【作者】 周琳璘；

【导师】 胡银岗；

【作者基本信息】 西北农林科技大学 ， 作物遗传育种， 2011， 博士

【摘要】 作物温光敏雄性不育系的利用为两系利用作物杂种优势提供了新的途径,然而,由于环境条件的复杂性,也加大了其生产利用的风险。揭示作物温光敏雄性不育系育性转换的分子基础,将为温光敏雄性不育系的合理高效利用提供理论基础,同时也为探索防控该类型不育系利用风险的措施,建立调控作物雄性不育育性转换的方法和技术奠定理论基础。YS型小麦温敏雄性不育系已应用于两系杂交小麦的选育,为了揭示YS型小麦温敏雄性不育系育性转换的分子基础,为其安全合理利用提供理论和技术,本研究对小麦TaMS-MADSbox和TaG3BP在YS型小麦温敏雄性不育系育性转换中的功能进行了较为深入的分析,旨在阐明其在该类型小麦温敏雄性不育系育性转换中的作用。本研究选取YS型小麦温敏雄性不育系A3017不育和可育条件下小孢子各发育时期(减数分裂期、单核期、单～二核期、二核期及三核期)的花药和三种K型小麦细胞质雄性不育系及其保持系(K3314A/3314B、KTsp732A/Tsp732B和KTm3314A/Tm3314B)小孢子发育在单核～二核期的幼穗为主要材料,对TaMS-MADSbox和TaG3BP在YS型小麦温敏雄性不育系育性转换中的功能进行分析,获得的主要研究结果如下:1 TaMS-MADSbox在育性转换中的功能分析利用RACE技术从A3017中克隆了TaMS-MADSbox的1,131bp的全长cDNA序列(GenBank登录号:JN874386)。序列分析表明TaMS-MADSbox编码269个氨基酸,具有MADS-box转录因子典型的K-box结构域。该基因编码蛋白的pI/Mw为9.30/30.35。同源比对表明该基因与小麦MIKC-type MADS-box转录因子WM2及小麦MADS-box转录因子WAG高度同源。采用Reverse transcription quantitative real-time PCR(RT-qPCR)技术对TaMS- MADSbox的表达模式分析表明,TaMS-MADSbox在A3017的育性转换关键时期(花粉发育的单核期～二核期)呈上调表达,且在不育条件下的表达量高于同时期可育条件下的表达量;TaMS-MADSbox在三种K型不育系幼穗中的表达量高于在其保持系中的表达量。表明该基因与小麦温敏雄性不育系的育性转换相关,该基因在育性转换关键时期的大量表达与其不育性密切相关。采用BSMV-VIGS技术,在可育温度条件下,有效地沉默了TaMS-MADSbox在A3017穗部的表达。沉默植株出现部分花药瘦小、不开裂等不育性状,自交结实率降低。其中植株A3017-M-4自交结实率为0,表现完全雄性不育。RT-qPCR检测结果表明,随着该基因的沉默程度的加深,植株自交结实率也相应降低。综上分析表明,该基因的适量表达对小麦雄性不育系的育性非常关键,过高或过低的表达都会导致其雄性育性的降低。2 TaG3BP在育性转换中的功能分析采用RACE技术从A3017中克隆了TaG3BP的全长cDNA序列。序列分析表明该基因在A3017中存在1,311 bp和1,308 bp两个拷贝,分别命名为TaG3BP1和TaG3BP2(GenBank登录号:JF830798和JF830800),分别编码436或435个氨基酸。序列分析表明该基因具有Ras-GTPase激活蛋白(G3BP)的典型结构域NTF2和RRM。根据获得的全长cDNA序列设计引物,在A3017中分离了TaG3BP1和TaG3BP2的完整基因组DNA序列(GenBank登录号:JF830799和JF830801)。基因组DNA与cDNA序列的比对分析表明该基因两种拷贝中均含有9个外显子和8个内含子,同一拷贝的外显子与cDNA编码区完全重叠。两种拷贝间在内含子区域有较大的差异,编码区无明显差异。采用GenomeWalker技术在A3017中获得了该基因814bp的5′侧翼序列,分析表明该序列具有启动子的典型结构及多个顺式调控元件。进一步在上述三种K型不育系及其保持系中分离了该基因的全长cDNA、基因组DNA及5′侧翼序列。序列多重比对分析表明,在不同试验材料间该基因同一拷贝的序列存在多处单碱基差异,但对编码蛋白无显著影响。RT-qPCR分析表明,TaG3BP在YS型小麦温敏雄性不育系A3017的育性转换关键时期(花粉发育的单核期～二核期)呈上调表达,且在可育条件下的表达量高于同时期不育条件下的表达量。该基因在三种K型不育系幼穗中的表达量低于在其保持系中的表达量。表明该基因与小麦温敏雄性不育系的育性转换相关,该基因在可育条件下的大量表达可能具有修复细胞质雄性不育系花药发育中的基因表达缺陷的作用,进而引起育性的恢复。利用原核表达载体系统成功诱导了TaG3BP编码蛋白的表达,并制备了该蛋白的多克隆抗体Anti-TaG3BP。以Anti-Beta-Actin (Rabbit) (Biosynthesis, Beijing, China)为内参抗体,通过Western blot分析该基因编码蛋白的表达模式,结果表明在两种育性条件下,A3017小孢子各发育时期的花药中均检测到了约50-kDa的目标蛋白条带,TaG3BP的编码蛋白在可育条件下各时期花药中的表达量均高于同时期不育条件下的表达量,而且该基因的编码蛋白在A3017育性转换的关键时期(单～二核期)呈上调表达。采用BSMV-VIGS技术,在可育条件下,使TaG3BP在YS型小麦温敏雄性不育系A3017的穗部表达得到了有效沉默。沉默植株出现部分花药瘦小,不开裂等不育性状,自交结实率降低。其中植株A3017-G-1自交结实率相对于对照植株显著降低。综上分析表明,该基因的上调表达对小麦温敏雄性不育系的育性恢复有重要作用,TaG3BP可能是育性转换中的重要调控基因。这也是首次对植物中的G3BP基因的功能分析。3小麦穗部BSMV-VIGS技术体系的初步建立本研究中通过对植株叶片的持续侵染,在可育条件下,在YS型小麦温敏雄性不育系A3017穗部实现了TaMS-MADSbox及TaG3BP的有效沉默。不仅在叶片部位观察到病毒侵染的表型,而且RT-qPCR分析表明其在穗部花药中的表达明显降低。初步建立了在小麦穗部特异表达基因功能研究的BSMV-VIGS技术体系,拓宽了该技术在植物基因功能研究中的应用。更多还原

【Abstract】 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.更多还原