Isolation And Function Analysis Of A Drought Induced Gene In Maize (zea Mays L.)

Water stress is one of the major environmental stresses that seriously affect plant growth and crop yield worldwide. In this study we cloned three drought induced genes ZmFBL2, ZmCKS2 and ZmBTF3b from our previous drought related SSH library.ZmFBL2 is a F-box protein, contein a F-box domain and LRR domain. F-box domain was fistly found at the N-terminal region of cyclin F. At the N terminus of F-box proteins contained a conserved F-box motif (about 40~50 amino acid) and at the carboxy-terminal, F-box proteins often contained highly variable protein-protein interaction domains, such as WD40 repeat (Trp and Asp), Leu-rich repeat (LRR), tetratricopeptide repeat (TPR) and kelch repeat. In the past few years, many F-box proteins have been cloned in higher plants such as Arabidopsis, petunia, rice and maize. These F-box proteins played essential roles in flowering development, self-incompatibility, light signalling and clock control, plant hormone response pathways and abiotic stress. In this study, we isolated an drought induced gene ZmFBL2, a new F-box gene in maize. Its structure and functions was analized.ZmCKS2 was predicted to contain a CKS (cyclin-dependent kinase regulatory subunit) structural domain, which was highly conserved in eukaryotes kingdom. ZmBTF3b encoded a putative transcription factor of 169 amino acids with a conserved NAC (nascent polypeptide-associated complex) domain.The main results were as follows:1. In this study we cloned the full-length cDNA of ZmFBL2, by using in silico and homology-based cloning techniques. The deduced protein of ZmFBL2 was predicted to contain a F-box structural domain, which was highly conserved in eukaryotes kingdom and a Leu-rich repeat (LRR) domains at the carboxy terminal. The promoter of ZmFBL2 was predicted to contain important regulatory elements including core promoter elements and drought, high salt, cold, SA, ABA, oxygen responsive, light regulation and embryo, endosperm, seeds development elements.2. Real-time quantitative PCR (qRT-PCR) analysis revealed that ZmFBL2 was highly expressed in leaves at seedling stage, ear leaves, roots at flowering stage and tassels and was up-regulated under dehydration, PEG, cold, NaCl, 2,4-D and ABA treatment while was down-regulated under SA .3. We cloned the full-length cDNA of ten MSK (maize skp1-like gene) genes by using in silico and homology-based cloning techniques. Yeast hybrid assay revealed that F-box protein interacted specifically with MSK1,MSK2,MSK9 and ZmMADS6,ZmMADS7 and the F-box domain was necessary.β-Galactosidase filter assays verified the reliability of the results.4. Ten days old lins grown in organic fertilizer was treated by 20% PEG 6000. Survival rates of transgenic Arabidopsis was higher than WT Arabidopsis. On MS medium with ABA and NaCl, survival rates of transgenic lines was also higher than WT. But there was no obvious difference on MS medium with mannitol.5. Two other drought induced genes ZmCKS2 and ZmBTF3b were cloned in this study. The deduced protein of ZmCKS2 was predicted to contain a CKS (cyclin-dependent kinase regulatory subunit) structural domain, which was highly conserved in eukaryotes kingdom. The promoter of ZmCKS2 was predicted to contain important regulatory elements including core promoter elements and drought, high salt, cold, ABA, light regulation, copper and oxygen responsive elements. Real-time quantitative PCR (qRT-PCR) analysis revealed that ZmCKS2 was highly expressed in ears while its expression was very low in roots and leaves at seedling stage and was up-regulated under drought or MeJA treatment but down-regulated under cold or ABA treatment. Under NaCl stress ZmCKS2 showed no obvious change. These results revealed that ZmCKS2 might be involved in multiple pathways of plants responding to different environmental conditions. The cloned ZmBTF3b gene encoded a putative transcription factor of 169 amino acids with a conserved NAC (nascent polypeptide-associated complex) domain. Transcriptional activity analysis showed that ZmBTF3b might be a functional transcription factor. Real-time quantitative PCR (qRT-PCR) analysis revealed that ZmBTF3b was highly expressed in silks, ears and tassels and was up-regulated under dehydration or PEG treatment while was down-regulated under cold, NaCl, ABA or SA. ZmBTF3b might have the transcriptional activation activity and play an essential role in response to abiotic stresses.