Improvements On The Methods Of Plant RNA Isolation And Northern Blot Analysis, And Effects Of Plastid Signal On Nuclear Gene Expression As Well As Leaf Morphogenesis

In this thesis, the methods of plant RNA isolation and Northern blot analysis were improved. And effects of plastid signal on expression of nuclear genes encoding different organelle-localized proteins and on leaf morphogenesis were studied.In the first part, an efficient and economic method for high quality RNA preparation from plant tissues was established. To avoid RNA degradation, sucrose, potassium chloride and Mg²⁺ were included in the extraction buffer. Plant tissues were lysed in the extraction buffer, then ribonucleases (RNAases) and other proteins were denatured and extracted by phenol/chloroform. After that, the DNA was selectively fractionated from RNA with sodium acetate (NaAc) (pH 5.6). The isolated RNA with this method gave good yield. Results of non-denaturing electrophoresis or formaldehyde agarose gel electrophoresis both showed higher amount of 25S ribosomal RNA (rRNA) than that of 18S rRNA. Northern hybridization gave sharp and clear signals. Both plastid gene and nuclear gene were amplified successfully by RT-PCR. These results show that, the RNAs isolated with this method are in good integrity and purity, and can meet the needs of most molecular biological experiments including gene cloning and expression analysis. In this method, phenol/chloroform were used to remove proteins and inactivate RNAases, NaAc (pH 5.6) was used to precipitate RNAs, thus largely reduced the experimental expenses.In the second part, factors which affect RNA staining sensitivity in the protocol of prior RNA staining with ethidium bromide (EtBr) before running agarose gels were systematically investigated The results showed that, the intensity of staining with prior" addition of EtBr is affected, not only by EtBr concentration as previously reported, but also by RNA amount, denaturation temperature and denaturation time. The sensitivity of prior staining RNA with EtBr is negatively correlated with Northern blot hybridization efficiency, therefore, for the design of a specific Northern protocol, the factors above which affect sensitivity of pre-staining RNA with EtBr must be considered simultaneously to get the best combination of staining sensitivity and efficiency of hybridization. When heterologous DN A probes was used to hybridize RNA pre-stained with EtBr, there is a more decrease of efficiency of Northern blot hybridization with homologous RNA probes as the concentration of EtBr in RNA samples increased. We proposed that, for the commonly and frequently used heterologous DNA probes in molecular biology study, the best overall performance of hybridization EtBr pre-stained RNA occurs when the EtBr concentration in prior staining is not in excess of 30 ug/mL, which is lower than the concentration of EtBr used with homologous RNA probes (50 ug/mL).In the third part, Lhcb gene expression and the content of chlorophyll (Chi) biosynthetic intermediates in Chl-deficiency mutant oilseed rape Cr3529 with defective chloroplast were investigated. The results showed that, higher amount of Lhcb mRNA presented in four-leaf stage mutant oilseed rape. But Lhcb expression was still light responsive, under the control of circadian and tissue-specific, indicating that the coordination expression of nuclear photosynthetic genes with the developmental status of plastids is perturbed in Cr3529. Results of Chi precursor content determination indicated that the Chi biosynthesis in Cr3529 was partially blocked at the early step and resulted in less accumulation of the substrate protoporphyrin IX and product Mg-protoporphyrin IX for first committed step of Chi biosynthesis. Roles of Lhcb expression in LHCII biogenesis and antenna size control are discussed. The comparison of porphyrin contents and transcript level for photosynthetic proteins in Cr3529 with previously published data in inhibitor andmutant studies suggests a specific function for levels of Proto IX and Mg-Proto in the regulation of nuclear photosynthetic genes. We proposed that their levels are sensed by catalytic subunit of Mg-chelatase, H subunit in a continuous manner, and this signal is transferred to nucleus, which in turn to coordinate the expression of nuclear photosynthetic genes with the functional state of chloroplasts.In the forth part, Effects of plastid signal on expression of nuclear genes coding for ,I plastid and non-plastid photosynthetical proteins as well as mitochondrial protein, fand on leaf morphogenesis were investigated. The transcripts from nuclear genes for plastid proteins, Lhcbl and RbcS, for peroxisomal glycolate oxidase, GLO and for mitochondrial alternative oxidase, Aoxl, were maintained at least to the normal level in mutant oilseed rape with defective-developmented chloroplasts. When the development of chloroplasts were inhibited by lincomycin, an inhibitor of chloroplast protein synthesis, the transcript levels for these three kinds genes were all decreased, while a lesser inhibition extent was exhibited for GLO. In mutant oilseed rape with inhibited-developmented chloroplasts, the expression of the three kinds genes were all de-repressed, the transcript levels for these genes were at least 2-fold higher than those in lincomycin-treated wild type oilseed rape. These results suggested that, Cr3529 carries a lesion in plastid signal with which to regulate expression of nuclear genes for plastid and non-plastid proteins and mitochondrial proteins, nuclear genes for different organelle-localized proteins are differently sensitive to plastid developmental and functional status. Cotyledon anatomy also showed an alteration of cell layers and cell arrangement in Cr3529 cotyledon. The different effects of expression of nuclear genes for different organelle-localized protein and leaf development in Cr3529 suggest that there is a difference between plastid signal regulate nuclear gene expression and plastid signal regulate leaf development.