| The utilization of thermo or photoperiod (or both) sensitive male sterility (TMS or PMS), either genic or cytoplasmic, is a break-through development for the utilization of heterosis in crop production. But the environmental conditions are complex, the risk for the utilization of TMS or PMS in production increased. A greater understanding of the mechanism of TMS or PMS will promote the proper use of those lines in crop production and could result in lasting benefits for food security world-wide. In this study, to investigate the function of TaMS-MADSbox and TaG3BP in fertility modulation of YS type thermo-sensitive cytoplasmic male sterile (TCMS) wheat line A3017, more function analysis were performed. The objective of this study was to reveal the molecular basis of fertility modulation and provide some theoretical basis and technical strategies for the safe utilization of this TCMS wheat lines.The anthers in the middle florets of YS-type TCMS wheat line A3017 at meiosis, uninucleate, uni-binucleate, binucleate and trinucleate stages of microsporogenesis, the young spikes at the critical developmental stage (uni-binucleate stages) of microsporogenesis of three kinds of K-type cytoplasmic male sterile (CMS) wheat lines and their maintainers, including the 1B/1R (K3314A and 3314B), non 1B/1R with 1B chromosome fragment from T. spelta (KTsp732A and Tsp732B), and non 1B/1R with 1B chromosome fragment from T. macha (KTm3314A and Tm3314B), were used in this study to characterize the function of TaMS-MADSbox and TaG3BP in fertility modulation of YS type thermo-sensitive cytoplasmic male sterile (TCMS) wheat line A3017. The main results were as follows:1. The function analysis of TaMS-MADSbox in the modulation of male fertilityThe full length cDNA sequence of TaMS-MADSbox with 1,131 bp was isolated from A3017 by 3′RACE and 5′RACE, including 58-bp of 5′UTR, 263-bp of 3′UTR and an 810-bp open reading frame (GenBank accession No. JN874386). TaMS-MADSbox was predicted to encode a protein of 269 amino acids, and the typical K-box domain of MADS box proteins were found in the deduced protein sequence of TaMS-MADSbox by the conserved domain analysis. The predicted protein has a theoretical pI of 9.3, and molecular weight of 30.35KDa. Phylogenetic analysis showed that TaMS-MADSbox is closely related to MIKC-type MADS-box transcription factor WM2 (CAM59041.1) and MADS-box transcription factor WAG (BAC22939.1) of wheat.The expression patterns of TaMS-MADSbox at various stages of microsporogenesis in anthers of A3017 grown under temperature regimes favoring either sterile pollen or fertile pollen development were investigated by Reverse transcription quantitative real-time PCR (RT-qPCR). The results indicated that the transcript levels of TaMS-MADSbox in anthers under conditions favoring male sterility were higher than under conditions favoring male fertility at all stages of microsporogenesis, especially at uni-binucleate stages. RT-qPCR anaylsis revealed the transcription level of TaMS-MADSbox in young spikes at the critical developmental stage (uni-binucleate stages) of microsporogenesis of the three K-type CMS wheat lines were higher than in their maintainers. The results indicated TaMS-MADSbox associated with the male fertility modulation of wheat.The transcription of TaMS-MADSbox was effectively silenced by BSMV-VIGS under male fertile conditions, which caused pollen abortion and the decrease of seedset rate in the infected A3017 plants.2 The function analysis of TaG3BP in the modulation of male fertilityThe cDNA, genomic DNA and 5’flanking sequences of TaG3BP were cloned from YS-type TCMS wheat line A3017, three kinds of K-type CMS wheat lines and their maintainers. Two cDNA of TaG3BP with 1,311 or 1,308 bp were assembled, encoding a protein with 436 or 435 amino acids residues, and designated TaG3BP1 and TaG3BP2. The conserved domain analysis of the TaG3BP deduced protein sequence indicated that there were the typical NTF2 and RRM domains of Ras-GTPase activating protein SH3 domain-binding protein (G3BP). Two genomic DNA sequences were obtained with 2843 bp and 2872 bp, respectively. Alignment of the two genomic DNA sequences with the two cDNA sequences obtained previously revealed that each of the two genomic DNA sequences aligned with a different one of the two cDNA sequences, and there were nine exons and eight introns included in both of the two TaG3BP genomic DNA sequences. Many variations were observed in the intron regions of the two genomic DNA sequences. An 819-bp 5′flanking sequence of TaG3BP was obtained by genome walking and analysis indicated the typical structures for a promoter, and some cis-acting regulatory elements were included in the sequence.RT-qPCR analysis indicated that the transcript levels of TaG3BP in anthers of A3017 grown under conditions favoring male fertility were higher than under conditions favoring male sterility at all stages of microsporogenesis, especially at uni-binucleate stages. And the transcription level of TaMS-MADSbox in young spikes at the critical developmental stage (uni-binucleate stages) of microsporogenesis of the three K-type CMS wheat lines were lower than in their maintainers also via RT-qPCR.The recombinant protein of TaG3BP was obtained by the IPTG induction, and used for the specific polyclonal antibody preparation of TaG3BP. ELISA analysis on the specific polyclonal antibody obtained indicated that the titer (the highest dilution factor) of the antiserum reached 1:10,000. Anti-Beta-Actin (Rabbit) (Biosynthesis, Beijing, China) was employed as an endogenous control to adjust all templates to be the same amounts. The western blot results showed a 50-kDa protein band in the anthers of A3017 at meiosis, uninucleate, uni-binucleate, binucleate and trinucleate stages grown under male sterile and fertile conditions. The protein expression levels were higher in male fertile anthers than in sterile anthers at all the stages, and the protein level greatly deceased in male sterile anthers at the trinucleate stage, but intensified in male fertile anthers at the same stage. The expression difference of TaG3BP between male sterile and male fertile anthers suggested that TaG3BP may play an important role in modulation of male fertility in the YS-type TCMS wheat line.The transcription of TaG3BP was also effectively silenced by BSMV-VIGS under male fertile conditions, which caused pollen abortion and the decrease of seedset rate in the infected A3017 plants.3 Application of BSMV-VIGS for silencing genes expressed in wheat spikesIn this study, TaMS-MADSbox and TaG3BP were successful silenced in spikelets by repetitive inoculations with BSMV-VIGS vector. Phenotypic changes were observed in leaves and also in anthers, which resulted in reducing selfed seedset rate that paralleled the transcriptional levels of TaMS-MADSbox and TaG3BP in the spikes. These results suggest that the effect of gene silencing by BSMV-inoculated on leaves could impact on the spikes. Therefore, BMSV-VIGS may be a powerful tool to help characterize the functions of genes expressed in the reproductive organs in wheat and may assist in constructing a more complete model of floral development. |
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