Clone And Expression Pattern Analysing Of The JH Related Enzyme Of Ulomoides Dermestoides(Chevrolat)

Juvenile hormone(JH)is a kind of important hormones in insects.The presence of the hormone and its drop degree are very important for the maintaining of larva state,the development of reproductive organs and diapause.Up on determining of the instar numbers,stadium of larva,developmental duration of pupa of Ulomoides dermestoides(Chevrolat),methods such as gene cloning,comparison and qRT-PCR were used to study the gene sequences and their expression characteristics of those enzymes related to the synthesis,transport,metabolism of JH,then we use HPLC to detect the JH ? titer,the results are as follows:1.The biological characteristics of various periods(from eggs to adults)of U.dermestoides(Chevrolat)were observed.It was determined that normally there was five instars at larval stage,the 6th instar larva was occurred only under some extreme environment.The stadium of ecch instar and the developmental duration of pupa were determined.2.By using the gene cloning and RACE methods,full length gene of JH acid methyl transferase(UdJHAMT),JH binding protein(UdJHBP),JH esterase(UdJHE)and JH epoxide hydrolase(UdJHEH)of U.dermestoides(Chevrolat)were cloned.The conserved sequence of the 3-hydroxy methyl glutaric acyl CoA synthetase(HMGS)and 3-hydroxy methyl glutaric acyl CoA reductase(HMGR)gene were cloned as well.3.Comfirmed the conserved sequence of UdJHAMT,UdJHBP,UdJHE and UdJHEH from U.dermestoides(Chevrolat)by Blastn and Blatstx;Deduced the amino acid sequences above by finder(Open Reading Frame Finder);Calculated isoelectric point and formula weight of these amino acid sequences by ProtParam;Multiple alignments is presented by GENEDOC;And established phylogenetic tree by CLUSTALW 2.Then we found that:in the consanguinity,Tribolium castaneum is the most closed and the inscets of Lepidoptera is the most far away.4.By using qRT-PCR method,the mRNA expression characteristics of those genes in different instar larva(4th,5th and 6th instar)and pupa-adult period were detected,found that:(1)UdJHAMT is a enzyme obviously related with the molting and metamorphosis of U.dermestoides(Chevrolat).At the strat point of each larval instar(pupa or adult),the expression quantity of its mRNA was significantly lower.As the larval instars and body size get larger,JHAMT expression will also become comparatively larger.The UdJHAMT expression quantity changed at different time of the same larval instar,or between different insect states.In pupal and adult stages,UdJHAMT often appeared down-regulated express,the overall level in pupal and adult stages was significantly lower than that in larval stage.(2)UdJHBP is a transport enzyme of JH.At the strat point of each larval instar(pupa or adult),the expression quantity of its mRNA was significantly lower.Then its expressive quantity began to rise,and in the middle period of each instars(or pupa and adult),it maintained higher expression level.When U.dermestoides(Chevrolat)is going to moulting(or metamorphosis),its expression quantity declined quickly.The UdJHBP expression quantity changed relatively smooth.There is no over expression,and no obvious transcriptional level inhibition as well.(3)Both UdJHE and UdJHEH are JH metabolic enzymes,and directly involve in the molting or metamorphosis of U.dermestoides(Chevrolat).At the strat point of each larval instar(pupa or adult),the expression quantity of UdJHE and UdJHEH gene mRNA was significantly higher.Especially at the critical period,when U.dermestoides(Chevrolat)is going to moulting(or metamorphosis),a large number UdJHE and UtJHEH gene mRNA expressed,then maked U.dermestoides(Chevrolat)going into molting,pupation or eclosion.(4)In the detection of mRNA expression of HMGS and HMGR,no evidence of the speed limit associated with JH was found.The changing range of these two enzymes expressed in the period of 5th instar larva was small.The was no special changes in the expression when the 5th instar larva of U.dermestoides(Chevrolat)is going to moulting(to pupation).5.The potency of JH? is got from HLPC.Chromatographic condition:methanol:H2O=75:25,flow rate of 1.0 mL/min and measured wave length of 218 nm,appearance time at 12min,and the equation of linear regression:y = 23538 x + 57.84,R2 = 0.999,a= 23538,b=57.84,R2=0.999.(1)The JH ? content in U.dermestoides(Chevrolat)lavars is reduced when the bettle molted.(2)When the bettle begin to pupate or to eclosion,the JH ? content is the lowest during the time pupate-adult.(3)The JH ? content in U.dermestoides(Chevrolat)have a direct correlation with the every developmental stage of the bettle.6.From the data of all the qRT-PCR data about the gene we got and the JH ?,we characted the relationship between the JH ? and the mRNA correlated genes about the UdJHAMT,UdJHBP,UdJHE and UdJHEH.Then we discusse the relationship between every two mRNA about enzyme.(1)The UdJHAMT expression have a direct correlation with JH ? titer of U.dermestoides(Chevrolat)(2)Gene expression pattern analysis:mRNA expression of UdJHAMT and UdJHBP showed a high degree of consistency.The expressive quantity rising or falling trend between UdJHAMT and UdJHE or UdJHEH are basicly opposite.In the initial period of normal developing larva,pupa and adult,the UdJHE and UdJHEH gene expression level is highly consistency.In adversity induced special 6th instar larva,UdJHEH and UdJHE express show consistency in a certain period of time,but also reflected UdJHEH and UdJHE are complementary each other in the degradation of JH ? in this special instar.(3)The JH ? content of U.dermestoides(Chevrolat)has direct correlation with UdJHAMT expression,and has concerned with UdJHE and UdJHEH.