BNY是新发现的小麦温敏雄性不育两用系.为了进一步确定BNY的育性敏感时期,对BNY和CK(温麦6号)的花粉育性及自交结实性进行了系统调查,结果表明,BNY不同蘖位、穗位、花位的花粉败育率存在差异,小分蘖败育率高于大分蘖,大分蘖高于主茎;同一穗不同部位的小穗花粉败育率差异不明显;同一小穗不同花位花粉败育率差异明显,下位小花败育率显著高于上位小花.自交结实率主茎穗高于大分蘖穗,大分蘖穗高于小分蘖穗,但均显著低于CK;主茎穗与大分蘖穗的自交结实率差异不显著,而小分蘖穗与主茎穗及小分蘖穗与大分蘖穗的自交结实率差异显著.

The male sterility transformation role of two-line male sterile line BNS was tested in southern ecological planting area by means of interval sowing method, and varieties Bainong-aikang 58, Nongda 211, Nongda 3688 and Yangmai 13 used as control  materials in Changsha, Hunan Province in 2006-2009. It was found that the male fertility of BNS was from completely sterility to highly-sterility, semi-sterility, and to normal fertility as the sowing date delaying. The pollen showed mainly typical abortion at the completely male sterility stage, completely sterile at the flowering phase during mid and late March. The male fertility converted in early April and being fertile in mid-April. The fertility transformation were related to the flowering habit to some extent.

于2006-2009年在湖南长沙采用分期播种方法以两系小麦不育系BNS和对照百农矮抗58、农大211、农大3688和杨麦13为试验材料，研究了BNS雄性育性在南方生态区域的转换规律。结果表明：随播种期的推迟，BNS雄性育性表现为完全败育→高度不育→部分可育→完全可育的育性转换规律；雄性完全败育阶段以花粉典型败育为主；3月中、下旬开花阶段，花粉完全败育；4月上旬开花阶段，雄性育性发生转变，4月中旬开花阶段，花粉可育。雄性育性的转换伴随着开花习性发生转换。

为探讨低温雄性不育小麦BNS的育性转换规律,2007-2009年通过分期播种试验,对BNS育性转换特性进行了初步研究.结果表明,BNS具有不育彻底、育性转换彻底的特性,育性变化趋势为:完全败育-半不育-完全可育.低温是影响BNS育性转换的主要原因,表现低温不育,高温可育.BNS感受低温的敏感部位为发育的幼穗;BNS育性转换敏感期在抽穗前5～18 d,即雌雄蕊分化期至四分体形成期;在温度敏感期内,温度低于8℃时表现彻底不育, 8～12℃是育性转换温度,温度高于12℃时表现完全可育.在豫北地区,9月23日-10月17日播种,表现彻底不育,可用于生产杂交种;11月18 日以后播种均表现完全可育,可以自交繁殖不育系种子.

Male sterility of wheat-breeding line 337S ( Triticum aestivum L.) is sensitive to both short day-length/low temperature and long day-length/high temperature. 337S was crossed with the common wheat variety, Huamai No. 8 and the F 1 was highly fertile. The F 2 population segregated in a 15:1 ratio for fertility/sterility in 243 individuals under long day-length/high-temperature. The two thermophotoperiod-responsive male sterile genes were mapped to chromosomes 5B and 2B using Simple Sequence Repeat (SSR) markers and bulked segregant analysis. Partial linkage maps around the sterility loci of chromosomes 2B and 5B were constructed using the 243 individuals in the F 2 population. One gene ( wptms1 ) for male sterility was flanked by the SSR markers Xgwm335 and Xgwm371 at a genetic distance in chromosome 5B of 4.1 and 24.4 cM, respectively. The second gene ( wptms2 ) was mapped between markers Xgwm374 and Xgwm120 at a genetic distance of 6.6 and 20.9 cM, respectively. The closest linked markers Xgwm335 ( wptms1 ) and Xgwm374 ( wptms2 ) explained 53 and 38% of phenotypic variation for the fertility. The SSR markers provide a useful tool to transfer the male sterile genes into elite wheat germplasm.

