拟南芥温敏雄性不育突变体atms1的获得及表型分析

从甲基磺酸乙酯（ethylmethane sulfonate,EMS）化学诱变建立的野生型拟南芥（Col-0）突变体文库中筛选到1株突变体.在低温16 ℃时,该突变体的雄性育性与野生型没有显著差异,花粉染色呈现100%可育.随着培养环境温度的升高,突变体花粉育性逐渐下降,因此,该突变体为一温敏雄性不育突变体,并被命名为atms1（ambient temperature-sensory male sterility 1,即环境温度敏感雄性不育1）.花药切片结果显示,在23 ℃以下,该突变体花药各个发育时期的形态与野生型花药没有显著的差异;而27 ℃处理1周后的突变体花药呈现多种表型：同一朵花中各个花药的发育时期出现显著分化,花粉母细胞胼胝体单薄,绒毡层发育滞后于同时期的野生型花药.遗传分析确定,atms1的不育表型是由单个隐性核基因控制的.     

拟南芥bHLH家族转录因子DYT1在花药发育过程中调控胼胝质降解(英文)

胼胝质的合成和降解是雄配子体减数分裂过程中的一个重要特征,对后期花粉成熟有重要作用.在此研究中,分离到了一个雄性不育突变体msl57,该突变体的绒毡层分化及胼胝质降解过程出现异常,导致花粉败育.图位克隆和遗传分析表明:MSl57基因与bHLI-I家族转录因子DYTI(At4g21330)是同一基因.因此,将ms157突变体改名为dyt1-2.反式激活作用实验揭示了DYTI的激活功能域位于基因的250 ～504bp之间.通过酵母双杂交实验发现DYT1蛋白在体内可以形成同源二聚体来执行其功能.RT-PCR及定量PCR分析表明胼胝质酶相关基因A6的表达在突变体背景下严重下调.因此,DVF1通过调控胼胝质的降解来影响花药发育过程.     

拟南芥At5g58100基因T-DNA纯合插入突变体PCR鉴定及表型观察

以拟南芥SPOT1功能缺失突变体spot1和野生型(WT)植株为材料,比较研究了它们在花粉发育和结实率方面的差异.spot1-1,spot1-2和spot1-3突变体都能形成可育的三核花粉,但用扫描电子显微镜(SEM)观察发现其花粉粒皱缩,花粉外壁存在纹理缺陷.用荧光显微镜观察了这些花粉粒的树脂半薄切片,发现花粉内壁无纤维素的自发荧光,spot1突变体成熟果荚中的种子数量显著少于野生型.对spot1-3突变体花药发育过程进行观察后发现,从花粉母细胞时期开始,突变体细胞壁组成与野生型已出现差异.10μmol·L~(-1)(物质的量浓度)茉莉酮酸既不能抑制黄化spot1幼苗的顶钩反应,又不能抑制1×10~(-6)(质量分数)乙烯利引起的夸张顶钩反应.上述结果提示SPOT1基因的功能与小孢子初生外壁、花粉内壁的发育,以及花丝伸长(长度)有密切关系,SPOT1可能与茉莉酮酸信号传导通路相关联.     

光信号途径组分HEMERA调控拟南芥的花药发育

雄蕊是植物的雄性生殖器官,对植物繁衍及作物的产量至关重要.光信号影响拟南芥生殖发育过程,但作用机制知之甚少.HEMERA(HMR)是拟南芥光敏色素途径的主要成员之一,通过介导光敏色素B(PhyB)的定位及光信号途径重要转录因子PIF的降解而在植物光形态建成过程中发挥重要作用,但其在生殖发育中的功能尚不清楚.本研究利用hmr突变体及转基因恢复植物进行研究发现:HMR功能缺失导致花药变小、花粉数目减少、花粉萌发率显著降低,绒毡层部分空泡化并提前降解;并且,其转基因功能互补能恢复这些育性问题,说明HMR可能通过参与绒毡层降解过程而调节植物雄性育性.     

通过转基因途径获得植物雄性不育

对利用转基因的方法获得植物雄性不育的研究进展进行了概述 .小孢子发育过程涉及花药发育到花粉粒成熟过程中一系列基因的表达 .这些基因表达的异常往往导致部分或全部的花粉败育 .利用绒毡层特异性启动子或花药特异性启动子驱动一种外来基因的表达可以导致雄性不育 .育性的恢复也可通过转基因技术来实现 .控制植物育性的基因工程将在优势育种中发挥越来越重要的作用 .     

