关键词: 东亚飞蝗; WntA基因, 发育生物学, 生长区, 翅芽, 整胚原位杂交

Abstract:  【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 1 101 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.

Key words: Locusta migratoria manilensis; WntA gene, developmental biology, growth zone, wing germ, whole-mount in situ hybridization