An Fe Deficiency Responsive With MAPK Cascade In Chlamydomonas Reinharditii

Iron is an important indispensable element in the organism and it is involved in electron transfe,r the cell respiration, DNA synthesis and as a cofactor in many enzymatic reactions in the cell.Iron is high iron form (Fe3+)in the environment and it cannot be directly absorbed by the body,soIron deficiency is the three major physiological diseases of agricultural production after Phosphorus reduction and nitrogen reduction.chlorophyll synthesis can reduce inhigher plants with reduced iron,and results in the decrease of photosynthetic rate influences the plant growth and development, cause huge economic losses.Human with reduced iron can lead to iron deficiency anemia,and too much can cause tissue damage; So the cell must have a set of strict regulation mechanism to adjust the assimilation and metabolism of iron, iron homeostasis of cell.This research is based on the iron-deficiency of Chlamydomonas Reinhardti and gives response to the mutant mu9 which was filtered by the defect mutant library, and the deleted gene Femu9p is a MAPKKK kinase. The missing of Femu9p gene is the reason that caused the iron regulatory genes such as FOX1, ATX1, FTR1 and FEA1 reduce significantly in expression. Therefore we can infer that MAPK signal pathway may play a role in deliver the iron-deficiency signal of chlamydomonas. Thus, we researched related kinases of MAPK, MAPKK, MAPKKK family of chlamydomonas. By using the RNAi technology on CrMAPK3, CrMEK1, CrMAPKKK13 to knockout gene. The transgenic algae strains report gene and iron regulation gene are as follows.Mitogen activated protein kinase (MAPK) is serine/threonine protein kinasepresent in the eukaryotes,The MAPK cascade is composed of MAPKKK,MAPKK and MAPK, the signal transmission through phosphorylation of MAPKKK-MAPKK-MAPK in turn,it plays an important role in eukaryotes differentiation, growth, development and apoptosis.At present,the study for MAPK pathway with iron deficieny just appears in the human cancer.Under the condition of iron loss,the research reports about Chlamydomonas reinhartii is less.Now 16 MAPK gene,9 MAPKK gene,27 MAPKKK genes of tne Chlamydomonas reinhartii are retrieved.CrMAPK3,CrMEK1 and CrMAPKKK13 are studied.The main study as follows:(1) The bioinformatics analysis of the Chlamydomonas reinhardti MAPKKK genes, MAPKK gene and MAPK gene.All the MAPK cascade gene in Chlamydomonas reinhardti have serine/threonine protein kinase domain structure.The core sequence of MAPK genes sequence is TXYVxTRWYRAPE (L/V).The core sequence of The MAPKK is VGTxxYMSPE.The MAPKKK gene sequence of core can be divided into two groups, G (T/S) Px (W/Y/F) MAPEV and GTxx MAPE (W/Y).(2)The Chlamydomonas reinhardti MAPK cascade pathway differentially expressed genes of RNA-seq analysis with-Fe-N, P, and 16? low temperature treament in 24hours.The heat map shows that with-Fe and 16? low temperature treament, most of the MAPK cascade pathway gene expression is low;And with-P and N condition treament, the majority of MAPK cascade pathway gene expression is higher. The Chlamydomonas reinhardti MAPK cascade gene expression level have a significant change in in-Fe-N-P and 16? low temperature.So that they may be involved in-Fe-N-P and 16? low temperature signal response and regulation.(3) Build CrMAPK3, CrMEK1, CrMAPKKK13 RNAi expression vector and converse Chlamydomonas 2A38, and conduct cytology and molecular biology detection in the transgenic algae strains.CrMAPK3 RNAi interference carrier transforms chlamydomonas 2A38, it can get 132 converters. ARS activity detection on the cultivated algae strains shows that there are a total of 88 converters which show colorless or pale blue, their color ratio is 67.7%. Contrasting the mRNA, genetically modified algae strains RNAil,RNAi37, RNAi62 CrMAPK3 were relatively decreased significantly. ARS enzyme activity decreased respectively 82%,85%,83%, ARS mRNA level reduced by 99%. mRNA level of endogenous CrFOX1, CrNRAMP2, CrATX1, CrFTR1, CrFEA1 dropped significantly. It shows that the CrMAPK3 gene of chlamydomonas is related with the regulation of iron metabolism related gene.CrMEK1 RNAi interference carrier transforms chlamydomonas 2A38, it can get 81 converters. ARS activity detection on the cultivated algae strains shows that there are a total of 64 converters which show colorless or pale blue, their color ratio is 79%. Contrasting the mRNA, genetically modified algae strains RNAi1,RNAi2, RNAi 18 CrMEK1 were relatively decreased significantly. ARS enzyme activity is decreased, ARS mRNA level is reduced. mRNA level of endogenous CrFOX1, CrNRAMP2, CrATXl, CrFTR1, CrFEAl dropped significantly. It shows that the CrMEK1 gene of chlamydomonas is related with the regulation of iron metabolism related gene.CrMAPKKK13 RNAi interference carrier transforms chlamydomonas 2A38, it can get 84converters. ARS activity detection on the cultivated algae strains shows that there are a total of 70 converters which show colorless or pale blue, their color ratio is 83.3%. Contrasting the mRNA, genetically modified algae strains RNAi4, RNAi 16, RNAi51. CrMAPKKK13 were relatively decreased significantly. ARS enzyme activity is decreased, ARS mRNA level is reduced. mRNA level of endogenous CrFOX1, CrNRAMP2, CrATX1, CrFTR1, CrFEA1 dropped significantly. It shows that the CrMAPKKK 13 gene of chlamydomonas is related with the regulation of iron metabolism related gene.(4) CrMAPK3 and CrMEK1 is positioned in the cytoplasm.