Male sterility is one of the major characteristics to be used in heterosis utilization of crops, in which thermo-sensitive or photo-sensitive male sterility is very important for two-line hybridization due to the conversion of their male fertility under special weather conditions. YS type thermo-sensitive male sterile wheat ( Triticum astivum L.) lines are applicable for heterosis use in the major wheat production areas of northern China. To investigate the molecular basis of male fertility conversion of YS type thermo-sensitive male sterile wheat lines, we constructed the sterile and fertile suppression subtractive hybridization (SSH) cDNA libraries respectively, using the cDNA of the male sterile or fertile young spikes from the same individual of one YS type thermo-sensitive male sterile wheat line A3017 under controlled male sterile or fertile conditions. Comparing the EST sequences between the two cDNA libraries, an EST (GenBank accession number: 36925702) highly similar to MADS box transcription factor gene was selected from the sterile SSH-cDNA library and used as the probe to search the dbEST. A pair of primers was designed based on the aligned sequence of highly homological EST sequences, and used to detect the expression difference of this gene between male sterile and fertile spikes via Reverse Transcriptase PCR (RT-PCR). The results showed that the expression of this gene in male sterile spikes was much higher than that in fertile spikes. Then the RT-PCR fragment amplified from the male sterile spikes was cloned and sequenced, a cDNA sequence with 666 bp and encoding 160 amino acids was obtained. The cDNA fragment contained the typical K-box domain of MADS-box, and designated as TaMS-MADSbox . The deduced amino acids were 94% similar to WAG (BAC22939), an MADS box transcription factor of wheat. The expression profiles of this MADS-box transcription factor gene in the male-sterile lines and their maintainers of three types of male sterile wheat lines were further analyzed via semi-quantitative RT-PCR. The result showed similar patterns that the expression of the MADS-box transcription factor gene in male-sterile lines was much higher than that in maintainers. It suggests that the expression of TaMS-MADSbox is related to the fertility conversion of male-sterile lines. The spikes are male-sterile under high-level expression of TaMS-MADSbox , while fertile under the low-level expression.

为了揭示YS型小麦温敏雄性不育育性转换的基础, 构建了该类型不育系A3017的不育和可育幼穗正、反杂交的两个SSH-cDNA文库。经文库比较, 在不育文库中筛选出一个与MADS-box基因同源的EST序列(GenBank登录号: 36925702)。以该EST序列的同源性比对和拼接结果为依据, 设计引物对该基因在可育和不育幼穗中的表达进行了RT-PCR分析, 结果表明, 该基因在不育幼穗中表达量较高, 可育幼穗中表达量很低。对不育幼穗中扩增出的cDNA片段进行克隆测序, 获得了666 bp的cDNA序列。序列分析表明, 该片段编码160个氨基酸, 具有MADS-box转录因子的典型结构域K-box, 被定名为 TaMS-MADSbox , 与一个小麦MADS box转录因子基因 WAG 的氨基酸序列的相似性为94%。进一步以3种不同类型的小麦雄性不育系和保持系的幼穗cDNA为材料, 利用半定量RT-PCR对该基因的表达模式分析发现也存在类似差异, 该基因在不育系幼穗中表达量较高, 而保持系幼穗中表达量较低。以上分析表明, 该MADS-box转录因子基因的表达与小麦雄性不育系的育性转化相关, 表达量高时表现雄性不育, 表达量低时表现雄性可育。

RNA editing of atp6 gene at different stages of pollen development was sequenced directly and from clones using the cytoplasmic male-sterile line ms(Kots)-90-110 (A) and its near-isogenic line (NIL, BC 5 F 2 ). The DNA sequences of ms(Kots)-90-110 (A) and the NIL had 99% identities with those of common wheat ( Triticum aestivum ) and T. timopheevi . There were 15 editing sites in the conservative regions of atp6 transcripts between male sterile and fertile lines, of which 13 occurred at the first or the second position of codons with alteration of amino acid type, and two occurred at the third position with no change of amino acid type. Codens at the sixth and seventh sites were cotranscripted. The frequency of the editing site was increased gradually as the developmental stage of anther proceeded. The restorer gene in BC 5 F 2 obviously increased the editing frequency at each editing site. Transcripts that were inadequately edited might affect the normal mitochondria function. RNA editing of atp6 is probably associated with CMS in wheat with Aegilops kotschyi cytoplasm.

以黏类小麦细胞质雄性不育系ms(Kots)-90-110(A)及其近等可育基因系BC 5 F 2 为材料，采用克隆测序与PCR产物直接测序方法，对黏类小麦线粒体 atp6 基因在花药发育各阶段的RNA编辑进行了分析。结果表明，小麦 atp6 基因保守区DNA序列在供试材料不育系及其近等可育基因系中完全一致，且与普通小麦和提莫菲维小麦 atp6 基因序列同源性为99%。两种方法测序分析 atp6 基因转录本保守区RNA编辑的结果规律相似。 atp6 基因共有15个编辑位点，其中13个发生在密码子的第一和第二位点上，这些位点的编辑都使氨基酸种类发生了变化；有2个发生在密码子的第三位点上，不引起氨基酸种类的变化；其中第6和第7位点是共转录的。随着花药发育时期的推移，各位点的编辑频率逐渐增高。不育系与其近等可育基因系相比，在引入核恢复基因后，各位点的编辑频率明显提高。编辑不充分的转录产物可能会影响线粒体功能的正常发挥，表明黏类小麦细胞质雄性不育与线粒体 atp6 基因转录本保守区的编辑有一定的相关性。

