CDNA Cloning And Expression Analysis Of Genes Involved In The Phosphatidylinositol Signaling Pathway From Maize

Phosphatidylinositol (PI) is a major phospholipid in eukaryotic cells. Early Studies have indicated phosphatidylinositol signaling pathway (PI pathway) plays a key role in plant growth, development and responses to environmental stresses. To begin the PI metabolic pathway, PI synthase (PIS) synthesizes PI from membrane-bound CDP-diacylglycerol (CDP-DG) and free cytoplasmic inositol. PI is then converted to PIP2 by sequential phosphorylation. PIP2 is hydrolyzed by phospholipase C (PLC), leading to the sustained production of inositol 1, 4, 5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 may be converted into differentially phosphorylated inositol molecules or myo-inositol, while diacylglycerol kinase (DGK) phosphorylates DAG into phosphatidic acid (PA). PA is then converted into diacylglycerol pyrophosphate (DGPP), which becomes CDP-DG as a result of a series of reactions before entering the next cycle in the pathway.Our research focuses on the molecular characterization and expression analysis of some genes involved in the PI pathway in maize.RNA was extracted from inbred line 90110 of maize and the cDNA was synthesized. Sequences of genes involved in PI pathway from Oryza sativa or Arabidopsis thaliana, were used to search the maize sequence database. Primers were designed according to the obtained sequences. cDNA sequences of ZmPIS2, ZmPLC2, ZmDGK1, ZmDGK2, ZmDGK3, ZmPLA2-1 and ZmPLA2-2, which contained full ORF (opening reading frame) and partial cDNA sequence of ZmPLC3 were obtained by RACE or in silico cloning combined with PCR. The BLAST searches with these cDNA sequences in the PlantGDB database led to the finding of some partial or full promoter sequences. Analysis with online software of PLACE showed that there were multiple cis-acting regulatory elements related to cold, drought and plant hormones.The RT-PCR primers were designed according to 3' or unconserved sequences of cDNAs of PIS gene family (ZmPIS, ZmPIS2), PLC gene family (ZmPLC, ZmPLC2, ZmPLC3), DGK gene family (ZmDGK1, ZmDGK2, ZmDGK3) and ZmPLD. RT-PCR analysis of cDNA from five tissues (root, stem, leaf, tassel, ear) of maize indicated that the expression patterns of these genes have specific expression patterns in different tissues. When their expression under treatment of cold, drought and plant hormones such as ABA, IAA, MeJA and GA3, were analyzed by Real-Time RT-PCR, it was found that that those gene families and isoforms involved in PI pathway varied in their expression patterns, which may be an indicator of their critical role in the vital processes.In addition, the plant expression vectors of ZmPIS2, ZmPLC2, ZmDGK1, ZmDGK2, ZmDGK3 and ZmPLA2-1 were constructed constructed for gene function analysis in this study. These efforts may contribute to future possible studies with transgenic techniques.