A polygalacturonase inhibitory protein gene (<Emphasis Type="Italic">BcMF19</Emphasis>) expressed during pollen development in Chinese cabbage-pak-choi

The level of polygalacturonase inhibitory protein (PGIP) genes involved in pollen development remains unclear. Characterization of the different PGIP genes that are expressed in pollen is necessary in understanding the similarities and differences of functions between the members of this gene family, as well as the underlying mechanism of pollen development. A gene-encoding putative PGIP, BcMF19 was successfully cloned on a cDNA-amplified fragment length polymorphism fragment after it was found to be up-regulated in the fertile flower buds of Chinese cabbage-pak-choi (Brassica campestris L. ssp. chinensis Makino) genic male sterile AB line (Bajh97-01A/B). The amino acid sequence of BcMF19 possessed the basic feature of PGIPs, containing an N-terminal signal peptide, several potential N-glycosylation sites, two disulfide bridges flanking both the N- and C-terminal regions, and 10 leucine-rich repeat (LRR) consensus sequences. Real-time RT-PCR verified the higher expression of BcMF19 in the fertile flower buds compared to the sterile flower buds. In situ hybridization showed that BcMF19 was exclusively expressed in the tapetal cells and microspores during anther development. These results indicate that BcMF19 is a novel PGIP gene that might be involved in pollen or tapetum development.     

Cloning and Characterization of the Microspore Development-Related Gene BcMF2 in Chinese Cabbage Pak-Choi (Brassica campestris L. ssp. chinensis Makino)

For the sake of providing some important information relevant to the study of the molecular mechanism of genic male sterility in plants, gene differential expression in flower buds at different developmental stages, as well as in rosette leaves, florescence leaves, and scapes was analyzed using cDNA amplified fragment length polymorphism (cDNA-AFLP) in the genic male sterile A and fertile B line of Chinese cabbage pak-choi. Following amplification of 125 pairs of primer combinations, 11 differential fragments were obtained, of which eight were from the B line and the other three were from the A line. Of 11 differential fragments, four were verified by Northern hybridization that were expressed preferentially in fertile flower buds. Results of GenBank BLAST showed that one fragment was with unknown function,whereas the other fragments have strong nucleotide sequence similarities with the polygalacturonase (PG)gene, the pectinesterase (PE) gene, and the polygalacturonase inhibitory protein (PGIP4) gene. Only fulllength cDNA from the differential fragment BcMF-A18T16-1 was amplified by rapid amplification of cDNA ends (RACE) and Northern analysis showed that this fragment was expressed only in medium and largesized flower buds of the B line. The full-length cDNA, designated as BcMF2 (Brassica campestris Male Fertile 2), was 1 485 bp long and was composed of a 1 263-bp open reading frame, which had 83% nucleotide similarity to a PG gene from Arabidopsis encoding polygalacturonase. Analysis of the basic structure of the protein revealed that it had one polygalacturonase active site (RVTCGPGHGLSVGS) at 256th site of amino acids and was classified as being a member of family 28 of the glycosyl hydrolases. The role of the BcMF2 gene on microspore development is discussed in the present paper.     

Cloning and Characterization of the Microspore Development-Related Gene BcMF2 in Chinese Cabbage Pak-Choi （Brassica campestris L. ssp. chinensis Makino）

For the sake of providing some important information relevant to the study of the molecular mechanism of genic male sterility in plants, gene differential expression in flower buds at different developmental stages, as well as in rosette leaves, florescence leaves, and scapes was analyzed using cDNA amplified fragment length polymorphism (cDNA-AFLP) in the genic male sterile A and fertile B line of Chinese cabbage pak-choi. Following amplification of 125 pairs of primer combinations, 11 differential fragments were obtained, of which eight were from the B line and the other three were from the A line. Of 11 differential fragments, four were verified by Northern hybridization that were expressed preferentially in fertile flower buds. Results of GenBank BLAST showed that one fragment was with unknown function, whereas the other fragments have strong nucleotide sequence similarities with the polygalacturonase (PG) gene, the pectinesterase (PE) gene, and the polygalacturonase inhibitory protein (PGIP4) gene. Only fulllength cDNA from the differential fragment BcMF-A 18T 16-1 was amplified by rapid amplification of cDNA ends (RACE) and Northern analysis showed that this fragment was expressed only in medium and largesized flower buds of the B line. The full-length cDNA, designated as BcMF2 (Brassica campestris Male Fertile 2), was 1 485 bp long and was composed ofa 1 263-bp open reading frame, which had 83% nucleotide similarity to a PG gene from Arabidopsis encoding polygalacturonase. Analysis of the basic structure of the protein revealed that it had one polygalacturonase active site (RVTCGPGHGLSVGS) at 256th site of amino acids and was classified as being a member of family 28 of the glycosyl hydrolases. The role of the BcMF2gene on microspore development is discussed in the present paper.     

