Digital Gene Expression Profile Analysis Of Maize In Response To Combined Stress Of High Temperature And Drought And Function Analysis Of ZmsHSP17.7

Maize is the main food crop, feed and industrial raw materials. High temperature and drought can occur in the whole growth period of maize, which is the major abiotic stresses decrease the yield and quality. Recently much works has been done about physiological, biochemical and molecular effect of high temperature and drought on maize. While there is a little systematic information about the combined stress. In this study, we analyze the gene expression profile of maize under stress of high temperature, drought and combination by high-throughput RNA sequencing. Differentially expressed genes(DEGs) were screened and annotated to elaborate the mechanism of maize in response to combined stress of high temperature. We cloned a small heat shock protein HSP17.7and analyze its function.The main results are as follows:From each sample under condition of control, high temperature, drought and combined stress, more than7million raw reads were sequenced, of which97.5to98.8percent were clean reads. Adaptor sequence is the major contaminant reads. About77.8to79.1percent of clean reads can be annotated according to the reference genome. The proportion of uniqueness with multi-position matched by clean reads(57.5%-58.1%) is slightly higher than that of unique match(41.9%-42.5%). The coverage degree of reads in most of the genes (99.6%) is below10%and that of a few genes(4.0%-7.9%) is very wide(30%-100%). The uniform distribution of reads in the genes suggested little self-degradation of mRNA and nonhomogenous fragmentation in library preparation. Reads classification and copies show high-level heterogeneity and partial redundancy. A few genes are expressed in high level, while most genes are expressed in low level. Differentially expressed genes(DEGs) were screened out according to standards of differentially expression>2folds stress than control, P-value<0.005and FDR<0.01. The number of up-regulated genes is more than down-regulated genes under stress of high temperature, drought and the combination. Nine hundred and ninety six genes is differentially expressed under stress of high temperature, of which612DEGs is up-regulated. Under stress of drought there are1097DEGs and795DEGs up-regulated. Under combined stress there are2025DEGs and1375DEGs up-regulated. One hundred and fifteen DEGs are shared under three kinds of stresses. There are75.0%DEGs in response to high temperature and61.5%DEGs in response to drought showed the same regulation pattern with combined stress. While42.1%up-regulated genes and44.2%down-regulated genes were specially regulated by the stress combination. The DEGs number under combined stress is larger than that of high temperature or drought. This increasement is not caused by accumulation of two kinds of single stress but new DEGs from the stress combination.GO and KEGG analysis shows that DEGs are involved in basal metabolism, secondary metabolism and hormone signal transduction pathways. The enrichment GO items and KEGG pathways from high temperature and drought stresses represent obvious difference. A few DEGs were significantly induced more than100folds by both stress. These differeces indicate the high complexity under combined stress. Content of proline can be increased by stress of high temperature, drought and the combined stress. But the gene expression profiles indicate great difference in regulation direction and regulation level of DEGs under three stress conditons. So the same physiological phynotype may result from different molecular basis.Referring to the maize genome sequence, a small molecular heat shock protein(sHSP) gene was cloned. The full length of474bp cDNA encodes a protein of158amino acids, of which the isoelectric point is predicted to be5.36and molecular weight of17745.86Da. Sequence similarity, homology and subcellular localization analyses showed the sHSP, named as ZmsHSP17.7, is cytosolic class I HSP. The expression anaylsis by northern blotting indicated that ZmsHSP17.7can be rapidly and ABA-independently induced by high temperature. Drought did not independently regulate the gene expression, but can enhance the regulation function from high temperature. Overexpression of ZmsHSP17.7gene can enhance tolerance of the transgenic Arabidopsis thaliana plants to multiple stresses of high temperature, drought and NaCl. It was suspected that ZmsHSP17.7gene to be member of the defense system against abiotic stress.