Molecular Identification And Effects Of Cadmium And Lead Stress On Pheretima Aspergillum

Guang-dilong?Pheretima aspergillum?is a traditional animal medicine in China.Many different species of earthworm and a wide range of sources were sprang up in Chinese herbal markets.With their similar appearance,it is difficult for consumers to quickly differentiate them.Furthermore,The excess levels of heavy metal has affected the quality of the products of Pheretima aspergillum.This study established the molecular identification technology of Pheretima aspergillum,and discussed the effects of heavy metal cadmium and lead stress on Pheretima aspergillum.1.We have established three molecular techniques for the identification of Pheretima aspergillum and its adulterants.The first method of identification was RAPD.Three primers can be selected as primers for identification of Pheretima aspergillum,primer DL05?5'-CATCCCCCTG-3'?,primer DL12?5'-TGAGCCTCAC-3'?and Primer DL18?5'-TTCCGAACCC-3'?,respectively;We optimized RAPD reaction system to obtain optimal reaction conditions:Mg²⁺concentration was 1.0 mmol/L,dNTPs concentration was 0.2 mmol/L,primer concentration was 0.3?mol/L,Taq DNA Polymerase concentration was1.5 U.The second method of identification was RFLP,which used the restriction enzyme Rsa I to digest the CO I fragment of Pheretima aspergillum and its adulterants.The results showed that the CO I fragment of Pheretima aspergillum obtained three fragments by Rsa I digestion,and the CO I fragments of Eisenia foetida,Amynthas obscuritoporus,and Pheretima guillelmi were digested to obtain two fragments.However,there was no recognition sites of Rsa I in the CO I fragment of Metaphire magna.So Pheretima aspergillum and its adulterants can be identified based on the size and number of fragments after restriction endonuclease reaction.The third method of identification was LAMP.Four sets of primers were designed using LAMP primer design software,FIP?CAACAGCCG CAGACCTTACTA-AACATAAGATTTTGACTTTTGCC?,BIP?GG ACAGTTTACCCCCCTTTAGC-GCTAAATGTAGTGAGAAAATTG CA?,F3?CATAGCATTCCCACGTCTA?and B3?ACCTAAAATTG ATGAGGCAC?,respectively.Pheretima aspergillum can be identified from its adulterants by LAMP technology.The LAMP reaction system was optimized to obtain an optimum reaction temperature of 63°C,and the optimum reaction time was 60 minutes.The detection limit of LAMP in Guang-dilong was 1000 times higher than conventional polymerase chain reaction2.We discussed the accumulation characteristics of cadmium and lead in Pheretima aspergillum,and studied the effects of lumbrokinase activity and hypoxanthine content on Pheretima aspergillum under the stress of cadmium and lead.Pheretima aspergillum has some tolerance and enrichment ability for cadmium and lead.With the increase of cadmium stress concentration on Pheretima aspergillum,the accumulation of cadmium showed an increasing trend,but did not show a specific time rule.When the cadmium accumulation reaches a limited amount,it will be discharged outward to reduce the heavy metal content in the body,and then continue to absorb and accumulate cadmium.With the increase of lead stress concentration,the enrichment of Pheretima aspergillum showed an overall increasing trend,the lead enrichment law of Pheretima aspergillum was the same within 5 weeks under different concentrations of lead stress.The accumulation of cadmium increased at first,then decreased,and increased at last.The effect of lumbrokinase activity on Pheretima aspergillum was closely related to the concentration and time of heavy metal stress.Compared with the control group,when single cadmium stress,the lumbrokinase activity of Pheretima aspergillum increased at first and then decreased.The lumbrokinase activity decreased when concentration of cadmium stress increased.Compared with the control group,when single lead stress,the lumbrokinase activity increased first and then decreased under low concentration stress,while decreased continuously under high concentration stress.Compared with the control group,under the combined stress of cadmium and lead,the lumbrokinase activity decreased at first,then increased,and decreased at last.The effect of the content of hypoxanthine on Pheretima aspergillum was closely related to the concentration and time of heavy metal stress.Compared with the control group,when single cadmium stress,the content of hypoxanthine of Pheretima aspergillum decreased at first,then increased,and decreased at last.There was no significant change in the content of hypoxanthine with different concentrations of cadmium stress.Compared with the control group,when single lead stress,the content of hypoxanthine increased first and then decreased.Compared with the control group,under the combined stress of cadmium and lead,the content of hypoxanthine in the stress of low and medium concentrations were decreased at first,then increased,then decreased,and then increased at last,while the content of hypoxanthine in the high concentration stress was decreased at first and then decreased.