Meiotic abnormality in dominant genic male sterile <Emphasis Type="Italic">Brassica napus</Emphasis>

Rs1046AB is a dominant genic male sterile (DGMS) Brassica napus line derived from Yi-3A. Until now the molecular mechanism of its male sterility is still unknown. In this paper, cytological observations demonstrated that all cells in sterile plants contained condensed nuclei at the beginning stage of meiosis; this implied that meiotic cells were degenerating. Although 31% (93/300) cells escaped from the state of nuclei condensation in buds about 3 mm in length (in such length, normal plants are at tetrade stage), no cells could pass the pachytene stage. Then pachytene-or zygotene-like chromatin/chromosomes sometimes congregated into two or more groups with different size, which resulted in the formation of micronuclei. A nucleoplasmic bridge could also be found in some meiotic cells. Even when the “microspore’s analogue” appeared in sterile buds about 4 mm in length (in such length, mature pollens could be detected in normal buds), the nuclei condensation and escaped cells with a pachytene-like chromosome still could be found in the sterile anthers. So it could be concluded that male sterility was caused by meiotic abnormality. According to our previous research, four genes related to cell cycle/DNA processing were identified in fertile plants. RT-PCR further confirmed that three DNA repair genes were partially or completely repressed in the sterile plants and were only expressed in the early stage fertile flower buds, i.e., the buds <3 mm in length. Therefore, DGMS of rapeseed was probably caused by the abnormality in the DNA damage repair system during meiosis. According to these results, some possible mechanisms of fertility control were discussed.     

Isolation and characterization of a male sterility gene homolog <Emphasis Type="Italic">BcMS2</Emphasis> from Chinese cabbagge-pak-choi that expressing in an anther-specific manner

A male sterility gene homolog, designated BcMS2, was isolated from flower buds using gene-specific primer pairs and was submitted to GenBank under accession number EF093533. Comparison of BcMS2 gene with MS2 from Arabidopsis thaliana and MS2Bnap from Brassica napus revealed some differences in gene structure and evolution. The full genomic DNA sequence of BcMS2 was 2,576 bp in length containing 8 exons and 7 introns, more than those of MS2Bnap but less than MS2. RT-PCR showed that BcMS2 gene expressed only in stage III flower buds of male fertile Chinese cabbage-pak-choi 'ZUBajh97-01B' and there were no detection in all organs of Polima cytoplasmic male sterility (CMS) line 'Bpol97-05A' and Ogura CMS line 'Bogu97-06A'. Furthermore, RT-PCR revealed that BcMS2 expressed only in anthers of male fertile material and there were no expression in sepals, petals, filaments and pistils. These results suggested that BcMS2 was an anther-specific gene and might be essential for the fertility of Chinese cabbage-pak-choi.     

Cytoplasmic effects on DNA methylation between male sterile lines and the maintainer in wheat (Triticum aestivum L.)

Male sterile cytoplasm plays an important role in hybrid wheat, and three-line system including male sterile (A line), its maintainer (B line) and restoring (R line) has played a major role in wheat hybrid production. It is well known that DNA methylation plays an important role in gene expression regulation during biological development in wheat. However, no reports are available on DNA methylation affected by different male sterile cytoplasms in hybrid wheat. We employed a methylation-sensitive amplified polymorphism technique to characterize nuclear DNA methylation in three male sterile cytoplasms. A and B lines share the same nucleus, but have different cytoplasms which is male sterile for the A and fertile for the B. The results revealed a relationship of DNA methylation at these sites specifically with male sterile cytoplasms, as well as male sterility, since the only difference between the A lines and B line was the cytoplasm. The DNA methylation was markedly affected by male sterile cytoplasms. K-type cytoplasm affected the methylation to a much greater degree than T-type and S-type cytoplasms, as indicated by the ratio of methylated sites, ratio of fully methylated sites, and polymorphism between A lines and B line for these cytoplasms. The genetic distance between the cytoplasm and nucleus for the K-type is much greater than for the T- and S-types because the former is between Aegilops genus and Triticum genus and the latter is within Triticum genus between Triticum spelta and Triticum timopheevii species. Thus, this difference in genetic distance may be responsible for the variation in methylation that we observed.