Cotton Dong A Male Sterile Line, And Keep On Anther Development In Mrna Differential Display

There are about 25000 unique genes expressed during anther development, of which 10000 are estimated to be specifically or preferentially expressed in pollen. Studying the control of gene expression in the anther development and cloning the tissue-specific genes and promoters is immensely important, not only for the understanding of gene regulation in the sexual reproduction of flowering plants, but also for its potential application in agriculture such as male-sterility in hybrid. The male sterile line Dong A is a pollenless mutant of cotton variety Dongting No.1 discovered in 1972. After more than twenty years of reproduction by backcross, the male sterile and the fertile individuals have a highly homogeneous genetic background except for the male sterility, which was under the control of a pair of recessive genes (msms). In order to identify the pollen-specific genes, cDNA-AYLP was used to detect variation in gene expression during the anther development between the two kinds of individuals. Main results are as followed: 1. The relationship between bud length and the stage of male gainetogeny was determined in Dong A. It showed that the buds of 5.0-7.0mm in length contained the pollen mother cells meiosis stage, and those of 7.5-9.0mm in microspore stage. 2. Using guanidine isothiocyanate method, the RNAs of anthers in meiosis 60 A WW~W~J mRNA ~rni~ stage and microspore stage were extracted from male sterile and fertile individuals, respectively, and then mRJ?As were purified for cDNA synthesis. On these cDNAs, 35 primer combinations were used and remarkable differences were detected between the male sterile line and fertile individuals. 64 differential bands have been successfully recovered and re-amplified. 3. Three differential fragments GHA27, GHA28 and GHA47 were rondarnly selected as probe to perform RNA dot blotting. It showed that 0HA27 transcripts accumulated in anther, pollen and corolla, but not in leaf, root and ovule; Gl-1A28 and GHA47 transcripts accumulated in anther and pollen, slightly accumulated in root, and not in leaf, corolla and ovule. 4. The three differential fragments were cloned and sequenced. BLAST analysis showed that GHA27 had a high similarity to several ADP-ribosylation factor cDNA clones from O.sativa, Salix baklco and Capsicum annuum. But GHA2S and GHA47 showed a very low level of similarity to the sequences of the data banks. 5. The 3?non-coding sequence of GHA27 was acquired by 3?RACE. Southern blot hybridization demonstrated that GHA27 exists as multi-copy in the G. Hirsutum L. Genome.