Functional Studies On Nuclear Restore Of Fertility Of CMS-S And Responsive Genes To Submergence Stress Of Maize (Zea Mays L.)

Utilization of heterosis is an efficient strategy for enhancing yield on unit area of maize (Zea mays L.). Cytoplasmic male sterility (CMS) has been used extensively in breeding for the production of a range of F1 hybrid crops because of its advantages on decreasing the cost of labor and improving seed quality. For using CMS in maize production by the greatest extent, it is necessary to theoretically elucidate the mechanisms of steility and restoration of fertility. The fertility of CMS-S is controlled by mitochondrial gene orf355lorf77 and nuclear gene Rf3/rf3. The chimeric gene orf35/orf77 had been cloned, and it was revealed that its expression effect on expression of other cytoplasmic and nuclear genes, but we have known little on the nuclear restorer of fertility Rf3/rf3, including its sequence and acting mechanism. In this study, the tassels from a male sterile line S-Mo17^rf3rf3 and its near isogenic line S-Mo17^Rf3Rf3 were selected to isolate microspore and pollen under different developmental stages; transcriptome was detected by cDNA microarray and suppression subtractive hybridization (SSH) in order to identify genes-associated with nuclear fertility restorer of maize CMS-S. And some primary results are listed as follows:1. The RNA from different tissues was extracted during different developmental stages of maize inbred S-Mol7^Rf3Rf3, and further to construct individual cDNA libraries respectively. Eight cDNA libraries had been made from young bud (containing embryos bud and coleoptiles), seedling with three leaves(containing roots), seedling with three leaves by submerging treatment (including roots), seedling with six leaves(including roots), interjacent meristem of stem, pooled anther, immature seeds after pollinating 5 days, and mature leaves. Sequentially, a normalized cDNA library designed by the MONL, containing more than 6x10⁴ recombinant, was achieved by the saturation hybridization between genomic DNA and plasmid DNA from individual cDNA libraries. The inserted fragment in the MONL varies from 0.4Kb to 4.0Kb, average inserted size is about 1.18Kb. Sequencing and bioinformatics analysis show that 71% sequences are unique ESTs, 12%ESTs are novel sequences which can not be found in existing DNA Database, only 23.9% sequences have annotated function in maize or other plant database, most of sequences, about 76%, were of unknown function. In the MONL, cDNA clones represent extensively maize genes. And abundance of cDNA clones from house keeping genes had been come down markedly accompany with enrichment of low-abundant genes.2. Inserted fragment from cDNA clones of the MONL were amplified by PCR, and run on agarose/EtBr gel, a total of 10830 high quality PCR products were selected and arrayed on nylon membrane in order to construct the microarray. For evaluating the quality of microarray, the pooled RNA from five distinctly tissues was hybridized with two set of cDNA microarray respectively. R2(square of Pearson correlation coefficiency) between signal value of corresponding dot from two set of microarray equals 0.9728, and R2 between signal value of duplicated dots on the same microarray equals 0.965, indicating that reproducibility of two sets of normalized data and duplicated dots within the same slide is good.3. The microspores and pollens in different developmental stages were isolated by sucrose density gradient centrifugation. Uninucleate microspore stages including pre-vacuolated(Y), multiple small vacuolated (SV) and single large vacuolated (LV) were purified from pre-emergent tassels by 20-60% sucrose density gradient centrifugation. Later pollen stages characterized by different degrees of starch filling or abortion (SF or CP) were isolated from emergent tassels by 20-75% sucrose density gradient centrifugation. The cDNA of SV, LV and SF1/CP1 from S-(Rf3) and S-(rf3) were prepared by reverse transcription respectively, and then hybridize with microarray. Hybridization using cDNA of SV, LV and SF1 revealed 7611,7494 and 7105 positive clones respectively, it demonstrated that number of gene expression in small vacuolated microspore is more than that in large vacuolated and starch filling pollen (117 and 506 clones ), number of gene expression in large vacuolated microspore is more than that in starch filling pollen (392). A tendency is that the number of expressed genes is decreased stage by stage from SV to SF, the result is accordant with physiological activity in different developing stages. 