Isolation And Characterization Of Genes Involved In Response To Waterlogging In Roots Of Maize Seedings (Zea Mays L.)

Waterlogging, caused by flooding, long periods of rain, and poor drainage, is a serious abiotic stress determining crop productivity worldwide alongside with drought, salt salinity and extreme temperatures. In the middle and lower Yangtze River area, the major corn-growing region of South China, seasonal rainfall greatly affects maize plantation, leading to yield losses. The goal of this study is to identify a serial of genes responsing to waterlogging in the late stage and to reveal the molecular basis of waterlogging tolerance by reverse genetics approach. Furthermore, cloning and characterization of important candidate genes and development of molecular markers based on these genes might provide new strategies to improve waterlogging tolerance of maize through marker assisted selective breeding. The main results achieved are as following:1. In this study, the transcriptome at the late stage of waterlogging was assayed in root cells of the tolerant inbred line HZ32 using suppression subtractive hybridization (SSH). A forward SSH library using RNA populations from four time points (12 h,16 h,20 h and 24 h) after waterlogging treatment was constructed to reveal up-regulated genes, and transcriptional and linkage data was integrated to identify candidate genes for waterlogging tolerance. Reverse Northern analysis of a set of 768 cDNA clones from the SSH library revealed a large number of genes were up-regulated by waterlogging. A total of 465 ESTs were assembled into 296 unigenes. Bioinformatic analysis revealed that the genes were involved in complex pathways, such as signal transduction, protein degradation, ion transport, carbon and amino acid metabolism, and transcriptional and translational regulation, and might play important roles at the late stage of the response to waterlogging. A significant number of unigenes were of unknown function. Approximately 67% of the unigenes could be aligned on the maize genome and 63 of them were co-located within reported QTLs. The late response to waterlogging in maize roots involves a broad spectrum of genes, which are mainly associated with two response processes:defense at the early stage and adaption at the late stage. Signal transduction plays a key role in activating genes related to the tolerance mechanism for survival during prolonged waterlogging. The crosstalk between carbon and amino acid metabolism reveals that amino acid metabolism performs two main roles at the late stage:the regulation of cytoplasmic pH and energy supply through breakdown of the carbon skeleton.2. In the SSH library mentioned above, there was a gene encoding prolyl 4-hydroxylase (P4H) induced in the response to waterlogging in HZ32. In animals, prolyl 4-hydroxylases (P4Hs) are regarded as oxygen sensors under hypoxia stress, but little is known about their role in the response to waterlogging in maize.A comprehensive genome-wide analysis of P4H genes of maize (zmP4Hs) was carried out, including gene structures, phylogeny, protein motifs, chromosomal locations and expression patterns under waterlogging. Nine zmP4H genes were identified in maize, of which five were alternatively spliced into at least 19 transcripts. Different alternative splicing (AS) events were revealed in different inbred lines, even for the same gene, possibly because of organ and developmental specificities or different stresses. The signal strength of splice sites were strongly correlated with selection of donor and receptor sites, and ambiguous junction sites because of small direct repeats at the exon/intron junction frequently resulted in the selection of unconventional splicing sites. Eleven out of 14 transcripts resulting from AS harbored a premature termination codon, rendering them potential candidates for nonsense-mediated RNA degradation. RT-PCR indicated that zmP4H genes displayed different expression patterns under waterlogging. The diverse transcripts generated from AS were expressed at different levels, suggesting that zmP4Hs were under specific control by post-transcriptional regulation under waterlogging stress in HZ32.3. The full length of zmbRLZ was isolated from HZ32 using the technique of rapid amplification of cDNA ends, showed that zmbRLZ was consisted of 282 amino acids and was homogy to the domain of BRLZ5 and bZIP from 118 to 182 amino acids. The expression of zmbRLZ was peaked in HZ32 at 4 h and was not changed in Mo 17 under waterlogging in the roots of seedings. The vector with GFP-zmbRLZ was construeted and transferred into onion epidermis using Agrobacterium, indicating that zmbRLZ was located in nucleus. The encoding sequence of zmbRLZ was cloned into the expression vector and expressed successfully as GST (Glutathione S-Transferase) fusion protein in E. coli. The zmbRLZ-GST fusion protein could bind specifically to the ARE sequence from adhl promoter in vitro by Electrophoretic Mobility Shift Assay, indicating that products of zmbRLZ-GST fusion protein had a function of ARE binding specificity. A marker "zmbRC/G" was developed based on the SNPs of the sequences between HZ32 and K12 and was mapped on the linkage map of a F2 (HZ32×K12) population and was located between the markers umc1743 and umc1107 indicated that zmbRLZ was located in the QTL of waterlogging tolerance.