Sduty On Cloning Of Eglin C?Guamerin Genes And Expression Pattern In Poecllobdella Manillensis Lesson

Poecilobdella manillensis Lesson(Hirudinidae Whitman,Poecilobdella Blanchard)is a kind of blood-sucking leeches as traditional medicine leeches.Hirudin secreted by salivary glands of P.manillensis can be used for a specific thrombin inhibitor that is one of stronger natural antithrombin material,properties of anticoagulant,anti-thrombus,dissolving thrombus,reducing blood lipid.During sucking blood,blood-sucking leeches transfer a broad range of bioactive substances into the host's wound to prevent premature hemostasis and blood coagulation,such as P.manillensis.At present,the researchs on blood-sucking leeches are mainly focused on the seeking of active substances and further study of pharmacological action.However,the studies on the changes of anticoagulant activity and the expression of blood-sucking related genes in blood-sucking leeches during sucking stages are poorly.In this study,we mainly studied the changes of anticoagulant activity after leech harvested and the expression patterns of blood-sucking related genes during sucking stages of P.manillensis.The main findings are as follows:(1)The effects of different harvesting and processing methods on the anti-thrombin activities of P.manillensis were conducted respectively under different treatments:processing methods(vacuum freeze drying,fresh homogenate,dried in the sun,cold-death,scalded to death,baked out with different temperatures),defferent parts(entire body,cephalon,pygidium,exudate)and greding body weights(?10,10?20,20?30,30?40,?40 g).The anti-thrombin activiyies of P.manillensis after harvesting and differrent processing methods were evaluated by directly titration on thrombin.The results indicated that processing methods significantly affected the anti-thrombin activities of P.Manillensis,and among the eleven groups,anti-thrombin activity of vacuum freeze drying of P.Manillensis(1303.56 U·g-1)was significantly highest than other groups(P<0.05),and baked out with 90?(15.44 U·g-1)was lowest.Anti-thrombin activity of the cephalon of P.Manillensis(226.42 U· g-1)was highest and the pygidium(102.12 U·g-1)was lowest;The anti-thrombin activities under different body weights were significantly different(P<0.05),and among the five groups,the body weight of ?10 g(328.86 U·g-1)was higher than other groups(P<0.05),and the body weight of>40 g(87.71 U·g-1)was lowest.In conclusion,harvesting and different processing methods had a significant impact on the anti-thrombin activities of P.Manillensis,the investigation has obtained the optimal processing method and greding body weight,and suggests that harvesting of the body weight of 20?30 g,and processing method of vacuum freeze drying first or dried in the sun second.(2)The activity of antithrombin was determined by direct titration of thrombin in six feeding treatment.The results showed that the antithrombin activities in the salivary glands and anterior suckers were significantly higher than other tissues(P<0.05).Meanwhile,before and after sucking blood,the antithrombin activities were significant differences in the tissues of P.Manillensis(P<0.05).(3)Results showed that 371 bp sequence was acquired with the largest open reading frame 264 bp,which encoding 87 amino acids is PME of P.Manillensis.Blast comparison was carried out on NCBI.The genes has the higher homology compared with the Eglin C genes of other leeches.There were significant differences in the relative expression of PME gene in the tissues of P.Manillensis(P<0.05)before and after sucking blood.(4)In this study,we described the temporal and spatial expression patterns of a leech homeobox gene,PMG1 and PMG2,during rice embryogenesis during sucking.Results showed that 281 bp sequence was acquired with the largest open reading frame 186 bp,which encoding 61 amino acids is PMG1 of P.Manillensis.Meanwhile,298 bp sequence was acquired with the largest open reading frame 204 bp,which encoding 67 amino acids is PMG2 of P.Manillensis.Blast comparisons were carried out on NCBI.The genes both had the higher homology compared with the Guamerin genes from other leeches.There were significant differences in the relative expression of PMG1 and PMG2 genes in the tissues of P.Manillensis(P<0.05)before and after sucking blood.