BNS is a new type of thermo-sensitive nuclear male-sterile lines in wheat ( Triticum aestivum L.). The mechanism of itssterility was studied using two-dimensional electrophoresis and MALDI-TOF-MS methods. Anthers of the sterile line (SL) and the conversion line (CL, fertile pollen rate higher than 50%) were obtained from plants with a series seeding dates, and two set of samples were used to compare the differentially expressed proteins which was extracted from the anthers at early stage of uninucleate and from late stage of uninucleate to binucleate stage of pollen development. The abundant expressions of proteins involved in respiratory and photosynthetic energy metabolism were identified in the CL, such as ATP synthase α and β subunit, NAD (P) Rossmann fold binding protein, cytoplasmic malate dehydrogenase, mitochondrial aldehyde dehydrogenase subunit, and Rubisco subunit protein. However, they were absent or down-regulated in the SL. Besides, a few abnormal proteins were detected in the SL, including sorbitol dehydrogenase, maturase K, histone H2B.2, Harpin-induced 1, and elongation factor TU. According to the functions of these differentially expressed proteins, we inferred that ATP synthase α and β subunits were most likely the source protein for the sterility of BNS. Their expressions are regulated by a temperature sensor that is located at upstream of the subunit gene operon in BNS. Mutation of the temperature sensor is determined by the temperature higher than a threshold, which may startup the transcriptions of α and β subunit genes. When the temperature is lower than the threshold, the expressions of α and β subunits are in very low levels, which affect the assembly of ATP synthase, and result in deficiency of ATP supply for the growth of microspore. This effect can be amplified in downstream physiological functions, such as development of respiratory and photosynthetic enzymes, which finally causes abnormal metabolism and pollen sterility.

BNS是一个新发现的温敏核型小麦雄性不育系, 它有良好的不育性和转换性, 被认为在小麦杂种优势利用上有重要价值。为探讨BNS的不育机制, 以不育系及其转换系的单核早期、单核晚期到二核期花药为材料, 用2-DE和MALDI-TOF-MS方法分离鉴定2个系的差异蛋白。结果发现, 在转换系中, ATP合酶α和β亚基、胞质苹果酸脱氢酶、线粒体醛脱氢酶亚基、Rubisco亚基等呼吸、光合能量代谢蛋白表达丰富, 但在不育系中这些蛋白缺失或下调。在不育系中还分离出山梨醇脱氢酶、组蛋白H2B.2、Harpin诱导子1、延伸因子TU等非小麦正常代谢蛋白。根据这些差异蛋白的功能, 推测ATP合酶α、β亚基蛋白最可能是BNS不育的源头蛋白, 它的表达可能受其上游的温度感应子调控。BNS的温度感应子隐性突变后转录启动温度阈值上调, 在较低温下, α、β亚基基因不表达或表达量小, 导致ATP合酶数量减少, 继而影响ATP合成。ATP数量减少, 使小孢子ATP供应短缺, 进一步影响到小孢子的发育, 使代谢异常, 花粉败育。

C412S is a novel type of thermo-photo-sensitive genic male sterile (TGMS) wheat ( Triticum aestivum L.) line, which is derived from the cross between TGMS line C49S and normal wheat line C412. To understand the fertility alternation of C412S, the courses of pollen mother cell (PMC) meiosis and microsporogenesis were observed using smear and squash microscopic techniques. Moreover, the expression characteristics of adenine phosphoribosyl-transferase gene ( APRT ) in young spikes were detected under sterile and fertile conditions using quantitative RT-PCR. When adjusting the environmental temperature and photoperiod at the formation and development stages of male sexual cell through series seeding dates, C412S showed continuous changes of fertility from complete sterility to high sterility, semi-sterility, lowe sterility, and normal fertility. C412S in normal PMC meiosis, however, showed a peak pollen abortion at later mononucleate stage of microsporogenesis, which mostly belonged to spherical abortion type. From PMC formation stage to mature pollen stage, C412S was sensitive in fertility, especially from PMC formation stage to PMC meiosis stage. Compared with C412 (control), C412S only had several base variations in a few loci of APRT1 gene, and the deducted amino acid sequences of C412S and C412 were completely identical. The RT-PCR analysis showed that the transcription level of APRT gene in the young spikes of sterile C412S plants was lower than that of fertile C412S plants from PMC formation stage to mono-nucleate microspore stage. Therefore, the transcription level of APRT gene in young spikes is probably related to the fertility alternation in TGMS wheat.

