The Cloning And Expression Analysis Of Heat Shock Proteins 70s In Different Strains Of Heated Shock Silkworms

Heat shock protein 70 (Heat shock proteins, Hsp70s) are stressed proteins which exist widely from bacteria to mammals in conditions such as heat stress, starvation and bacterial infection. Not only they highly expressed in stress conditions, but also they be present consititutively under normal conditions. It is widely accepted that the induceble Hsp70s are expressed highly against stressful environment to resist heat. and the constitutive Hsc70s are expressed for basic resistance when at high-temperature.The expression and functions of Hsp70s may indicate very diffrently in different species because of the multiple different mumbers in each specie. There are 17 Hsp70s and 30 pseudogene in human with induceble Hsp70s such as Hsp70-1a, Hsp70-1b, Hsp70-1t, Hsp70-6 and consitutitive Hsp70s including Hsp70-5, Hsp70-9 and so on. In fruitfly, there are at least 12 Hsp70s members such as the induceble Hsp68 and Hsp70s as well as the consitutitive Hsc70-1, Hsc70-2, Hsc70-4, Hsc70-3. In 2009, Fabian M found that the Hsc70-3 of endoplasmic reticulum expressed higher in fruitflies when at high temperature than that in normal environment. The function of the Hsp70s members have been studied much in humans, fruit flies and yeast. But the expression of each member of Hsp70s is relatively researched less, especially the expression of Hsp70s in silkworm is scarely studied and the report about functions is still blank. It is necessary to studied Hsp70s in silkworms.In this thesis, the Hsp70s were cloned electronicly in silkworms and named according to the name of Hsp70s in other species. All the Hsp70s were also sited exactly on chromosomes.There are at least 10 Hsp70s. including 4 induceble Hsp70s and 6 constitutive Hsp70s. Hsp70A, Hsp70B, Hsp68 and Hsp70-14 are intronless belonging to induceble Hsp70s. Hsc70-4, Hsc70-3, Hsc70-5, Hsc70-2, Hsc70-cs and Hsc70Cb all contain introns which are expressed constitutively. Hsc70-3 is expressed constitutively in the endoplasmic reticulum and Hsc70-4 is expressed constitutively in the cytoplasm. Hsp70-14 is a protein of only 52KD and has a non-standard C motif end. In addition, there is a Hsp70-w named pseudogene on the chromosome 11 in silkworm, which is transcribed (CK517125), but can not be translated. The induced Hsp70B and Hsp70A are concentrated on chromosome 27, with multiple copies. Hsp70A and Hsp70B are chained on the genome, but in the opposite direction. Constitutive Hsp70s are located in different chromosomes, generally only one copy.Silkworm is a specie of Lepidoptera insects, with thousands years of economic useness. There are a large number of strains in gene library with different traits providing lots of good research materials. Our experiments contain three different strains including the cs-10 of thermotolerance,cs-11 of thermosensitive and cs-7 of close isogenic strain which is descendant of cs-10 and cs-11. The expression of Hsp70s in silkworm when at high temprature is systematicly analysised. Hsp68 and Hsp70B are expressed induceblely . Hsc70-3 is a heat shock protein in the endoplasmic reticulum which is constitutived expressed. Hsc70-4 is a constitutive heat shock protein expressed in the cytoplasm. The four Hsp70s of the three strains were cloned and sequenced. The analysis of the sequence for each member in using MegAlign software showned that each member of Hsp70s in the three strains are highly similar with only a few amino acids are different. Quantitative real-time PCR (qRT-PCR) is used to test the level change of their expression when the three strains of silkworms are cultrued at 40℃. The induceble Hsp70s including Hsp68 and Hsp70B and the constitutives including Hsc70-3 and Hsc70-4 are all up-regulated. This means that the four members of Hsp70s are all involved in the heat resistant, but the amount of expression of each member is changed greatly which indicating that there are certain relationships between the amount of Hsp70s expression and the ability of resistanting heat. Furthermore, the four Hsp70s mumbers showned dramatically differently at different being heated shock time, these indicate that although they all belong to the same family of Hsp70, but the abilities of them in heat resisting are not similar and the mumber of Hsp70s may cannot be replaced by another one. At the same time, Northern is used for analysing the expression of Hsp70B and Hsc70-3 in the three strains. The reserch about the expression of Hsp70s in silkworms is the base of further study for the mechanism of heat-resistant and can offer data for the function research of Hsp70s.