Cloning And Expression Analyses Of Genes Related To Signal Transduction Under High Temperature Stress In Pyropia Haitanensis

Pyropia haitanensis is an important economic macroalgae distributed in southern coast of China.In recent years,the frequent occurrence of rotten thallus caused by global warming has seriously restricted the sustainable development of the P.haitanensis culture industry.Therefore,it's very important to study the response mechanisms of P.haitanensis to high temperature stress and breed new varieties.P.haitanensis strain Z-61 was chosen as materials.This study cloned the full-length sequences of genes related to signal transduction under high temperature stress.We also analysed the signal transduction response to heat stress at levels of gene expression and physiology,aiming to provide theoretical basis for breeding new varieties and genetic engineering of P.haitanensis.The main results are as follows:1.Through the blast of NCBI database,the unigenes of 2 CaM,6 MAPK,2 MAPKK and 1MAPKKK were found in the P.haitanensis global transcriptome.These 11 genes were named as PhCaM1,PhCaM2,PhMAPK1,PhMAPK2,PhMAPK3,PhMAPK4,PhMAPK5,PhMAPK6,PhMAPKK1,PhMAPKK2 and PhMAPKKK1.The full-length sequences of PhCaM1,PhCaM2,PhMAPK1,PhMAPK2,PhMAPK3,PhMAPK4,PhMAPKK2 and PhMAPKKK1 were obtained by the rapid amplification of cDNA ends?RACE?technology or direct PCR.2.The concentrations of Ca²⁺in the cytoplasm of P.haitanensis responding to high temperature stress were measured using the fluorescence method.Between 5min to 20min of heat stress,the intracellular Ca²⁺concentration showed a rapid and strong increase and subsequently return to baseline levels.3.The MDA contents in P.haitanensis cells demonstrated a downward trend after an increase at initial stage of high temperature stress treatment,while the increase of MDA content was moved up by addition of Ca²⁺chelator EGTA,illustrating that Ca²⁺could alleviate the oxidative damage of P.haitanensis under high temperature stress treatment.4.The results of real-time quantification PCR of CaMs,MAPKs and HSPs genes showed that each gene family actively responded to heat stress.The expressions of PhHSP100 and PhHSP22 were related to Ca²⁺/CaM signal transduction.In addition,there may be two MAPK signal cascade pathways in response to high temperature stress.According to these studies,the Ca²⁺/CaM and the MAPK signaling transduction pathways of P.haitanensis play a key role in response to high temperature stress.