AFLP And Biochemical Character Analysis Of Male Sterile Induced By Space Flight In Maize

Anther and pollen development is one of the most important areas in plantdevelopment biology.With the development of microspore and anther, several of genesparticipate in it.Once a gene is defective, it will result in male sterility. Male sterile mutantshave been identified in many higher plants.Studying male sterile mutants is an efficientstrategy, not only for the molecular mechanisms of plant anther and pollen development,but also for the heterosis utilization.1996, a male sterile mutant controlled by one single recessive gene was screened in theoffspring of the hybrid-chuan Dan No.9 which was induced by space flight.There was nopollen formation in the mutant.Experimental materials were from a male fertile and malesterile segregating population with six generation sib-mating and the segregating ratio was1:1as expected.In order to analysis this mutant, studying on the genome DNA, cDNA andbiochemical characters were carried out.The main results concluded as follows.1. The difference between male fertile and male sterile was analyzed by DNA-AFLPtechnology.Total 56 primer combinations were used and 4 differential fragments wereeluted, cloned, sequenced. They were named Zmd1-302, Zmd2-287, Zmd3-246 andZmd4-227, respectly.Sequencing analysis through internet Blast searching showed thatZmd1-302 was homology to a est (gi33771201) which was from a maize sperm library anda DNA sequence (AC187265)which was located at the third chromosome 3.04 in maize.And, Zmd2-287 showed 97% similarity with a maize BAC clone (AC191267); Zmd3-246showed 99% similarity with a maize BAC clone (AC 1877992).2. Flanking sequences of Zmd1-302 and Zmd1-287 were obtained using the sequenceinformation of AC187265 and AC191267.Then, 4 special primers were designed toidentity male fertile and male sterile, but the amplified bands were same in 8 male fertileand male sterile individuals. AFLP marker unsuccessfully converted into dominant SCAR markers.3.16 primer combinations were used to analyze the differential expression between theanthers of male fertile and male sterile by cDNA-AFLP.Total 9 special cDNA fragmentswere obtained and they were named as Z1～Z9. The key fragments were studied bysemi-quantitative RT-PCR at 4 development stages of male fertile and male sterile anther.The sequence comparison against the database revealed that 4 of them had homology togenes with known functions,the similarity of others were below 50%.The clone Z3,whichwas 54% homologous to the chalcone synthase N-terminal domain containing protein(Oryza sativa) was expressed only when the tassel have just emerged in male fertile andwas completely absent in male sterile.The clone Z5 showed 91% similarity with putativeprotein kinases AFC3, PK12.The expression of Z5 was complication, not only in theprocess of tassel emerging in male fertile,but also in male sterile when tassel had emergedcompletely.The clone Z8, which was 78% homologous to putative glycine decarboxylaseH-protein (GDC, H-protein) was expressed only in male fertile, and with thedevelopmental of the anther, its expression was significantly increased.4. The contents of soluble sugar, starch, protein and free proline were comparedbetween anthers of male fertile and male sterile at 4 differential developmental stages. Theresults showed that all contents were lower in male sterile than in male fertile. With theanther developing, the contents of starch, soluble protein and free proline increasedobviously in male sterile, but have not founded increased in the anther of male sterile,especially the content of free proline.