Cloning And Functional Analysis Of ZmRH2 In Zea Mays L.

The full-length cDNA of ZmRH2, which expresses differently in near isogenic linesS-Mo17^rf3rf3 and S-Mo17^Rf3Rf3, has been cloned in our lab. On this foundation, the cloningof ZmRH2 genomic DNA was carried out and functional analysis was done by forwardgenetics, reverse genetics and bioinformation. Primary results are listed as follows:1. Cloning of ZmRH2 genomic DNA. A 5,965 bp-length DNA fragment wasobtained using PCR and IPCR., which contains a 2,040 bp-length upstreampromoter region, and a 700 bp-length 3' uncoding region. The sequence ofZmRH2 was submitted to GenBnak (Accession number: EF537575). Sequencesof ZmRH2 are the same in S-Mo17^rf3rf3 and S-Mo17^Rf3Rf3.2. Bioinformational analysis of ZmRH2. Amino acid sequense analysis of ZmRH2was done with Blastp. The result displayed that ZmRH2 showed highlyhomology with eIF4Aâ…¢, which the amino acid positive is 95% betweenZmRH2 and Np-eIF4Aâ…¢, 88% between ZmRH2 and Hs-eIF4Aâ…¢. Alignmentanalysis among eIF4Aâ…¢from various species revealed that ZmRH2 possessednot only the nine motifs of DEAD-box RNA helicase family, but also the eightmotifs which are special for eIF4Aâ…¢. So we presumed that ZmRH2 was theeIF4Aâ…¢of maize. In addition, ZmRH2 was also homologous with Fall ofepiphyte, and they had similar conservative motifs. The 3D configuration ofZmRH2, which was constructed with comparative protein modelling, fit thefunction of eIF4Aâ…¢.3. Copy number analysis of ZmRH2. Southern blotting was done, with 3' uncodingregion of ZmRH2 as probe. The result showed that ZmRH2 had single copy inboth S-MO17^rf3rf3 and S-MO17^Rf3Rf3 genome, and blotted bands were identicalin both lines.4. Stress-response analyses of ZmRH2. Young shoots with three leaves were treatedwith high-salinity, drought, cold and wound stress. Semi-quantity RT-PCR wasused to measure expression of ZmRH2. The result showed that transcription ofZmRH2 begined to elevate 1-2h after treating, which indicated that ZmRH2 wasan early responsive gene to these stress treatments.5. Subcellular distribution of ZmRH2. The vector with GFP-ZmRH2 fusion wasconstructed and transfered into onion epidermis using Agrobacteriumtumefaciens. Transformed onion epidermises were observed with fluorescence microscope at 48h after transformation. The result showed that there wasfluorescence in nucleolus, which indicated that ZmRH2 located in nucleolus.6. Knockout analysis of At-eIF4Aâ…¢. RNAi vectors for ZmRH2 and At-eIF4Aâ…¢were constructed. And Arabidopsis was transformed with RNAi vector forAt-eIF4Aâ…¢. Harvested seeds were selected with Hygromycin B, but no one wasthe positive line. The possible reason is that At-eIF4Aâ…¢is a necessary gene,once konckeded out, it would lead cells die.