| There are many regenerative phenomena in animals in nature,such as regeneration in leeches,regeneration of body parts of starfish in invertebrates,regeneration of appendages in vertebrates,regeneration of gecko tails,and etc.Many reproduction phenomena have also been found in many crustaceans.For example,in Macrobrachium rosenbergii,it was found that the lost limbs can re-grow first to a new bud and eventually developed into new limbs.The study and exploration of limb regeneration in M.rosenbergii has been rarely reported.Therefore,the objective of this study was to carry out a preliminary transcriptomic study of limb regeneration of M.rosenbergii and its differentially expressed genes involved.The basic expression function of some differentially expressed genes during limb regeneration was investigated.M.rosenbergii belongs to the most abundant species of arthropods and its limbs have strong regeneration ability.It is an ideal model organism for regeneration research.In order to find important genes regulating the early regeneration of M.rosenbergii,we first performed artificial limb amputation on normal shrimps,and used transcriptome sequencing technology to detect and analyze the gene expression differences in the early and late stages of the regenerating limbs.The experiments were performed using the Hi Seq sequencing platform for sequencing.The numbers of Clean reads obtained in normal(control),pre-experimental(1)and post-experimental(experimental 2)regenerated tissue samples were: 62,114,332,48,469,208 And 62,800,124;The total number of Unigenes after quality control and statistics was 87,783,and the average length was 1,118.19.A total of 33,058 Unigenes were annotated with one or more databases through functional annotation of the Blast search databases.The Unigene GO functional annotation results showed that the number of Unigenes annotated by the biological process was 48,171;the number of cellular components annotated to Unigenes was 36,018;the number of molecular function annotations to Unigenes was 21,383.Analysis by DEGseq and DESeq software showed that 16,949 genes were differentially expressed in experimental group 1compared with the control group;comparing to the experimental group 2 with the controlgroup,8,291 genes were differentially expressed,;in experimental group 2 compared with experimental group 1,18,495 genes were differentially expressed.Comparative transcriptomic analysis of limb regeneration of M.rosenbergii was used to explore genes involved in the regulation of limb regeneration and to provide certain biological information for the molecular mechanism of limb regeneration in M.rosenbergii.Based on statistical analysis of the annotation information of differential genes,the candidate genes that may be involved in the limb regeneration of M.rosenbergii were belong to the CHH/MIH/GIH family neuropeptide with gene numbers 3555,8110,36549,16122 for further experiments.Transcriptome data analysis revealed 4 genes with gene numbers 8110,16122,3555,and 36549 were that show significantly different expression pattern and might be involved in the regulation of regeneration,and a.An eyestalk gene(i.e 112722)was included in the study.In this early part of this project,RT-PCR and RACE techniques were used to clone the c DNAs of the five genes(Gene # 8110,16122,3555,36549,and 112722).In addition,real-time fluorescence quantitative PCR was used to detect the expression and distribution of genes 8110,16122,3555,36549,and 112722 in different tissues of M.rosenbergii;the expression and distribution at the base of different stages during the severed limb of M.rosenbergii;after the severed limbs of M.rosenbergii.The expression of these gene during different stages of the molting cycles;the eyestalk expression and distribution of at different stages after the severed limbs of M.rosenbergii.Experimental conclusion: Gene No.3555 is the molt inhibiting hormone(MIH)of M.rosenbergii,Gene # 8110 is the CHH member of M.rosenbergii,and genes 3555 and 8110 have been previously cloned by other researchers;Gene # 36549,16122 No.112722,no complete c DNA sequence,these five genes may participate in the regulation of limb bud regeneration of M.rosenbergii,of which Genes # 8110 and 36549 appeared to have a much greater effect on limb bud regeneration through a series expression studies reported in this study.In addition to these CHH family members,other genes identified in the transcriptome may also be involved.In conclusion,several eyestalk CHH/MIH/GIH family genes(i.e.# 8110,36549)have identified and may require further detail study to verify their involvement in limb regeneration in M.rosenbergii. |
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