美国白蛾Wnt-1基因的基因组编辑

Genome Editing of Wnt-1 in Fall Webworm (Hyphantria cunea)

【目的】为了从分子水平上揭示美国白蛾飞行能力的机制,利用基因组编辑技术对翅形成相关基因Wnt-1进行功能研究。【方法】根据美国白蛾的基因组和转录组信息设计引物,克隆得到HcWnt-1基因CDS区全长,利用在线软件分析HcWNT-1蛋白的结构特征;通过RT-PCR确定该基因在其生长发育过程中的时间表达模式,并通过免疫组化方法确定HcWNT-1蛋白的时空表达模式;通过CRISPR/Cas9技术对HcWnt-1基因进行编辑,观察胚胎期突变体表型和基因型特点,利用直接PCR和阳性克隆测序检测HcWnt-1基因突变。【结果】HcWnt-1基因核苷酸序列大小为1 221 bp,编码407个氨基酸,HcWNT-1蛋白含有螺旋-转角-螺旋DNA结合基序和24个保守的半胱氨酸残基,并且高度保守的Motif分散分布于整个序列。转录水平和翻译水平验证表明:美国白蛾HcWnt-1基因在胚胎早期高表达,且其表达水平随着体节分化和附肢发育而呈时序性变化;24 h时,早期胚带形成,转录水平出现第1个表达高峰,HcWNT-1蛋白表达主要集中于原头区;随着胚胎的发育,HcWnt-1基因表达水平逐渐下降,HcWNT-1蛋白表达沿着前后体轴由头部向尾部逐渐延伸;在144 h,转录水平出现第2个表达高峰,HcWNT-1蛋白表达主要集中于附肢。美国白蛾HcWnt-1基因的突变造成了胚胎期99.8%的个体死亡(注射1 000头),突变率为62.5%,PCR测序结果显示2个靶点之间存在片段删除,最大删除片段为423 bp;大部分突变体不能形成胚胎,少数能形成胚胎的个体体节形成异常和附肢发育受阻。【结论】美国白蛾胚胎发育类型符合短胚带和中间胚带型;CRISPR/Cas9基因组编辑技术可以对美国白蛾基因组进行高效编辑,可为林业以及非模式害虫基因功能研究提供理论支持;HcWnt-1基因敲除影响美国白蛾体节分化和附肢形成,说明该基因对于美国白蛾胚胎发育至关重要。此外,HcWnt-1突变造成美国白蛾胚胎期死亡,可作为未来美国白蛾遗传防治的靶标基因。

[ Objective] The fall webworm （Hyphantria cunea） is an invasive species, and has been causing severe damage to forestry and agricultural production. It is urgently needed to effectively control the pest population by an environmentally-benign technique. To reveal the mechanism of its strong migration capability which plays an important for its spreading, we analyzed the function of Wnt-1, an essential gene in wing development, by using the genome editing method. [ Method] The full length cDNAs of HcWnt-1 were obtained by using the primers designed based on the genome data and transcriptome data of H. cunea. Online softwares was used to predict the structure characteristics of HcWNT-1 protein. RT-PCR and Immunohistochemistry were used to explore the expression pattern of HcWnt-1. The CRISPR/Cas9 was applied to induce mutations of HcWnt-1, the phenotypic and genotypic characteristics of embryonic mutants were observed, and further direct sequencing were used to detect the mutation events. [ Result ] The complete coding sequence of HcWnt-1 is 1 221 bp, which encodes a protein of 407 amino acids, characterized by helix-turn-helix DNA binding motif and 24 cysteine residues, and the highly conserved motifs are scattered throughout the sequence. During the embryogenesis stages, HcWnt-1 mRNA was highly expressed at early stage, WNT-I protein expression showed sequential change during the embryonic development stages. The early embryos formation was completed in 24 h, at which the first peak was detected at transcriptional level and the HcWNT-1 expression was mainly concentrated in acron. The expression of HcWnt-1 declined as development progressed and the expression of HcWNT-1 elongated from head to tail along the anterior-posterior body axis. HcWnt-1 expression presented the second peak at 144 h, which occured in the appendages. CRISPR/Cas9-based mutagenesis of HcWnt-1 led to a high mortality rate （99.8 % ） and a mutation efficiency of 62.5% at embryonic stages following injection of Cas9 mRNA and sgRNAs with 1 000 eggs. Defective phenotypes as missing segments and appendages were observed in unhatched larvae. [ Conclusion ] The results have revealed that the segmentation model of H. cunea belongs to short and intermediate germ band. CRISPR/Cas9 system is a powerful genome manipulation tool in the invasive pest, and would be a promising tool for gene functional research in forestry and non- model insects. Loss-of-function of HcWnt-1 inhibited the segmentation and appendage development, demonstrating that HcWnt-1 plays an important role in embryonic development of H. cunea. Furthermore, HcWnt-1 was proved as a lethal gene, and it could be used as an appropriate target for future genetic control of H. cunea.