| Gracilaria lemaneiformis is an important economic red alga, which can produceagar and also can be used as feedstuff for abalone aquaculture, with the value oflarge-scale cultivation. Although the heat-resisting cultivars-981and07-2promotethe development of G.lemaneiformis cultivation, high sea temperatures will restrainthe growth and lead to decay, even cause death in the actual cultivation process. Tosolve these problems, breeding of a new strain with better character and stronger heatresistance is important. Consequently, research the mechanism oftemperature-response will lay basis for breeding. Heat shock proteins (HSPs) are theprotein family which can increase the ability in the stress tolerance when the bodysuffers the stress. They play a significant role in reducing the harm of stress on thebody. Studying HSP will lay basis for understanding the response mechanisms of G.lemaneiformis to heat stress and provide insights for selecting new strain of G.lemaneiformis that can endure the stress of much higher temperature.Based on the partial hsp90sequence obtained from the suppression subtractivehybridization (SSH) cDNA libraries of G. lemaneiformis, full DNA sequence of heatshock protein90gene was cloned. The full length of hsp90DNA was2605bpincluding an ORF of2100bp and the upstream regulatory sequence of505bp. hsp90encodes for669amino acids containing conserved N-terminal ATPase domain,intermediate domain and a conserved5peptide-MEEVD. From Blast analyzing, thesimilarity about sequence of hsp90in G. Lemaneiformis and Chondrus crispus is thehighest，up to87%. After further comparison, no differences were found between thesequences of hsp90in the wild type,07-2and981. Southern blotting proved that therewere two copies of hsp90in the genome of wild type,07-2and981.The sequence is different between hsp70-1, hsp70-2cloned by our lab and the hsp70reported in GenBank. The reported partial hsp70sequence named hp70-3wasconsidered as the third new hsp70gene in G. lemaneiformis. So the hsp70-3gene wascloned. The full-length of hsp70-3DNA is2780bp including an ORF of1867bp，theupstream regulatory sequence of416bp and the downstream regulatory sequence of495bp. Hsp70-3encodes for621amino acids containing conserved N-terminalATPase domain and C-terminal substrate binding domain. From Blast analyzing, thesimilarity about sequence of hsp70in G. Lemaneiformis and Gracilaria tenuistipitatais the highest,up to91%.This study tried to find the appropriate reference gene from ten candidate genes,including actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH),elongation factor1(EF1), eukaryotic translation initiate factor (eIF), histone (H2B),18S ribosomal rRNA (18S rRNA), internal transcribed spacer2(ITS2), cytochrome coxidase (COX), cytochrome b-5reductase (CR) and phycoerythrin (PEB) of G.lemaneiformis under temperature stress. The results are as followed: a) The moststability reference genes are eIF and ACT at32℃with different heat shock time. b)The best reference genes are GAPDH, ITS2,CR, and18S rRNA at8℃with differenttreated time. c) The reliable reference genes are eIF, EF1and ACT for differenttemperature treatments. d) For different generations, the most effective referencegenes are GAPDH and ACT.The mRNA transcriptions of the hsp90and hsp70-3are analyzed at hightemperature (32°C) of wild type，07-2and981using Real-time PCR. The resultsshow that when three types of G. lemaneiformis are exposed to high temperaturestress, the transcription levels of hsp90are increased again after the first increase andthen decrease. Hsp90is expressed highest in07-2and followed by981, while inwild type the expression level of hsp90is the lowest, which indicates that the amountof hsp90expression is consistent with the ability of temperature resistance in the threedifferent G. Lemaneiformis. The transcription levels of hsp70-3are decreased in07-2and increased in981and wild type under heat stress. The expression level of hsp70-3is highest in wild type. Compared with the transcription levels of hsp70-1, hsp70-2detected by Yinghui Gu, hsp70-3is considered as the main factor of HSP70family that responses to high temperature stress in wild type of G. Lemaneiformis. Forcultivar981of G. Lemaneiformis, under high temperature stress, the transcriptionlevels of hsp70-1, hsp70-2and hsp70-3are all increased, indicating a variety ofHSP70proteins together in protecting cells from high-temperature injury, which maybe one of the reasons for the high temperature tolerance of981. Although cultivar07-2of G. Lemaneiformis has the highest heat-resistant ability, the transcriptionlevels of hsp70-3are decreased under heat stress, indicating that other elementsresponse to temperature stress may play a more important role.In this paper, hsp90and hsp70-3are cloned and the transcription patterns of themunder heat stress are analyzed for the first time, which will help us to illuminate theresponse mechanism to heat stress of G. lemaneiformis and also provide usefulsequences for screening new strain of G. lemaneiformis with stronger heat-resistantability. |
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