b-4 mutant,we performed RNAi experiments.The result showed that only knocking down the Bm-mah,the egg color turned red,other genes don’t,Therefore,we determined that the Bm-mah gene is control gene for the b-4.5.Bm-mah gene and Bm-re gene may joinly regulate the color formation of silkworm eggThe expression of Bm-KMO,Bm-white,Bm-scarlet,Bm-cardinal and Bm-re in the eye pigment synthesis pathway was investigated in the dazao after the interference gene of mah,the results showed that the expression of red egg control gene Bm-re was significantly reduced.We further investigated the expression of Bm-mah and Bm-re in Dazao,b-4 and re,found that the expression patterns of Bm-mah and Bm-re genes were similar,the expression levels of two genes were significantly lower in b-4 and re mutants during the critical period of egg color formation.In summary,this study has gradually locked the candidate gene of b-4 through molecular location,temporal expression patter and expression difference analysis,and sequence analysis of genes,and conducted functional research on the candidate gene through RNAi,finally,proved that Bm-mah is the control gene of b-4,which encodes an amino acid transporter with 11 transmembrane domains,is a new gene in insect ommochromes metabolism pathway.This study provides important clues to further explore the key steps downstream of insect eye pigment synthesis pathway.更多还原