Cloning And Environmental Response Of Heat Shock Protein 90 And Rhodopsin Gene Of Prorocentrum Donghaiense

Red tide is a hot issue in the current global marine environment.Prorocentrum donghaiense is a common red tide algae species in coastal waters of China.At present,the research on molecular adaptation mechanism of P.donghaiense in various stress environments is not thorough enough.In this study,two genes of P.donghaiense were cloned and expressed,and we hoped to understand the molecular adaptation mechanism of P.donghaiense in various environmental conditions.The heat shock protein 90(HSP90)is a molecular chaperone,plays essential roles in cell's machinery for protein folding,keeping structural integrity and helps to protect cells from environmental stress.Based on early laboratory work,specific primers were designed to obtain the 5? and 3? of HSP90 by RACE.The full length of HSP90 is 2443 bp from assemble 5? and 3? sequence.This sequence was submitted to Gen Bank under Accession No.KX899924.HSP90 contains a 5?-UTR of 111 bp,a 3?-UTR of 199 bp,and a complete open reading frame(ORF)of 2133 bp.The ORF encodes a polypeptide of 711-amino-acid residues with a molecular weight of approximately 85.81 k Da and an isoelectric point of 4.95.Phylogen etic analysis revealed that Pd HSP90 belongs to the dinoflagellate clade and is closely related to HSP90 s from P.minimum(Accession No.ADP65795.1)and P.micans(Accession No.AAR27546.1).Real-time q PCR reveled that the expression of HSP90 reached the maximum at 10 ? under temperature stress.When the temperature lower than 20 ?,the expression of HSP90 was gradually reduced as the temperature rises;when the temperature was above 20 ?,the expression of HSP90 was gradually increased.In nitrogen-deficiency experiment,real-time q PCR reveled that the expression of HSP90 reached the maximum at 88 ?M nitrogen(P < 0.05).In phosphorus-deficiency experiment,the expression of HSP90 reached the maximum at 3.6 ?M phosphorus(P < 0.05).With the decrease of nitrogen or phosphorus concentration,the expression of HSP90 trend to increased and then decreased.Under the heavy metal of copper stress,the expression of HSP90 reached the maximum at 32.25 ?M copper(P < 0.05).The expression of HSP90 reached the maximum and then decreased.The above results indicate that stress conditions such as temperature,nutrient and heavy metals can cause HSP90 expression to counter the negative effects of environmental stress.The main function of rhodopsin gene is to capturing light energy and converting it to ATP.We used the RACE to get the 5? and 3?sequence of rhodopsin.The full length of rhodopsin is 1104 bp by assembling 5 ? and 3?.This sequence was submitted to Gen Bank under Accession No.KY399746.Rhodopsin contains a 5?-UTR of 106 bp,a 3?-UTR of 221 bp,and a complete open reading frame(ORF)of 777 bp.The ORF encodes a polypeptide of 259 amino-acid residues with a molecular weight of approximately 28.79 k Da and an isoelectric point of 5.83.Phylogenetic analysis revealed that rhodopsin belongs to the dinoflagellate clade and is closely related to rhodopsins from Pseudo-nitzschia granii(Accession No.AJA37445.1).Real-time q PCR showed that the expression level of rhodopsins gene showed a tendency to increase at first and then decrease with the prolongation of illumination time in the LL cycle;The expression of rhodopsin decreased gradually in the DD cycle,there was significant difference(P < 0.05)with control group;In the dark-light cycle,the expression of rhodopsin decreased gradually in the night,and increased rapidly after restoration of light;Under the color spectrum,the expression level of rhodopsin was the highest in green group,and there was significant difference(P < 0.05)with white group(control);Under the condition of phosphorus stress,the expression level of rhodopsin decreased with the decrease of phosphorus concentration.When phosphorus concentrations below 7.2 ?m,the expression of rhodopsin showed significantly different with control(P < 0.05)Furthermore,the expression vector and expression bacterial stra in of rhodopsin were prepared,and the spectral characteristics of the protein were analyzed,and found the maximum absorption of this protein located at 320 nm.These results provide a new insight for understanding the adaptation mechanism of P.donghaiense under environmental stress,and have some reference for studying the adaptation mechanism of other dinoflagellates.