Molecular Cloning And Expression Analysis Of Ubiquitin-activating Enzyme And Ubiquitin-conjugating Enzyme Genes From Gracilaria Lemaneiformis Under Heat Shock

Gracilaria lemaneiformis is an important economic red alga which can produceAgar. The original production areas are Shandong Province and Liaoning Province.The heat-resisting types-981and07-2which were bred from the wild G.lemaneiformis, has been transplanted and cultivated in the waters of South China.Meanwhile this brought huge economic benefit and social, ecological benefits. But inthe natural waters of the South, the G. lemaneiformis still can not successfully lastthrough the summer. So, it is urgent to breed new materials with better character andstronger heat resistance. Therefore, the response mechanism of G. lemaneiformisunder high temperature stress should be studied immediately. Numerous studies haveshown that, the Ubiquitin/26S proteasome pathway (UPP) is the peculiar and efficientprotein degradation of organism which can increase the heat-resistant ability when thebody suffers the hyperthermy. Ubiquitin-activating enzyme and ubiquitin-conjugatingenzyme are the first and second enzymes of the UPP. The research of the twoenzymes will lay foundation for present the role of the UPP in the G. lemaneiformisheat-resisting.The ESTs of ubiquitin-activating enzyme gene and ubiquitin-conjugatingenzyme gene were screened from the differentially expressed cDNA library of G.lemaneiformis under high temperature stress, which indicated that UPP played animportant role in the response of G. lemaneiformis to heat stress. In this research, wildtype,981and07-2G. lemaneiformis were used as materials, the full-length sequencesof ubiquitin-activating enzyme gene and ubiquitin-conjugating enzyme gene werecloned. The results show that, the full length of DNA sequence of ubiquitin-activatingenzyme is1206bp, containing only one exon. There is an open reading frame (ORF) sequence with981bp which encodes326amino acids and a calculated molecularmass with35.4kD. And the sequence of amino acids contains conservative ATPbinding sites (LYDRQIRLWGLE, ELAKNVLLAGV, LKEMN, VVACI) and theubiquitin-activating domain (VVCAI…LMTEAC, VFLDLGDEYSYQ, AIVGGMWGRE). The sequences of ubiquitin-activating enzyme are the same among wild type,981and07-2G. lemaneiformis. The sequence of ubiquitin-conjugating enzyme geneis882bp, containing four exons and three introns. There is an open reading frame(ORF) sequence with444bp which encodes147amino acids and a calculatedmolecular mass with16.5kD. And the sequence of amino acids contains conservativeubiquitin-activating domain (GSICLDIL), the ubiquitin-conjugating domain(RIYHPNIN, KVLLSICSLL, DDPLV) and the ubiquitin-ligase recognition sites(KRI, YPF, WSP). The sequences of ubiquitin-conjugating enzyme gene are the samebetween wild type and981G. lemaneiformis.Under heat shock (28℃,32℃and36℃), Realtime-PCR was taken to test theexpression of ubiquitin-activating enzyme and ubiquitin-conjugating enzyme in wildtype,981and07-2G. lemaneiformis. The results show that: in wild type G.lemaneiformis, at28℃, expressions of ubiquitin-activating enzyme andubiquitin-conjugating enzyme were up-regulated to clear the damaged protein in thebody, while at32℃and36℃, expressions of the two enzymes were below normallevel, which may be due to the deactivation of the enzymes for transcription andtranslation under high temperature. The results suggested that for wild type G.lemaneiformis, the protein synthesis and folding was directly damaged above28℃.And the damage could not be repaired by UPP. So the metabolism of body could notbe completed. In981G. lemaneiformis, the expressions of ubiquitin-activatingenzyme and ubiquitin-conjugating enzyme were higher than the normal level at6hand72h at28℃. At32℃, both the duration time of expression and the expressionlevel of the two enzymes were better than28℃. While at36℃, the expression levelsof the two enzymes increased in the early of heat shock and then decreased undernormal level. This is consistent with that the high temperature resistance ability of981G. lemaneiformis is stronger than that of wild type G. lemaneiformis. In07-2G. lemaneiformis, at36℃, the expressions of ubiquitin-activating enzyme andubiquitin-conjugating enzyme increased significantly, which indicated that under hightemperature of36℃,07-2G. lemaneiformis still could up-regulate these two enzymesgenes’ expression to clear damaged protein. This is consistent with that the hightemperature resistance ability of07-2G. lemaneiformis is stronger than that of981G.lemaneiformis.This research mainly studied the expression of two key enzymes(ubiquitin-activating enzyme and ubiquitin-conjugating enzyme) of the UPP underheat shock, the results showed that the two enzymes could be up-regulated efficientlyto clear damaged protein under heat shock. In addition, the expression pattern wasdirectly associated with the high temperature-resistant ability of G.lemaneiformis.This research lays the theoretical basis for revealing molecular mechanism of G.lemaneiformis to resist heat shock and may provide the genetic information forbreeding heat-tolerant new species.