Research On The Calmodulin Signaling System Under Heat Shock In Gracilaria Lemaneiformis

Gracilaria lemaneiformis is an important agar-producing seaweed. In order toimprove the heat-tolerant ability and extend the period of cultivation, it is important toresearch the molecular response mechanism to high temperature stress in G.lemaneiformis. Previous studies of SSH under heat shock have shown that calmodulin(CaM) plays an important role in heat stress response of G. lemaneiformis. In thispaper, cam and a kind of calmodulin-binding protein (CaMBP)—myo-inositol-1-phosphate synthase(MIPS) were first cloned from G. lemaneiformis.Then the transcriptions of them were analyzed under heat shock by real-time PCR.This work lays foundations for researching the molecular response mechanism of G.lemaneiformis in heat shock, and provides the genetic information for breeding theheat-resistant strain of G. lemaneiformis.Firstly, a variety of calmodulin (CaM) genes were cloned from the wild type and981G. lemaneiformis in this paper.Wild G. lemaneiformis contains three kinds of cam:cam1, cam2and cam3.981G. lemaneiformis contains two kinds of cam: cam1andcam2. cam1in two kinds of G. lemaneiformis are exactly the same. The length ofcDNA sequence of cam1is526bp and the length of ORF is450bp, encoding150amino acids. The length of the DNA sequence of cam1is832bp, containing fourexons and three introns. There is only one copy in genomic DNA of wild type and981G. lemaneiformis. Compared with cam1, cam2is different in the two kinds of G.lemaneiformis. In the wild G. lemaneiformis, the length of cDNA sequence of cam2is600bp and the length of ORF is447bp, encoding149amino acids. In the981G.lemaneiformis, the length of cDNA sequence of cam2is474bp and the length of ORFis447bp, encoding149amino acids. Both of the cam2in the two kinds of G.lemaneiformis have no introns in their DNA sequences. The genetic similarity is78%,and the similarity of amino acid sequence is87%. The length of cam3DNA sequenceis809bp. Analyzed by the software, the cam3sequence contains two introns and threeexons, and the length of cDNA sequences is447bp. But right now, the gene can onlybe amplified from the genomic DNA of wild G. lemaneiformis, and can not beamplified directly from cDNA, indicating that cam3may be silent in the transcription process.Secondly, the transcriptions of cam in wild G. lemaneiformis and981G.lemaneiformis under heat shock were analyzed by real-time PCR. The results showthat, whether at28°C or32°C, the transcription level of cam1in981G.lemaneiformis is higher than that in wild G. lemaneiformis, indicating that cam1in981G. lemaneiformis is more effective under high temperature stress, which maybecauses the981G. lemaneiformis to be better heat-resistant. The transcription patternof cam1and cam2in981G. lemaneiformis are not the same. At28°C heat shock, theoverall transcription level of cam2is lower than cam1, but at32°C heat shock, theirtranscription patterns are similar. The results indicate that in the signal transduction of981G. lemaneiformis, CaM1may play a major role under ordinary heat stress, whilethe transcription of CaM2might upregulate only at higher temperature and longerheat shock time. In addition, cam2of wild G. lemaneiformis could only be amplifiedby nested PCR reaction, so it is difficult to analyze its transcription by real time PCR.In comparison,981G. lemaneiformis may have more kinds of CaM involved in signaltransduction under heat shock, which may be directly related with the thermostabilityof981G. lemaneiformis.As reported, CaM can be activated only after combination withcalmodulin-binding protein (CaMBP), and then regulate the signal transductionpathway. In our previous work, the EST of a kind of calmodulin-binding protein(CaMBP)—myo-inositol-1-phosphate synthase (MIPS) was obtained by Yeast TwoHybrid System. On that basis, the whole sequence of mips was first amplified in thispaper. The sequence is the same in two kinds of G. lemaneiformis and has highhomology compared with other species. The length of mips DNA sequence is2067bp.There is no intron in this sequence. The open reading frame is1621bp, encoding540amino acids. By analyzing the amino acid sequence of MIPS, the CaMBD(CaMBinding Domain) of MIPS is predicted at the site from the212th amino acid to the236th amino acid, which is according with the structural characteristics of CaMBD inother species. The interaction between MIPS and CaM was confirmed by Yeast TwoHybrid System, which proved that MIPS could interact with CaM as a kind ofcalmodulin binding protein. Then the transcription of CaM and MIPS in two kinds ofG. lemaneiformis under heat shock of32°C was analyzed by real-time PCR. Thetranscription level showed a cyclical upward trend, but the transcription trends ofCaM and MIPS were not identical. The results imply that although MIPS may be a kind of downstream binding protein of CaM, its transcription level does not fluctuatewith the transcription level of CaM. CaM may be not the only upstream activatingsubstance of MIPS. The two proteins might have independent activity, and play rolestogether to withstand high temperature stress in G. lemaneiformis. In addition, thetranscription level of CaM in981G. lemaneiformis is higher, which might relatedirectly with the heat resistant superiority of981G. lemaneiformis.