转录因子ZnF-706在鳞翅目昆虫中的进化格局及在家蚕中的功能

Evolutionary pattern of the transcription factor ZnF-706 in Lepidoptera and its function in Bombyx mori

【目的】探讨家蚕Bombyx mori的潜在驯化基因——转录因子ZnF-706在鳞翅目(Lepidoptera)昆虫进化过程及家蚕驯化过程中的分子进化格局;并基于CRISPR/Cas9家蚕基因组编辑平台,探讨ZnF-706基因在家蚕中的功能。【方法】首先分析了家蚕ZnF-706序列特征,并利用已发表芯片数据调研该基因在家蚕幼虫组织中的表达格局;利用Phylogenetic Analysis byMaximum Likelihood (PAML)分支检验方法,分析该基因在鳞翅目不同类群中的分子进化格局。基于已发表的家蚕-野桑蚕Bombyx mandarina群体基因组多态性数据,对ZnF-706进行基因区域人工选择信号分析;对ZnF-706基因上游2 kb的调控区域进行单核苷酸多态性位点频率检测,发掘在家蚕群体中固定下来的突变位点;针对突变位点所在区域进行转录因子结合活性预测。利用CRISPR/Cas9基因编辑技术敲除ZnF-706基因,获得纯和突变体;以野生型家蚕为对照,检测突变体的茧重及蛹重变化。【结果】家蚕ZnF-706的编码蛋白具有典型的锌指蛋白结构域。ZnF-706在家蚕5龄第3天幼虫各组织中广泛表达,尤其表皮、脂肪体和生殖腺中有很高的表达量;该基因在鳞翅目、蚕蛾总科(Bombycoidea)及天蚕Antherea yamamai 3个分支中均呈现快速进化信号,在家蚕中有强烈的人工选择信号。该基因所在基因组区域的家蚕-野桑蚕种群分歧度参数Fst明显升高,家蚕群体中的群体多样性π明显降低,表明它位于一个选择扫荡区域内;该基因在家蚕-野桑蚕中的9个SNP位点存在于上游调控区,并位于转录因子结合活性区域内。该基因的纯合家蚕突变体ΔZn F-706生存力减弱,并且茧重以及蛹重与野生型家蚕相比都显著降低。但与黑腹果蝇Drosophila melanogaster中不同的是,家蚕中该基因的突变并不致死。【结论】ZnF-706可能在鳞翅目尤其是泌丝昆虫中进化,并在家蚕驯化过程中受到选择压力,提示其对于特征性状茧丝的变异可能发挥作用。该基因可能通过对丝蛋白基因的直接调控,或通过影响家蚕的生长发育而间接地影响茧丝性状。本研究不仅为探究家养动物人工选择机制提供了来自昆虫类材料的独有证据,也为后续深入开展家蚕重要经济性状的转录调控研究提供线索。

【Aim】This study aims to explore the molecular evolution pattern of the putative domestication gene,the transcription factor ZnF-706 in different lepidopteran groups and in the domestication process of the silkworm Bombyx mori,and to explore its function in the silkworm by knockout of ZnF-706 via the CRISPR/Cas9 genome editing system.【Methods】We analyzed the gene structure of ZnF-706 and investigated its tissue expression pattern in the day-3 5th instar larvae of the silkworm based on the published silkworm microarray data.Using phylogenetic analysis by Maximum Likelihood(PAML),we tested the evolution pattern of ZnF-706 in Lepidoptera.Based on the published genetic polymorphism data of the B.mori and Bombyx mandarina populations,we screened the artificial selection signal of ZnF-706 genic region.After analyzing the upstream 2 kb region of this gene,we detected the fixed SNPs in the domestic silkworm and predicted transcription factor binding motif in the region covering these SNPs.By knockout of this gene using CRISPR/Cas9 genome editing,we obtained the homozygosis mutant.The variation of cocoon weight and pupal weight of mutants was detected by using wild-type silkworm as the control.【Results】The encoded protein of ZnF-706 has a typical zinc finger protein domain.ZnF-706 is ubiquitously expressed in the silkworm larva,especially highly expressed in the cuticle,fat body and gonads.It evolved rapidly in three clades,namely Lepidoptera,Bombycoidea and Antherea yamamai.Strong artificial selection signals in the region covering this gene were detected,with significantly elevated population divergence(Fst)between the B.mori and B.mandarina populations and decreased population diversity(π)in the B.mori population,suggesting that it is located in a selective sweep.Nine fixed SNPs in the regulatory region of upstream of this gene were detected,and they were located in the motifs with transcription factor binding activity.Homozygous loss-of-function mutants ofΔZnF-706 showed weaker viability and significantly lower cocoon weight and pupal weight,as compared with the wild-type silkworm.But unlike in Drosophila melanogaster,the homozygous mutants of ZnF-706 in B.mori were not lethal.【Conclusion】ZnF-706 evolves rapidly in Lepidoptera,especially silk-producing insects,and is also under artificial selection during silkworm domestication,suggesting that it might be associated with cocoon silk.It may affect silk cocoon traits by direct regulation of silk protein gene,or by indirect regulation through affecting the growth and development of the silkworm.This study provides not only unique evidence from insect domestication for exploring the mechanism of artificial selection,but also cues to the further research on the transcriptional regulation of economic traits of the silkworm.