| Ga1-S is a gametophytic mutant involved in maize cross incompatibility. The presence of the dominant allele Ga1-S in silks prevents fertilization by pollen carrying the recessive allele ga1. Successful pollination relies on pollen-pistil interactions by continued communication and coordination between the pollen and stigma. The process depends on a congruous and compatible recognition of the pollen and on appropriate conditions for pollen grains adhesion, pollen hydration, pollen tube germination, and the newly formed pollen tube subsequently penetrates the stigma, grows down through the transmitting tract of the style, and directional growth toward the ovules. In this process, the pollen tube interacts with several different types of cell, so it becomes an ideal model system for studying changes of gene expression patterns in response to cell-cell interactions. However, the molecular mechanisms of these events involved in pollen-pistil interactions are largely unknown in maize. And the physiological basis of cross incompatibility is poorly understood in maize.In this investigation, fist we compared the process of pollen tube growth in three crosses by fluorescence microscopy; second we constructed BC1F1 population for fine mapping the gene Ga1-S, and the tightly linked markers to Ga1-S would help marker-assisted introgression and fine mapping would greatly facilitate the isolation of Ga1-S; finally we performed transcriptomic profiling and proteomic profiling of Ga1-S silks pollinated by Ga1-S and ga1 pollens in an attempt to isolate genes involved in cross incompatibility. The main results are as follows:1 By fluorescence microscopy pollen tube growth observation indicated ga1 pollen tube growth is obstructed in 401D (GG) pistil, Cross incompatibility resulted from pollen tube growth arrest around 2-10h after pollination. We compared pollen tube growth among three crosses: 401D (GG) selfing (compatible), 401D(GG)pollinated with W22pollen (ga1) (incompatible) and W22 (ga1ga1)pollinated with 401Dpollen (G) (compatible), and the results indicated that pollen grains germinated well and pollen tubes were able to enter the transmitting tract. Pollen tubes grew much faster in compatible cross than that in incompatible cross. Compatible pollen displayed a linear growth of about 10mm h-1 while incompatible pollen 2.5mm h-1. At 20h interval, pollen tubes reached ovule in compatible crosses, while pollen tubes were found in the silk segment 5.5cm distal to the pollination area in the incompatible cross and no pollen tube reached ovule area. 2 Since F2 population is a distorted segregating population for Ga1-S, we construct a BC1F1population W22// W22/401D. And Ga1-S has been previously reported on chrosome 4, SSR markers on this chromosome were used to screen the BC1F1 population W22// W22/401D. Ga1-S was initially detected by all SSR on chrosome 4 short army in public database and linked to SSR umc2410 on BAC AC206720 and SSR umc1294 on BAC AC190633. Consequently, to fine map the target gene, new SSR markers were developed from the BACs between this regien . The closest markers we found AC184840-2 that is 2.31cM to the target gene on the centromere side, and AC184772-1 which didn't found exchange event on the telomere side. There are 11 BACs between AC184840 and AC184772 (AC184840, AC194254, AC203042, AC191393, AC184113, AC180823, AC205353, AC214441, AC196002, AC210943, AC184772). The genetic region mapped spanning the Ga1-S locus was estimated 2.31cM in length.3 We performed genome-wide transcriptional profiling of 401D (GG) pistil tissues 5 hour after pollination (5HAP) with G and g pollen respectively, using the Solexa deep sequencing technology. The profiling analysis revealed numerous changes in gene expression attributable to the Ga1-S, which resulted in the down-regulation 737 genes and up-regulation 641 genes. Gene Ontology analysis revealed that the differentially expressed genes (DEGs) were significantly the structural molecule activity (GO: 0005198) in DEGs (p<0.05), and included 66 genes involved in transcription, transport, and cell organization. 31genes were up-regulated and 35 genes down-regulated, and we selected 17 genes which may related with cross-incompatible, included Mitochondrial carrier-like protein,Nucleic acid binding protein,Ubiquitin fusion protein,CIPK-like protein,containing leucine-rich repeat receptor-like protein kinase,RALF precursor and so on. The results of this study suggest that the regulation of reinforcing, modifying, or remodeling the structure of the transmitting tract and the pollen tube tip play a critical role in pollen-pistil interactions during pollen tube elongation in maize pistils, and the inhibition of these functions maybe the molecular mechanism of how Gametophytic factor1-Strong (Ga1-S) presence in stylar tissue completely prevents ga1 pollen to accomplish fertilization.4 First we compared proteomic pfofiles of total silk proteins of 401D (GG) 10 h after pollination (10HAP) by G and g pollens. Total proteins were extracted by TCA/Acetone, separated by two-dimensional gel electrophoresis (2-DE) and analyzed through MALDI-TOF-MS mass spectrometry. The results indicated that in the silk proteome of crosses GG×GG and GG×gg , there were 25 differentially expressed proteins, including 15 specifically expressed in GG×GG, and 10 specifically expressed in GG×gg. And 12 of them were annotated in various databases by MALDI-TOF-MS and MASCOT analyses. Proteins 11, 12, 14, 18, 22 and 24 presumably play important roles in the maize cross incompatibility.Secondly compared the 401D pollen/silk proteome 1 hour after self-pollination (1HAP) with the proteome 2 hour after self-pollination (2HAP)。The pollen/silk proteome profiles were different between the 1hour and 2 hour pollination intervals. The majority of differentially expressed proteins were up-regulated; down-regulated and speciafically expressed proteins were also found in the 2 hour pollination proteome profile. There were 28 differentially expressed proteins, including 6 specifically expressed ones, 19 up-regulated and 3 down-regulated ones. And 23 of them were identified homologous to known proteins in various databases by MALDI-TOF-MS and MASCOT analysis. The predicted functions of the 23 proteins were related to cell wall biogenesis (21.4%), defense/stress responses (17.9%), cellular organization, protein synthesis and transcription. Secretory peroxidase, expansin, pectin methylesterase inhibitor, glutathione S-transferase and unknown protein 4 presumely play important roles in early pollen-silk interactions in maize.
|
PDF Full Text Request