东亚飞蝗WntA在胚胎发育阶段的时空表达

Spatiotemporal expression of WntA during the embryonic development in Locusta migratoria manilensis(Orthoptera:Locustidae)

【目的】探讨东亚飞蝗Locusta migratoria manilensis Wnt基因家族中WntA编码蛋白序列特征及其在胚胎发育阶段的时空表达谱,为进一步开展LmmWntA的功能研究及挖掘东亚飞蝗其他Wnt基因家族成员奠定基础。【方法】PCR克隆并利用邻接法(neighbor-joining,NJ)鉴定东亚飞蝗Wnt基因家族基因LmmWntA;通过同源序列多重比对分析LmmWntA氨基酸序列特征;利用整胚原位杂交技术对LmmWntA在东亚飞蝗产卵后(after egg laying,AEL)发育至12,24,35,46,56和65 h以及3,3.5,4,4.5,5,5.5,6.5,8,8.5,9.5和11 d共17个连续胚胎发育阶段进行转录信号筛查。【结果】克隆获得东亚飞蝗LmmWntA(GenBank登录号:MW052768),CDS全长1101 bp,编码336个氨基酸;LmmWtnA与头索动物、昆虫、有爪动物及环节动物WntA蛋白共同聚为WntA亚家族单系群;LmmWntA中段和C端与比对物种WntA蛋白序列保持了较高同源性,仅在N端信号肽区域出现差异,LmmWntA与膜翅目(Hymenoptera)西方蜜蜂Apis mellifera AmWntA蛋白聚为姊妹群,氨基酸序列一致性为59.05%。LmmWntA最先表达在东亚飞蝗35 h AEL胚胎期末端生长区并在该区域持续表达至4 d AEL胚胎期,同时在新生体节每节腹部形成条纹状表达;在46 h AEL胚胎期视叶后半区持续表达至8.5 d AEL胚胎期,该区域未来发育成复眼;在56 h AEL胚胎期脑持续表达至5.5 d AEL胚胎期;在65 h AEL胚胎期每节腹部的条纹状表达信号逐渐转移至腹中线两侧,在触角基部有明显的表达信号;在5.5 d AEL胚胎期表达信号进一步转移至腹神经;从3 d AEL胚胎期开始在腹侧体节远轴端表达,后期转移至上颚、足关节及末端;伴随4.5 d AEL胚胎末端开始内陷形成肛道,LmmWntA在内陷肛道的腹面及前端表达,最多内陷至腹部第7体节;至9.5 d AEL胚胎期时LmmWntA在翅芽盘处表达。【结论】LmmWntA在东亚飞蝗胚胎发育阶段动态表达,推测LmmWntA参与东亚飞蝗胚胎后端体节生长、神经系统(脑和腹神经)、复眼、触角、消化系统后端(肛道)、颚、胸部附肢(足和翅)等重要组织和器官的发生和形成。本研究结果为进一步开展LmmWntA功能缺失研究奠定发育生物学基础。

【Aim】To explore the sequence characteristics of WntA-encoding protein in the Wnt gene family of Locusta migratoria manilensis and its spatiotemporal expression profile during the embryonic development,and lay a foundation for the further functional research of LmmWntA and mining other Wnt gene family members of L.migratoria manilensis.【Methods】The Wnt gene family gene LmmWntA of L.migratoria manilensis was cloned by PCR and identified by neighbor-joining(NJ)method.The amino acid sequence characteristics of LmmWntA were analyzed by homologous sequence multiple alignment.Whole-mount in situ hybridization was used to screen the transcriptional signals of LmmWntA in 17 consecutive embryonic developmental stages of L.migratoria manilensis after egg laying(AEL)to 12,24,35,46,56 and 65 h,and 3,3.5,4,4.5,5,5.5,6.5,8,8.5,9.5 and 11 d.【Results】The full-length CDS of LmmWntA(GenBank accession number:MW052768)of L.migratoria manilensis was cloned and is 1101 bp,encoding 336 amino acids.LmmWntA and WntA proteins of cephalochordates,insects,clawed animals and annelids were clustered into a monophyletic group of WntA subfamily.The middle region and C-terminus of LmmWntA maintain high homology with the WntA protein sequences of the corresponding species,except for the difference in the N-terminal signal peptide region,and LmmWntA was clustered with the AmWntA protein of Apis mellifera in Hymenoptera as a sister group with the amino acid sequence identity of 59.05%.LmmWntA was first expressed in the terminal growth region of the 35 h AEL and continued to be expressed in this region until the 4 d AEL,forming stripe expression in the abdomen of each segment of the neonatal segment,and continued to be expressed in the posterior half of the optic lobe,which develops into compound eyes in the future,from the 46 h AEL to the 8.5 d AEL of L.migratoria manilensis.LmmWntA was continuously expressed in the brain from the 56 h AEL to the 5.5 d AEL.At the 65 h AEL,the stripe-like expression signal of LmmWntA in each segment of the abdomen was gradually transferred to both sides of the midline of the abdomen,and there was a clear expression signal at the base of the antennae.At the 5.5 d AEL,the expression signal of LmmWntA was further transferred to the abdominal nerve.From the 3 d AEL,LmmWntA was expressed at the distal end of the ventral somite,and later transferred to the palate,foot joint and end.As the end of the 4.5 d AEL embryo began to invaginate to form proctodeum,LmmWntA was expressed in the ventral and anterior end of the invaginated proctodeum,and most of them invaginated to the 7th somite of the abdomen.At the 9.5 d AEL,LmmWntA was expressed at the wing germ disc.【Conclusion】LmmWntA was dynamically expressed during the embryonic development of L.migratoria manilensis,speculating that LmmWntA is involved in the development and formation of important tissues and organs such as the posterior segment growth of embryo,nervous system(brain and abdominal nerve),compound eye,antennae,posterior end of digestive system(proctodeum),jaw,chest appendages(leg and wing)of L.migratoria manilensis.The results of this study lay the foundation of developmental biology for further research on LmmWntA deficiency.