美国白蛾Wnt-1基因的基因组编辑

发布时间：2018-02-20 21:42

本文关键词： 美国白蛾 CRISPR/Cas Wnt- 基因组编辑免疫组化体节形成附肢发育　出处：《林业科学》2017年03期 　论文类型：期刊论文

【摘要】：【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。
[Abstract]:[objective] in order to reveal the mechanism of flying ability of American white moth at molecular level, the function of Wnt-1, a gene related to wing formation, was studied by genomic editing technique. [methods] Primer was designed according to the genomic and transcriptional information of White moth. The full-length CDS region of HcWnt-1 gene was cloned, the structural characteristics of HcWNT-1 protein were analyzed by on-line software, the time expression pattern of the gene during its growth and development was determined by RT-PCR, and the temporal and spatial expression pattern of HcWNT-1 protein was determined by immunohistochemical method. The HcWnt-1 gene was edited by CRISPR/Cas9 technique to observe the surface type and genotypic characteristics of embryonic mutant. Direct PCR and positive clone sequencing were used to detect the mutation of HcWnt-1 gene. [results] the nucleotide sequence of HcWnt-1 gene was 1 221 BP, encoding 407 amino acids HcWNT-1 protein containing helico-turn-helical DNA binding motif and 24 conserved cysteine residues. The highly conserved Motif was distributed throughout the whole sequence. Transcriptional and translational level verification showed that HcWnt-1 gene was highly expressed in early embryonic stage, and its expression level changed chronically with somatic ganglion differentiation and appendage development for 24 h. The expression of HcWNT-1 protein was mainly concentrated in the primordial region, and the expression level of HcWnt-1 gene gradually decreased with the development of embryo, and the expression of HcWNT-1 protein gradually extended from head to tail along the axis of anterior and posterior body. At 144h, the second peak of HcWNT-1 expression at transcription level was mainly found in the appendages. The mutation of HcWnt-1 gene caused the death of 99.8% individuals at embryonic stage. There is a fragment deletion between the target points, The maximum deleted fragment was 423 BP; most of the mutants could not form embryos. A few individuals who can form embryos have abnormal somatic formation and obstructed appendage development. [conclusion] the embryonic development types of American white moth are consistent with the short embryo band and intermediate embryo band type CRISPR-Cas9 genome editing technique, which can be used to efficiently edit the genome of the American white moth. HcWnt-1 gene knockout may affect the somatic node differentiation and appendage formation of the moth, which may provide theoretical support for the study of gene function of forestry and non-model pests. In addition, HcWnt-1 mutation resulted in embryo death, which could be used as a target gene for genetic control of White Moth in the future.
【作者单位】：国家林业局森林保护重点实验室中国林业科学研究院森林生态环境与保护研究所;中国林业科学研究院华北林业实验中心;中国科学院上海生命科学研究院植物生理生态研究所昆虫发育与进化生物学重点实验室;
【基金】：林业公益性行业科研专项(201504302)
【分类号】：S763.42

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