以选育的小麦 ( Triticum aestivum L.) 温光敏核不育系 C412S 为试材，以 C412S 回交转育时的受体亲本 C412 为常规品系对照，通过分期播种试验研究了其 育性转换特性。用涂抹压片法观察减数分裂和小孢子发育进程，用半定量 RT-PCR 技术分析不育与可育条件下不同发育时期的幼穗中腺嘌呤磷酸核糖基转移酶基因 ( APRT ) 的表达水平。结果表明，通过调整播种期改变雄性发育的温光条件， C412S 表现出完全不育 — 高不育 — 半不育 — 正常可育的育性转换特性。 C412S 花粉败育的高峰在单核小孢子晚期，主要表现圆败型不育。 C412S 的育性敏感期是从花粉母细胞形成期到成熟花粉期，其中最敏感的时段是花粉母细胞形成期到减数分裂期。与对照相比， C412S 的 APRT1 基因序列有个别碱基变异，但编码氨基酸序列没有变化。在花粉母细胞形成期至单核期，与晚播可育条件相比，早播不育的 C412S 幼穗中 APRT 基因转录水平下调，因此认为，其育性转换与幼穗中 APRT 基因转录水平有一定关系。

Real-time quantitative PCR is often used to analyze the target gene expression variation. The most commonly used methods to analyze data from real-time quantitative PCR include two types: absolute quantification and relative quantification. To avoid the complex of absolute quantification and simplify the relative quantification, the methods of 2 -ΔΔCT , 2 -ΔCT and standard curve were used to analyze the OsRDB1 expression variation under different chemical and hormone treatments in this study. The ratio of gene expression variation was calculated with the slope of standard curve. The results revealed that the 2 -ΔΔCT method had to be introduced a housekeep gene as a control, with a complicated calculation and the relative ratio was deviated by PCR amplification efficiency. The 2 -ΔCT and standard curve methods were convenient and saved chemicals used in the quantitative PCR, but the results were liable to the effect of the veracity of the RNA concentration. The modified method of standard curve introduced the curve slope to eliminate the effect of amplification efficiency, and the result of standard curve method most closely revealed the diversity of original copy.

利用实时荧光定量PCR方法分析目标基因转录本之间的表达差异，目前常用的分析实验数据的有绝对定量和相对定量方法。为了克服绝对定量方法的繁琐和简化相对定量的分析方法，分别利用2 -ΔΔCT 、2 -ΔCT 和标准曲线法对水稻 OsRDB1 基因在不同化学试剂和激素处理下的表达差异进行了分析，发现2 -ΔΔCT 必须引入标准的看家基因作为参考，计算方法繁琐，计算结果受扩增效率影响；而利用2 -ΔCT 和标准曲线法计算方便，节省定量PCR试剂，但其结果易受到RNA浓度测量准确率的影响，其中标准曲线法引入斜率消除扩增效率的影响，最能测得实际的原始拷贝数差异。

以红莲(HL)型水稻细胞质雄性不育系A、保持系B及杂种一代F1为材料,首次比较研究了红莲型水稻线粒体atp6基因转录本的编辑位点及各位点的编辑频率.结果表明atp6基因的转录本有18个编辑位点,其中有15个发生在密码子的第一和第二位点上,这些位点的编辑最终会导致氨基酸种类的变化.18个编辑位点在A、B和F1中没有差异,但各位点的编辑频率在引入了核恢复基因的条件下发生了较大的变化,完全编辑的比例增加.这些结果首次证明HL型细胞质雄性不育与线粒体atp6转录本的编辑有一定相关性,编辑不充分的转录产物最终会干扰线粒体功能的正常发挥.

本研究以BNS小麦为试材,采用分期播种和春季播种试验,研究BNS不同播期的幼穗分化进程及其温光与育性的关系,旨在进一步明确BNS育性转化的穗分化敏感时期.结果表明:(1)随播期推迟,BNS二棱期至药隔期的穗分化天数和不育度均逐渐减小,且二者呈极显著正相关(r=0.940～0.942>r(0.05/0.01)=0.658/0.765).10月1日～10月9日播种,穗分化天数为52～59d,不育度99%～100%.春播试验天数为19～22 d,自交结实率为63%～78%.(2) BNS温度敏感期为雌雄蕊原基分化期和药隔期,日均温与不育度相关密切(r=-0.915-0.818>r(0.05/0.01)=0.658/0.765),高度不育时平均温度为8.4～12.6℃.(3) BNS光照敏感期为药隔期,日均光长和总光照与不育度相关极显著(r为-0.799,-0.784,0.854,0.954>r(0.05/0.01)=0.658/0.765),高度不育时日均光照长度为6.6～6.8 h.(4)春播试验中雌雄蕊分化期和药隔期的日均温、光(14.2～16.0℃、7～8 h)高于诱导高度不育的温光条件,故表现可育.因此BNS温度敏感期为雌雄蕊原基分化至药隔期,光照敏感期为药隔期,是温敏为主的低温短日生态雄性不育小麦.