The Identification And Cloning Of Pheretima Aspergillum(E Perrier)Metallothionein

1ObjectiveThe Chinese medicine "Guang Dilong" is the dried body of Pheretima aspergillum(E Perrier) which originated in Family Megasoolecidae. The quality of Guang Dilong is the best of the four earthworms that recorded by Chinese Pharmacopoeia used as Chinese medicine.The international market share of" Guangd Dilong" is disrupted for its exceeding concentration of heavy metals.Earthworms that live in the wild always enrich high concentration of heavy metals throught MT-2. So MT-2is an important gen in the process of the accumulation and metabolism of heavy metals in earthworms. Earthworms produce metallothionein in the stress of heavy metals. By binding to heavy metals, Metallothionein function as "antidote" to help earthworm survive in the stress of heavy metals. In this study, we cloned and expressed MT-2so as to help finding the inducing Characteristic of MT-2. And we also made gen walking study to obtain the sequence of the promoter of MT-2.Finnaly, this study aim to clarify the inducing mechanism of MT-2, which may help making solution in relieving the enrichment of heavy metals and improve the quality of "Guang Dilong".2Method2.1The collection and laboratory induction of Pheretima aspergillum(E Perrier)Pheretima aspergillum(E Perrier) were collected in the wild,identified and divided into different boxes in laboratory. After adapting to lab condition, Pheretima aspergillum(E Perrier) were exposed to different concentration of Cd.2.2Total RNA extraction and SqRT-PCRThe total RNA of earthworms prostate is extracted. And then SqRT-PCR and gel electrophoresis were performed. The results of gel electrophoresis was analyzed by Quntity One so as to investigate the Relevance between mRNA expression abundance and Cd concentration.2.3Cloning of gen MT-2 cDNA of MT-2was amplified by PCR, PCR product was purified and cloned to pGEM-T easy vector. the colonies were screened by blue/white screening and PCR. Positive colonies were sequenced and analysed.2.4Construction of MT-2prokaryotic expression plasmidObtain the ORF of MT-2by PCR, PCR product is purified, double-digested with EcoR Ⅰ/HindⅢ and then inserted into the expression vector pET-32a(+) which is digested with the same enzymes, then transform the recombinant plasmid with E. coli DH5a. The positive colonies were identified by PCR and restriction digest of isolated plasmid DNA and then performed sequencing to the positive plasmid.2.5Expression of MT-2as fusion proteinThe MT-2prokaryotic expression plasmid were extracted from E. coli DH5α,and transformed with E. coli BL21.The cell were plated on LB plates containing Ampicillin. Positive colonies were identified by PCR and restriction digest of isolated plasmid DNA. Expression of MT-2gene was investigated in different E. coli BL21carrying prokaryotic expression plasmid under the induction of IPTG SDS-PAGE was used to analyze the results of protein expression. The expression products were analysed by activity testing.2.6Tail-PCRThe total DNA of earthworms was extracted. Taking total DNA as template, Tail-PCR was performed. The results of gel electrophoresis was analyzed and then sequencing the bane within the predicted size.3Results3.1SqRT-PCRBy inducing experiment, SqRT-PCR of MT-2were Successfully performed. The results of SqRT-PCR showed that the mRNA expression abundance were uploaded by Cd induction.3.2Cloning of MT-2By blue/white screening and gel electrophoresis. pGEM-T easy-MT is Constructed. The results of sequencing showed that the ORF of MT-2was an approximately237bp fragment, coding78amino acid.3.3Construction of MT-2prokaryotic expression plasmidBy PCR, double-digested and sequencing, positive clones of pET-32a(+)-MT-2was identified. The predicted size of fusion protein expressed was approximately25KDa. 3.4Expression of MT-2as fusion proteinDifferent inducing time, concentration and temperature were performed in the prokaryotic expression. And the best inducing condition was generated. An approximately25KDa fusion protein was expressed. The fusion protein existed both in the the supernatant and the precipitation while mostly they were Soluble protein. The results of activity-testing showed that the fusion protein was of high activity of original MT-2protein.3.5Tail-PCRTail-PCR of MT-2were successfully performed. The gel electrophoresis showed an complete single bright bane which consistent with the expected size.4ConelusionThis study researched the correlation between MT-2and heavy metals through SqRT-PCR. The cloning and prokaryotic expression plasmid was constructed while the fusion protein containing MT-2function was expressed. Tail-PCR of MT-2were successfully performed. The gel electrophoresis showed an complete single bright bane which consistent with the expected size.But,the Tail-PCR product failed in the sequencing. There might be two reasons. For one thing Tail-PCR adapted two primers of different length, the Tm of which differ greatly. This might lead to difficult combination to the samples in the formal sequencing program and result in failure. For another thing, the purified PCR product of gel electrophoresis might contain different fragment with the same size, which might also lead to failure sequencing. For the first reason, T-A clone would help solve the problem. For the later one,we can only screen more pure bane by more Tail-PCR process.