拟南芥转录因子TDF1的原核表达及多克隆抗体制备

在拟南芥花药发育过程中,MYB家族转录因子TDF1在调控绒毡层发育及后期功能上起到关键的作用．利用PCR方法扩增TDF1基因并克隆到原核表达载体pET-32a．将表达载体TDF1-pET32a转入大肠杆菌BL21（DE3）,用IPTG成功诱导表达了分子量约为56KD的TDF1融合蛋白．该融合蛋白主要以包涵体形式存在,分离出包涵体后进行可溶性处理作为抗原免疫家兔．制备出的多克隆抗血清经ELISA测定效价为1：2560,Westernblot检测表明该抗血清与TDF1融合蛋白识别良好．TDF1抗体的制备有助于进一步从生化水平上研究TDF1在花药发育中的功能．     

转录组分析揭示BAM1/2与SPL调控拟南芥花药发育的功能关系及调控网络

花药的正常发育对于植物正常繁衍和农作物产量都有着至关重要的作用.转录因子SPL及受体蛋白激酶BAM1和BAM2均在调控孢原细胞分化的过程中发挥重要作用.但迄今对其功能关系及下游信号途径仍缺乏深入研究.本文利用高通量转录组测序手段,通过对拟南芥spl、bam1、bam2、bam1bam2突变体花药的转录组对比分析,明确了:1)BAM1和BAM2功能冗余地调控花药绒毡层发育、脂质转运、花粉壁形成等方面,但两者在调控部分基因表达上存在亚功能化;2)SPL与BAM1/2共同调控791个花粉发育、脂类转运和细胞壁形成等过程相关基因的表达,同时BAM1/2特异调控326个脱落酸、水杨酸、茉莉酸信号途径以及水分、防御等胁迫刺激相关基因的表达,而SPL还单独调控3 789个基因的表达,主要参与到生长素合成和信号响应、水分、温度、光等相关的胁迫刺激.以上结果为生殖发育领域相关研究提供了重要参考.     

拟南芥bHLH家族转录因子DYT1在花药发育过程中调控胼胝质降解

胼胝质的合成和降解是雄配子体减数分裂过程中的一个重要特征,对后期花粉成熟有重要作用．在此研究中,分离到了一个雄性不育突变体ms157,该突变体的绒毡层分化及胼胝质降解过程出现异常,导致花粉败育．图位克隆和遗传分析表明：MS157基因与bHLH家族转录因子DYT1（At4g21330）是同一基因．因此,将ms157突变体改名为dyt1-2．反式激活作用实验揭示了DYT1的激活功能域位于基因的250～504bp之间．通过酵母双杂交实验发现DYT1蛋白在体内可以形成同源二聚体来执行其功能．RT—PCR及定量PCR分析表明胼胝质酶相关基因A6的表达在突变体背景下严重下调．因此,DYT1通过调控胼胝质的降解来影响花药发育过程．     

拟南芥atfes1a突变体种子的热敏感性分析

根据基因芯片数据等生物信息学资料,我们锁定一些未知的且被高温诱导的基因,并获得了一个推测编码蛋白含armadillo/beta-catenin repeat(简称ARM)结构域的突变体salk-021784,该突变体缺失AtFes1A基因,该基因编码一种受高温诱导的ARM 蛋白.利用抗体检测了在拟南芥种子发育过程中AtFes1A的热诱导表达特征, 通过表型分析发现,热处理后的突变体种子萌发率明显低于热处理后的野生型,表明AtFes1A蛋白与拟南芥耐热有关.     

水稻籼粳杂交F_1植株育性与花粉育性的关系

以2个广亲和品种(轮回422,02428)分别与籼、粳稻配组及粳/籼、籼/粳、粳/粳配组,检测了杂种F1代的结实率及镜检亲本F1代的花粉活性.试验结果表明:除测49外,亲本花粉可育性均较高,杂种F1代的花粉育性因亲本不同而有所差异,以广亲和品种与籼、粳稻配组所得的杂种F1代的花粉可育性最高;籼粳杂种F1代的花粉败育率与植株不结实率的相关系数达0.8380.     

拟南芥雄性不育突变体opw(only pollen wall)候选突变基因的克隆

在T-DNA插入突变体Salk_059463株系的群体中,筛选到两株雄性不育突变体,对TDNA序列上的一对引物进行PCR鉴定,结果表明:其基因组中没有T-DNA插入.遗传分析表明这两株雄性不育突变体由同一单个隐性基因控制,引起不育的主要原因是从花药发育的第8期开始,小孢子细胞质内容物逐渐减少直至消失,到花药发育的第12期,药室内的小孢子只剩下一个花粉壁空壳,故该突变体命名为opw(only pollen wall).利用图位克隆的方法对OPW基因进行了定位,结果表明OPW基因位于第二条染色体上分子标记T28M21和T3G21之间的12 kb区间内,该区间内一共有21个基因注释.通过克隆区间内的基因并测序发现opw-1突变体基因组中At2g40140基因编码序列的外显子在第289和第290个碱基之间插入了一个A碱基,而opw-2突变体基因组中At2g40140基因编码序列的外显子在第412和第413个碱基之间插入了一个T碱基,造成的编码序列移码使第424至第426碱基成为终止密码子,故At2g40140是编码OPW的候选基因.     

