Express Pattern Of Metalloprotease-a New Type Of Calmodulin Binding Protein Under Heat Stress In Gracilaria Lemaneiformis

Gracilaria lemaneiformis is an important commercial alga for production of agar. Theoriginal production areas are Shandong and Liaoning in China. G. lemaneiformis has becomethe third largest cultivation alga following Saccharina and Pyropia. To promote thedevelopment of cultivation of G. lemaneiformis, researchers have bred new varieties with hightemperature resistant ability-the cultivar981and07-2. However, with the increase ofcultivation time, these two strains also faced the problem of degradation. Therefore, it isurgent to study the response mechanism of G. lemaneiformis to high temperature stress.The ESTs of metalloproteinase of Gracilaria lemaneiformis (GlMP）was screened fromyeast two hybrid system of G. lemaneiformis using CaM as the bait under high temperaturestress, which indicated that GlMP may play a role in the CaM signal transduction in heatresponse of G. lemaneiformis. In this paper, the metalloproteinase (GlMP) gene was clonedand the expression pattern of GlMP under high-temperaturein G. lemaneiformis was explored.The research layed the foundation for study the molecular mechanism of heat response of G.lemaneiformis and provided the available genetic information for breedingnew strains.In this research, the wild type, cultivar981and07-2of G. lemaneiformis were used asmaterials. The full cDNA and genomic DNA encoding GlMP were obtained by using rapidamplification of cDNA ends (RACE) and genome walking techniques respectively. ThecDNA sequence of GlMP consisted of546nucleotides, containing an open reading frame(ORF) of462nucleotides, a5’ UTR of36bp and a3’UTR of48bp. The ORF of GlMP waspredicted to encode153amino acids, including the zinc metalloprotease HEXXH consensusmotif ofmetalloproteinase.The DNA sequence of GlMP is1123bp, including an intron of71bp and a promoter of542bp. There is no difference in the composition of sequence of GlMPof the wild type, cultivar981and07-2. Southern blotting indicated that there are two copiesof GlMP in the three strains of G. lemaneiformis. The interaction between GlMP and CaM was confirmed by Yeast Two Hybrid System, which proved that GlMP could interact withCaM as a kind of calmodulin binding protein.By comparing GlMP and CaM expression levels of the three kinds of G. lemaneiformisunder heat stress, GlMP in wild type expressed higher than that in981and07-2, CaM in981expressed higher than the rest of the two strains, while the heat resistant properties of thethree kinds of G. lemaneiformis is07-2>981>Wild. The results showed that heat resistantperformance is not determined by single factor, which may relate to a variety of hightemperature resistance factors. The expression level of both GlMP and CaM are higher in32°C than in28°C, which indicated that the two factors may act positively in response to heatstress.By constructing a yeast two-hybrid library,39kinds of CaM bingding proteins(CaMBPs)were screened. The result of the biological analysis showed that the proteins were proteases atmost, participating in a variety of hydrolysis, oxidation, reduction, decarboxylation reaction.And the secondary structure of the proteins is consistent with the characteristics of CaMbinding dormain (CaMBD). According to the CaMBP database, GlMP has a CaMBD in82-123animo acid sites. According to the prediction, the open reading frame was divided intofour parts-Front, Most, Last, Whole. The yeast two-hybrid results showed that the colonies ofMost and Whole parts could turn blue, while the Front and Last parts could not turn blue,which was consistent with the expected results and further demonstrated that GlMP was aCaMBP.This study investigated the CaM signaling system of G. lemaneiformis under hightemperature stress and layed the theoretical basis for revealing molecular mechanism of G.lemaneiformis to resist heat shock.