| Macrobrachium nipponense is mainly distributed in China,Japan,the Korean Peninsula,and northern Vietnam,which is one of the main freshwater aquaculture species in China.M.nipponense has many excellent breeding characteristics and high economic value.However,there are still fatal problems such as molting failure and sloughing rigidity in the breeding of M.nipponense.These problems will lead to slow growth and even death of M.nipponense,causing serious losses and restricting the rapid development of the M.nipponense farming industry.Therefore,it is of great significance to study the regulatory mechanism of the molting of M.nipponense and its influence on its growth and development.Regarding these issues,we screened the Halloween family genes CYP307α1,CYP306α1,CYP302α1,CYP305α1 and other genes with significantly different expression levels in the molting regulation pathway in the transcriptome of M.nipponense established by the research group.Previous studies have shown that the Halloween gene family plays an important regulatory role in the synthesis of 20-hydroxyecdysone(20E)in arthropods.Therefore,the CYP306α1 gene with significantly different expression levels was selected as the research object in this pathway,and the gene was cloned by cDNA end rapid amplification technology,and phylogenetic analysis was carried out to clarify its evolutionary origin;real-time fluorescence quantitative PCR(qRT-PCR)technology to study the expression pattern of CYP306α1 gene in different tissues,embryonic development and larval development of adult male and female shrimp,and combined in situ hybridization(ISH)technology to locate the distribution of CYP306α1 gene mRNA in gonadal tissue;finally,RNA was used Interference(RNAi)technology to study the effect of CYP306α1 gene on the molting and growth and development of M nipponense.The main findings of this paper are as follows:(1)The total length of the CYP306α1 gene cDNA of M.nipponense is 2108 bp,encoding 600 amino acids.The amino acid sequence encoded by the Mn-CYP306α1 gene has the typical conserved domains of the Halloween family:P/G rich,Helic C,Helic I,Helic K,PERF,and Heme-binding.Phylogenetic analysis showed that the CYP306α1 gene of M.nipponense was evolutionarily consistent with the arthropod CYP306α1 gene,and the Mn-CYP306α1 gene belonged to the Halloween gene family.(2)The expression pattern of Mn-CYP306α1 mRNA in M nipponense was investigated by fluorescence quantitative analysis.The results showed that:in male,the expression of Mn-CYP306α1 gene was the highest in the testis,less in the eye stalk and heart,and the expression in other tissues(hepatopancreas,gill,muscle,brain ganglion)is very low.In female,Mn-CYP306α1 has the highest expression in ovary,lower expression in eye stalk,cranial ganglion and heart,and extremely low expression in other tissues(hepatopancreas,gill,muscle).Further research on the expression of Mn-CYP306α1 in different stages of ovarian development showed that the level of Mn-CYP306α1 gradually decreased from ovarian stage Ⅰ to Ⅳ,and decreased rapidly in stage Ⅴ(P<0.05).Mn-CYP306α1 gene was expressed in all stages of embryonic development,and there were significant differences(P<0.05).The expression level was the highest in gastrulation stage(GS),followed by nauplii stage(NS),and the expression levels were lower in other stages.There were also significant differences in the expression of Mn-CYP306α1 gene in each stage of larval development(P<0.05).The expression of larvae(PL25)on the 25th day after metamorphosis was the highest,followed by the expression of PL10,and the expression of PL1 again.In addition,the expression levels of the remaining stages were lower.This indicated that CYP306α1 was expressed to different degrees in the whole life cycle of M.nipponense and in different tissues of male and female.It was speculated that CYP306α1 may have different degrees of influence on embryonic development,growth,metamorphosis and reproduction of M.nipponense.(3)Mapping of Mn-CYP306α1 in the gonadal tissue of M nipponense by in situ hybridization.In the ovary,Mn-CYP306α1 mRNA signal expression was found in the cell membrane,yolk granule,and nucleus.Mn-CYP306α1 mRNA was first highly expressed in the oocyte nucleus in the ovary of M.nipponense,and with the development of the ovary,the expression of Mn-CYP306α1 mRNA decreased significantly in the nucleus and slightly increased near the cell membrane,which is consistent with Mn-CYP306α1.The qRT-PCR results of genes at different stages of ovarian development were consistent.Mn-CYP306α1 mRNA signal was found to be highly expressed in spermatocytes,spermatogonia and spermatozoa in the testis.(4)In the 30-day interference experiment of M nipponense injected with Mn-CYP306α1 dsRNA,it was found that the expression level of Mn-CYP306α1 mRNA in the gonad tissue decreased by 96.01%and 83.64%on the 4th and 7th day of interference,respectively(P<0.05),indicating that Mn-CYP306α1 dsRNA can effectively reduce the expression of Mn-CYP306α1 mRNA in gonadal tissue.At the same time,on the 4th and 7th day,the content of ecdysone(20E)in M nipponense also decreased by 34.63%and 22.74%,respectively,which was significantly lower than that of the control group.Secondly,while interfering with the Mn-CYP306a1 gene,the molting situation of M.nipponense was observed every day and its body weight was weighed once every 10 days.It was found that the molting frequency of the experimental group was significantly reduced by 41.75%compared with the control group in the second week.The decreasing rate of molting frequency remained around 40%in the following two weeks(P<0.05).On the 20th and 30th days,the weight gain of the experimental group of M.nipponense decreased by 7.36%and 14.42%,respectively,which was significantly lower than that of the control group(P<0.05).The results showed that RNAi of Mn-CYP306a1 gene could significantly reduce its expression in gonadal tissue and reduce the synthesis of 20E,thereby inhibiting the molting and growth of M.nipponense.This study shows that the Mn-CYP306a1 belongs to the Halloween gene family and is tissue-specific.It is expressed in different degrees throughout the life cycle of M.nipponense and in different tissues of male and female shrimp,and has different degrees of influence on it.Mn-CYP306a1 plays an important role in promoting the synthesis of 20E in M.nipponense,thereby affecting its molting and growth,which can provide a reference for studying the function of CYP306a1 gene in crustaceans. |
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