拟南芥雄性不育突变体 pmi1 的遗传与定位

通过甲基磺酸乙酯（简称EMS）诱变拟南芥Col－0种子获得一株隐形雄性不育突变体pollen mitosis1,pmil．遗传分析结果显示突变体为配子体遗传．细胞学观察发现突变体花粉粒发育出现异常：细胞塌陷,细胞核在单核靠边期后和花粉第一次有丝分裂（简称PMI）前的时间段发生降解．通过图位克隆方法将该基因定位在分子标记T19L18和T1D16之间,物理距离55Kb．测序分析证明这55Kb区间中的ERECTA基因的编码区在3067bp的位置发生单碱基替换,C／G变为A／T．由此说明ERECTA基因在拟南芥从单核小孢子到二细胞花粉的发育过程中起着重要作用．     

Isolation and characterization of 4 gametophytic male sterile mutants in Arabidopsis thaliana

In flowering plants, male fertility depends on the formation and development of normal male gametophytes or pollen grains. However, little is known about the molecular mechanisms that regulate the processes. Here, we report the identification of four novel independent Arabidopsis gametophytic male sterile mutants, apam1, apam2, apam3 and apam4 (Arabidopsis pollen abortion mutant). The four mutants that were generated by the insertions of geneand enhancer-trap Ds transposon elements were defective in pollen development. Genetic analysis results showed that all four mutations resulted in the loss of male gametophytic function, but did not affect female gametophytic function, and the Ds elements were linked to the mutations tightly in all four mutants. Localization of the Ds insertion sites by thermal asymmetric interlaced PCR (TAIL-PCR) showed that the Ds elements were inserted in four different loci distributed on three chromosomes, chromosomes II, III and V. In summary, the apam4 is allelic to AHA3, while the other three were located in places where there are no genes that have been known to be involved in pollen development, suggesting that they are novel mutations involved in pollen development.     

Identifying neutral allele Sb at pollen-sterility loci in cultivated rice with Oryza rufipogon origin

Pollen sterility is commonly found in the intra-specific hybrids of indica and japonica rice, which is one of the main constrains for the utilization of heterosis between indica and japonica. Six loci controlling the pollen sterility of F1 between indica and japonica have been identified from previous studies. Neutral alleles at each locus are potential to overcome the F1 pollen sterility associated with the locus. Therefore, exploitation and utilization of neutral alleles are of significant importance. The present research was based on fine mapping of the F1 pollen-sterility gene Sb and the abundant genetic diversity of Oryza rufipogon Griff. indigenous to Gaozhou, Guangdong Province （referred to as Gaozhou wild rice）. Crosses were made using Taichung65 （with the genotype of Sb^jSb^j and referred to as El） and its near-isogenic line of F1 pollen sterility gene Sb（with the genotype of Sb^iSb^i, E2） as female parents, and 12 different accessions of Gaozhou wild rice as male parents. F1 pollen fertility was examined to identify the materials having the neutral alleles at the F1 pollen-sterility locus. Segregation of 4 molecular markers tightly linked with the Sb locus was analyzed in the F2 populations derived from the FlS carrying the neutral gene. The pollen fertility related to the 3 genotypes of the molecular markers was also checked by statistical test to determine whether it was consistent with the hypothesis. The results showed that the pollen fertility of two F1s from one accession of Gaozhou wild rice （GZW099） with E1 and E2 was （89.2±21.07）% and （85.65±1.05）%, respectively. Both of them were fertile and showed no significant difference by t-test. Segregation of the 3 genotypes of the 4 molecular markers followed the expected Mendelian ratio （1：2：1） in the F2 populations. There was no significant difference for the averaged pollen fertility of the plants related to the 3 genotypes, suggesting that no interaction exists between the alleles at the Sb locus in GZW099 and Taichung65 or E2. Evidentially, GZW099 carried the neutral gene （named Sb″Sb″） at the Sb locus, which provides valuable theoretical basis and resources for further studying and overcoming the sterility of indica-japonica hybrids.     

遗传定位一个花粉发育相关的拟南芥新基因SPG1

通过EMS诱变获得一个拟南芥突变体shrunken pollen grain1,spg1.根据横切片的观察结果,与野生型相比较,突变体观察到异常的花粉粒.遗传学的实验表明:spg1突变现象由一个隐性的单基因控制.通过图位克隆的方法,spg1突变位点定位在3号染色体的分子标记3-AL138638-6632和3-AL353865-6814之间大约427kb的区间内.生物信息学的分析表明,在这个区间内没有任何已知的与育性相关的基因.以上结果说明SPG1是一个控制拟南芥雄性不育的新基因.     

Zfy gene expression patterns are not compatible with a primary role in mouse sex determination

The Y chromosome determines maleness in mammals. A Y chromosome-linked gene diverts the indifferent embryonic gonad from the default ovarian pathway in favour of testis differentiation, initiating male development. Study of this basic developmental switch requires the isolation of the testis-determining gene, termed TDF in humans and Tdy in mice. ZFY, a candidate gene for TDF, potentially encodes a zinc-finger protein, and has two Y-linked homologues, Zfy-1 and Zfy-2, in mice. Although ZFY, Zfy-1 and Zfy-2 seem to map to the sex-determining regions of the human and mouse Y chromosomes, there is no direct evidence that these genes are involved in testis determination. We report here that Zfy-1 but not Zfy-2 is expressed in differentiating embryonic mouse testes. Neither gene, however, is expressed in We/We mutant embryonic testes which lack germ cells. These observations exclude both Zfy-1 and Zfy-2 as candidates for the mouse testis-determining gene.