鄱阳湖泥鳅黑斑形成观察及mitfa表达分析

Observation of Black Spot Formation and mitfa Expression Analysis of Misgurnus anguillicaudatus from Poyang Lake

本研究观察了孵化后1~60日龄泥鳅(Misgurnus anguillicaudatus)中色素细胞的形成、分布和主要类型。孵化后3 h,在泥鳅卵黄囊内首次观察到幼体型黑色素细胞;从仔鱼期到21日龄,泥鳅体表可见幼体型黑色素细胞;从22日龄的幼鱼到成鱼阶段,泥鳅体表可见成体型黑色素细胞。虹彩细胞首先在1日龄泥鳅仔鱼的眼睛中出现,直到12日龄才在体表被观察到。7日龄稚鱼体表出现黄色素细胞。在孵化后2~21d内,泥鳅的黑色素细胞为幼体型黑色素细胞,其形状由星状变化成雪花状,进一步变化成黑点状。从22 d开始,3种花斑类型的泥鳅体表形成不同形态的成体型黑色素细胞。在大花斑泥鳅体表,菊花状黑色素细胞有规律地聚集成大花斑;在小花斑泥鳅体表,圆形和树枝状黑色素细胞聚集形成小花斑;在无花斑泥鳅体表,树突状黑色素细胞分布均匀。利用生物信息学和实时荧光定量PCR(qRT-PCR)方法对泥鳅色素沉着相关基因mitfa进行了分析,结果显示,小眼畸形相关转录因子a基因(microphtalmia-associated transcription factor a,mitfa)编码的MITFa蛋白含408个氨基酸,相对分子质量为45.68kDa,预测等电点为7.16,包含MITF_TFEB_C_3_N、bHLH-Zip和DUF3371结构域。与各种鱼类的mitf基因序列相比,mitfa保守性较好,序列相似度较高(58.8%~83.2%)。qRT-PCR结果显示,mitfa mRNA在胚胎发育的受精卵阶段达到峰值,背部皮肤的表达水平高于腹部皮肤。本研究初步探究了鄱阳湖泥鳅色素和体表花斑的形成及mitfa基因的表达,为进一步了解泥鳅体色形成的遗传机制奠定了基础。

Color pattern plays a vital role in animal survival and communication.The type,distribution,and pigment state of pigment cells,and the reflective ability of iridophores determines body color.It varies adaptively in response to external environmental changes and physiological states.The skin pigmentation pattern reflects the number and arrangement of chromatophores.Some fish with rich color patterns,including egg spot patterns,blotch patterns,melanism,horizontal stripe patterns,and vertical bar patterns,have been studied increasingly.This study observed the formation,distribution,and main pattern of chromatophores in 1–60-day-old Misgurnus anguillicaudatus after hatching.Larval melanocytes were first observed in the yolk sac of loach larvae at 3 h post-hatching.From the larval to juvenile stage at 21 days,larval melanocytes appeared on the loach body surface.From the juvenile stage at 22 days to the adult stage,adult melanocytes appeared on the loach body surface.Iridocytes were first observed in the eyes of one-day-old larvae but not on the body surface until they were 12 days old.Xanthophores appeared on the body surface of seven-day-old juveniles.At 2–21 days post-hatching,the melanocytes in the loaches were larval,and their shape changed from star-to snowflake-shaped before forming a black spot.From 22 days,different morphological adult melanocytes formed on the body surface of the loaches with three types of black spots.Chrysanthemum-shaped melanocytes regularly aggregated into large black spots on large black spot loaches.Round and dendritic melanocytes gathered to form small black spots on small black spot loaches.Dendritic melanocytes were evenly distributed on non-black spot loaches.The pigmentation-related mitfa gene was obtained from M.anguillicaudatus using the rapid amplification of cDNA ends(RACE)approach with the SMARTer RACE 5´/3´Kit User Manual according to the manufacturer’s recommendations and was analyzed using bioinformatics and quantitative methods.The results showed that the mitfa gene encoded a protein with 408 amino acids with a calculated molecular mass of 45.68 kDa and an estimated isoelectric point of 7.16.MITFa contained MITF_TFEB_C_3_N,bHLH-Zip,and DUF 3371 domains.MITFa was well-conserved compared to MITF of various species with a higher degree of sequence similarity with other fishes(58.8%-83.2%).The qRT-PCR results showed that the mitfa mRNA was expressed at all stages of embryonic development and reached a peak value at the fertilization stage.mitfa expression was detected in all examined tissues of the three types of loaches,and the highest level of expression was detected in both muscle and dorsal skin(P<0.01).This study explored pigmentation formation and mitfa expression,serving as a foundation for gaining further insight into the genetic mechanism of body color formation in M.anguillicaudatus.