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Studies on the Transcription and Process of atp6-orfH79 and the Function of Fertility-restoration Related Genes in CMS-HL Rice

Author: WangKun
Tutor: ZhuYingGuo
School: Wuhan University
Course: Genetics
Keywords: Oryza sativa Honglian type cytoplasmic male sterility Restorer gene Bacterial two hybrid system GRP protein atp6-orfH79
CLC: S511
Type: PhD thesis
Year: 2009
Downloads: 100
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Abstract


The CMS rice generally could be categorized into three types, namely, WA-type, Honglian(HL)-type and BT-type. The HL-type CMS rice was discovered in the last century’s 70s by Wuhan University. Currently, it had been promoted and applied to large cultivated area of hybrid rice. The molecular mechanisms for CMS and fertility restoration of Honglian CMS system were performed in this paper. The results were shown as follow.1. With Northern blot, primer extension, CR-RT-PCR and RACE methods, a detailed analysis was made about CMS associated gene of HL-type rice sterility line Yuetai A(YTA) and hybrid F1 Honglian you-6(HL-6) on transcriptional and post-transcriptional level. Based on this, a comparative study between HL-type and BT-type at the molecular level was done. From the results, some similarities were found. However, there did exist some mentionable differences in transcription mode of CMS associated gene in sterility line and cleavage sites functioned by restorer gene in hybrid F1 between BT-type and HL-type CMS rice. Main results are as follows: (1) Transcript initiation sites 1117 and 1124 in the intergenic region of H-atp6 were found, the length of novel transcripts were 483 and 476 bp. Density gradient centrifugation assay verified that the 483 and 476 bp of transcipts would be associated with polysome, which implied that the novel transcripts possessed the ability of translation. (2) There might be 3 of cleavage sites on the atp6-orfH79 transcript by the restorer gene(s) in 9311. one was at the-11--13 bp upstream of atp6, the other two were at 1132 and 1170 of the intergenic region of atp6-orfH79 transcript.(3) a phylogenetic research was carried through based on DNA sequences of intergenic region of CMS associated gene among 33 rice accessions with orfH79, the results showed that the B-atp6 and H-atp6 respectively belonged to different subgroups.2. In order to study the molecular mechanism of sterility and fertility restoration in Honglian CMS rice, the total RNA was extracted from the Young Panicle of F1 hybrid rice by Trizol reagent. The mRNA was purified from total RNA by Poly-A Tract mRNA Isolation Kit and the double-stranded(ds) cDNA was synthesized after reverse transcription. The fragments longer than 400 bp in length were fractionated by Sephrose CL-2B Column, and then ligated into the pTRG vertor. The ligation product was transformed into XL 1-Blue MRF’Kan Library Pack Competent Cells to construct the bacterial Two-Hybrid system library. Analysis showed that the primary library contained 1.03×106 pfu/mL, and approximately 100% of the clones were positive recombinants and about 97% of the cDNA fragments inserted were longer than 800 bp in length. The capacity of the amplified library was 2.14×1012 pfu/mL.3. Many researchers thought that the restorer gene(s) play their function of processing transcript in the form of complex. For clarifying the mechanism of fertility restoration by restorer gene Rf5 in CMS-HL rice, we conducted a detailed analysis on the RF5 and GRP protein which was a candidate interactor with RF5. The main findings were as follows:(1) Recombinant protein of RF5 and GRP were expressed and purified in E. coli. The secondary structure of these two proteins were analyed by circular dichroism and the results showed that the secondary structures predicted by CD matched the characteristic of PPR and RRM motif. (2) The qualitative and quantitative interaction between GRP and the intergenic RNA of H-atp6 in vitro was acquired by gel shift and SPR assays. Furthermore, the more precise bind region in two small sections was identified by yeast three hybrid assay.

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