| When the earthworm(Eisenia foetida)is cut off by external forces(such as ploughs,natural enemies,etc.),it is considered to be the best material for regeneration due to its rapid and constantly updated ability.The pluripotent genes is very important for the regulation of cell status,and it has obvious effect on earthworm regeneration.Most studies have found that exogenous retinoic acid(RA)has delayed and interfered with the regeneration of invertebrates,and the action period is very strict,but the specific mechanism is not clear yet.The material in this study is the earthworm(Eisenia foetida).Using the method of realtime fluorescence quantitative PCR(qRT-PCR),Western blot(WB)and transcriptome method,detected the expression of the pluripotency sox2 in different parts of the earthworm exposed to RA and the expression of regeneration in different time.We found that RA effected ten gene superfamilies in earthworm tail tissue regeneration.The main results are as follows:1.In this study,we compared the regeneration of different parts of the amputated earthworm(Eisenia foetida)and the effects of RA on the regeneration of different parts.The body segment is the head tissue is the first 25 paragraphs,the midpiece is 26-51 paragraphs,and the tail tissue is 52-95 paragraphs.The concentration of retinoic acid was 0.1mmol/kg,and the sampling time was days 0,3 and 7.The result showed that the regeneration of the head tissue and tail tissue is slightly faster than that of the midpiece.The buds appeared late by treated with retinoic acid,so RA can delay and inhibit earthworm regeneration.2.In this study,the expression levels of the multipotent gene Sox2 were detected by qRT-PCR in different body segments(head,midpiece and tail),as well as the expression level of the multipotent gene sox2 in the development of the mature earthworm in different body segments.The results showed that the expression of Sox2 gene was highest in the 7th day in different body segments.After RA stress,the expression level of Sox2 gene was significantly decreased(p<0.05).The results showed that RA significantly inhibited the expression of Sox2 gene.3.In this study,the expression levels of the multipotent gene Sox2 were detected by western blot(WB)in different body segments(head,midpiece and tail),as well as the protein level of the multipotent gene Sox2 in the development of the mature earthworm in different body segments.The results showed that the expression of Sox2 protein was highest in the 7th day in different body segments.After RA stress,the expression level of Sox2 protein was significantly decreased(p<0.05).The results showed that RA significantly inhibited the expression of Sox2 protein.4.In this study,the expression levels of all genes were detected and the expression level of the genes after the RA stress was detected through the analysis of transcriptomics at different periods in the tail regeneration of earthworms.The results showed that ten gene superfamilies were the same as the Sox2 gene expression pattern.They are the Sox,Fox,Pax,Pou,Six,Hox,Klf,Wnt,Col families.These gene families are all achieve high expression in day 3 or day 7,and the expression of the ten gene families was significantly reduced after RA stress(p<0.05).The results showed that RA significantly inhibited the expression of ten genes superfamilies related to the omnipotent and cell differentiation.To sum up,the genes of ten gene families have been significantly increased in the regeneration of earthworm(Eisenia foetida),and these genes have certain similarities in expression patterns.RA can significantly inhibit the generation of the blastema in earthworm regeneration,possibly by inhibiting the expression of Sox2 gene and Sox2 protein and the ten gene families that are similar to the Sox2 gene expression.This study revealed the effect mechanism of RA on earthworm regeneration,and the effects of RA on the regeneration of invertebrate animals were provided by the molecular level. |
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