The Character And Molecular Mechanisms Of Fertility Conversion Of YM Type Thermo-Sensitive Male-Sterile Wheat Line

Utilization of heterosis is one of the important approach to improve wheat yield and quality. Complicated process of seeds product were reduced and seed purity were enhanced effectively by using of thermo-photoperiod sensitive male-sterile line. Breeding of thermo-photoperiod sensitive male-sterile wheat which can be used in production has important significance. YM type thermo-sensitive male sterile wheat were bred by transfering chromosomal 1BS fragment of T. macha into K type male-sterile wheat and it had thermo-sensitivity speciality. At present, the reports of male sterile mechanisms about YM type thermo-sensitive male-sterile wheat were few. In order to knowing the male sterile and fertility conversion mechanisms of this line, experiments were conducted in the following aspects: the fertility of YM3314 was investigated by the experiments of sowing in different periods and spikes of ratooning tillers in Yangling, Shaanxi to realize thermo-sensitivity speciality and temperature conditions of fertility conversion; By Semi-quantitative reverse transcription polymerase chain reaction(RT-PCR), expression profiles of a few of MADS-box genes were analyzed; suppression subtractive hybridization (SSH) were applied to construct cDNA library and comparison analysis proceeded on the gene expression profiles, The results were as followed:1. Thermo-sensitivity speciality and the critical phase of fertility conversion of YM type thermo-sensitive male-sterile wheat line YM3314The developing periods fertility of YM3314 was investigated by the experiments of sowing in different periods and spikes of ratooning tillers in Yangling, Shaanxi. Related analysis was carried between selfed seedset rate and climate elements from booting to abloom time . The results were showed: YM3314 was sterile highly or completely sowing in autumn and fertility was restored stably sowing in spring; The correlation between the fertility of YM3314 with different sowing dates and average temperature per day from booting to heading time was positive and very significant; Between fertility and average relative humidity of booting time was negative and reached significant level; The important thermo-sensitive phase was 5 days before booting to 5 days after heading; It was fertile in excess of 18.18℃and sterile when the temperature was less than 18℃.2. Expression profiles analyses of MADS-box genes in the young spikes with different developing periods of YM type thermo-sensitive male-sterile wheatYoung spikes RNA with dinucleate stage of three type of male-sterile lines and their maintainer lines included YM and YS types and K type of non-1B/1R was used by Semi-quantitative RT-PCR. Three fragments were produced by WAP1, TaMADS12 and TaVRT-1 respectively. Comparison analyses proceeded on the gene expression profiles and the results were showed that the fragments produced by WAP1 and TaVRT-1 were different expression amount in the sterile lines and maintainer lines. The expression amount of WAP1 was a little higher in YM type Thermo-sensitive male-sterile line KTm3314A than its maintainer line and the expression amount of TaVRT-1 was higher in sterile line than maintainer line, too.Total RNA of the young spikes of uninucleate stage and dinucleate stage of YM3314 which were sowed in different dates was used by Semi-quantitative RT-PCR. The primers WAP1, TaMADS12, TaMADS51 and TaVRT-1 could produced evaluated fragments. The fragments produced by these four primers had different expression profiles in the young spikes of uninucleate stage and dinucleate stage which were sowed in autumn and spring respectively. It was implied that these fragments of MADS-box genes had some connection with male sterile or fertility conversion.3. cDNA libraries of the young spikes of uninucleate stage of YM3314 which were sowed in autumn (sterile conditions) and spring (fertile conditions) by SSH. Sequence homology analyses were conducted via BLAST (Basic Local Alignment Search Tool) program in NCBI (National Center for Biotechnology Information) against non-redundant Protein database , non-redundant nucleotide database and dbEST of Genbank117 and 118 positive clones from cDNA library of sterile young spikes (B library) and cDNA library of fertile young spikes (K library) were randomly selected and sequenced, respectively. The vector, primers, adaptors in the sequences were eliminated. After screening repeat and redundant sequences, the sequences which length were no more than 100bp were eliminated, too. 60 ESTs were acquired in the B library, BlASTx results in nr-protein database revealed that 37 ESTs (occupied 61.67%) were highly homologous with known proteins involved in primary metabolism (13.51%), energy metabolism (24.32%), cell growth/division(2.70%), protein synthesis(10.81%), protein destination and storage(2.70%), transport (13.51%), signal transduction (5.41%) and disease resistance and defenses (8.11%), ESTs of unknown function occupied 18.92%; 76 ESTs were acquired in the K library, BlASTx results in nr-protein database revealed that 34 ESTs (occupied 44.74%) were highly homologous with known proteins involved in primary metabolism (5.88%), energy metabolism (23.53%), transcription (11.76%), protein synthesis (11.76%), protein destination and storage(2.94%), transport (8.82%), cell structure (2.94%) , signal transduction (14.71%) and disease resistance and defenses (2.94%), ESTs of unclear function occupied 14.71%. It was showed that the genes involved mainly primary metabolism, energy metabolism and transport in the B library were important in the expression of male sterile; the genes involved mainly energy metabolism, transcription, protein synthesis and signal transduction in the K library had important function of regulation in the process of fertility conversion between male sterile and fertile.