| Gene duplication is an important mechanism for the formation of new genes, which play an important role in the genetic evolution and biological evolution. Retrocopies, which were formed by the RNA-based gene duplication, were usually considered to be no function. However, some experiments have screened a large number of functional retrocopies among the genome of fruit flies, zebrafish and some other mammals and plants, and their function have been confirmed by experiments. Caenorhabditis elegans is the first to be sequenced in the multicellular animals; it has become the model organism to the research of developmental biology, neurobiology, cell apoptosis, diseases aging, et al, and has become the ideal experiment material in the research of comparative genomics and system evolution. Previous work had screened the retrocopies in the genome of Caenorhabditis elegans, and43retrocopies were found in these work. According to the public expression database and RT-PCR experiment, it speculates that most of the retrocopies can express and may have certain function to some extent; however, no experiment has been conducted to analyze the biological functions and the expression difference in C. elegans different developmental period of retrocopies.The retrocopy ssp-31is affiliated to the family of SSP, which has12members. According to the results of the RNA interference experiments to the SSP family members on wormbase, it speculates that this family has some certain function in fat metabolism, reproduction and development, et al. What’s more, the fat metabolism is usually associated with the reproduction and development of C. elegans, so it can speculate that ssp-31may play a role in fat metabolism, reproduction, development, et al. However, it has no related research to the gene function of ssp-31. It’s the first time to research the expression and function of the retrocopy ssp-31of C. elegans.The research had amplified the331bp gene fragment of ssp-31, which shows that the gene can surely be expressed; and then, the gene expression quantity in different growth development period of C. elegans (Egg, L3, L4, and Adult) is analyzed by real-time PCR. The results showed that ssp-31had high gene expression quantity in the egg and adult period and had low gene expression quantity in L3and L4period, which had significant differences (p<0.05), so the gene can be speculated as a maternal gene, which play a role in the development of eggs, oviposition behavior, reproductive development of adult, et al.The research analyzed the biology function of ssp-31gene by RNA interference in the next step. First,331bp of the ssp-31gene was cloned, and was connected with the nematode RNAi expression vector L4440after sequenced, and as a result, the RNAi recombinant expression vector L4440-ssp-31was constructed successfully. Then, L4440-ssp-31was transformed into competent Ecoli. HT115cells and the expression of ds RNA were induced by IPTG. Finally, ssp-31of C. elegans was silenced by feeding Ecoli. HT115could express dsRNA. In the RNAi nematodes, the following phenomena were observed:(1) ssp-31RNAi nematodes layed an average of255±8eggs compared with278±7eggs for the control group nematodes (p<0.05), live progeny numbers increased from219±11in control group to249±88(p<0.05), incubating rate of eggs increased from (78.5±1.5)%in control group to (97.8±0.9)%(p<0.001);(2) Spawning period shorten from6-8days in control group to5days;(3) Oviposition amount of the first day and the second day increased from8±5and60±6in control group to33±5(p<0.01) and92±7(p<0.01), respectively; oviposition amount of the forth day and the fifth day decreased from81±2and37±5in control group to31±11(p<0.01) and3±3(p<0.001), respectively;(4) Spawning peak period decreased from3days(the second day, the third day and the fourth day) in control group to2days(the second day and the third day);(5) From the fat staining images and the gray value analysis, the research had found that body fat content in interference group increased extremely significantly (p<0.001);(6) It had no anomalies in other aspects of C. elegans, such as the morphological characteristics, the movement way and the growth of eggs and larva.The experimental results above showed that ssp-31gene silencing reduced the egg laying number of the nematode by shortening the spawning period and spawning rush hour, this maybe caused by overdose of polyunsaturated fatty acids (PUFA) in C.elegans. Besides, the increase of the live progeny in C. elegans may be caused by the changes in the expression quantity of lipid metabolism-related genes expression level (such as nhr-64); or this perhaps because the directional sperm motility was promoted by the increased PUFA, and led to the increase of egg hatching rate (such as fat-2, fat-3). In conclusion, retrocopy ssp-31of C. elegans has certain effect in regulating and controlling the fat metabolism and reproductive capacity of C. elegans. |
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