398 clones which were greater than 4-fold difference were revealed between LV and SF, 115 clones were revealed among LV and SV, account for about 3.7% and 1.06% of total clones on microarray respectively. Comparing gene expressions between S- (Rf3) and S- (rf3) , 166 clones whose expression changed equal to or greater than ±4-fold represented 127 unique tentative genes(UTGs). These UTGs were assigned into 9 functional categories based on their biology function. They representtranscripts for a broad spectrum of biochemical, cellular and signaling processes, ranging from cell component and pollen development (15%), signal transduction (13%), metabolism (15%), transcript and transcript factor (5%), protein synthesis and fate (13%), transport facilitation (4%) and cell defense and apoptosis (7%). Among the categories, 27 out of 127 were related to unknown function protein (20%), 10 out of 127 were novel ESTs (8%).4. Suppression subtractive hybridization between S-(Rf3) and S^rf3 gametophyte revealed 67 up-regulated expression clones in S-(Rf3) which represent 13 genes or gene families. Three out of 13 genes encoding PG, expansin and allergen were discovered in microarray data too. Query on gene function showed that 13 genes act primary in later stages of pollen development, involve in signal conduct, pollen maturation, pollen tube germination and etc. Apart from genes encode maize 25S rRNA, 26S rRNA and a unknown functional protein of Arabidopsis, no function gene which was upregulated expression in S-(rf3) was revealed. And genes, which involved in carbohydrate metabolism, starch biosynthesis and transporter, were not discovered too.5. Four apoptosis inhibitors (cystatin > Vdac2 > BI-1 and 14-3-3), two apoptosis-associated genes (translationally controlled rumor protein-like protein and leaf-senescence-related protein) and genes-associated with Ca2+ signal cascade and with small G protein signal cascade expressed differentially in pollen between S-(rf3) and S-(Rf3). Northern blot revealed the same phenomena and spatial and temporal expression patterns of cystatin, vdac2, Bl-land 14-3-3. Oligonucleosomal cleavage of total DNA is earlier in S-(rf3rf3) than that in S-(Rf3Rf3) anthers, DNA fragmentation is appeared in S-(rf3) pollens too. In S-(rf3) pollen, the abnormal expressions were discovered on genes which locate on the inner membrane of mitochondria and associate with aspiratory metabolism, such as Cox II, Cytochrome cl, ATP synthase B-subunit and inorganic pyrophosphatase, and on genes which involve into H+ transfer such as thioredoxin m2 and dihydroflavanoid reductase, sequentially resulted in disruption of respiratory metabolism and dysfunction of mitochondria.6. Six out of fifteen interested ESTs from maize were located on chromosome 2. The six ESTs might encode PP2C, CDPK, CDPK-related protein kinase, Tim9, S-AdoMetDC and translationally controlled tumor protein-like protein (TCTP) respectively. S-AdoMetDC has been discovered 2 copies on chromosome. Interestedly, DNA sequences of CDPK, CDPK-related protein kinase, Tim9 and Translationally controlled tumor protein-like protein were located on 2.09bin, especially, Translationally controlled tumor protein-likeprotein, Tim9 and CDPK were flanked on umcl525 which linked tightly with Rf3.7. Taken together these results, we suggest that expression product of orf355lorp7 in S-(rf3) microspore function as a signal to induce abnormally expression of respiratory chain-associated genes, and cause disruption of electron transport and mitochondrial dysfunction. Energy deficiency was appeared in pollen, and mitochondrial apoptosis was triggered. The mechanism of Rf3 might potentially regulate accumulation of nuclear and mitochondrial gene transcripts directly or indirectly to inhibit multiple PCD pathways in S-type cytoplasm allowing the normal developmental pathways to unfold.Another area focused in our research is that identification and cloning of early stage responsive genes of maize to submergence stress. Submerging stress is one of the primary stress factors to maize in the southwest of china during the spring season. The essential of submergence is anaerobic stress. During submerging stress, the gene expressions in stress early stage are play a key role to regulate the expressions of genes in late stage of stress treatment which can are translated into anaerobic polypeptides (ANPs), and to control alterations of cellular and molecular adaptations in maize roots directly. In the study, a cDNA microarray resource has been development to study the low-oxygen stress response. 32 cDNA clones, which represent 24 non-redundant genes, expressed 3-fold higher under submerging stress. The transcriptional level is markedly altered after 2 hours submerging treatment. Early-responsive genes under submerging stress are involved in signal transduction, glycolysis, photosynthesis, transcription and translation, lipid and energy metabolic pathway. Three novel cDNAs were identified and submitted to public DNA DataBase (from CF569035 to CF569037). A cDNA (AY515607) encoding the putative zinc finger protein (ZmZF) was cloned and sequenced; its biological function is also inferred. The expression of a SKPl/ASKl-like protein, 20S proteasomes subunit a-3 and ZmZF increased more than 3 fold after 2h low-oxygen. Based on their expression pattern and function, we hypothesize that regulation mechanisms on translation level also play a critical role to low oxygen responsive in early submerging stage.