拟南芥雄性不育突变体ms1142的遗传定位与功能分析

经EMS诱变野生型拟南芥（Arabidopsis thaliana）群体筛选得到一株雄性不育突变体ms1142,突变体的果荚短小,不含种子。细胞学观察和扫描电镜结果表明,突变体花药发育过程中,花药中小孢子外壁异常、破裂,最后没有花粉形成。遗传分析表明,该突变体为隐性单核基因突变所致;利用图位克隆的方法将MS1142基因定位于第1条染色体的BAC克隆F16P17上44kb区间内,目前尚未见该区间内有雄性不育基因的报道。以上结果结合生物信息学分析表明,MS1142是一个新的调控花药发育的关键基因。该工作为花药发育关键基因MS1142的克隆及功能分析奠定了基础。     

拟南芥雄性不育突变体ms1142的遗传定位与功能分析

经EMS诱变野生型拟南芥(Arabidopsis thaliana)群体筛选得到一株雄性不育突变体ms1142, 突变体的果荚短小, 不含种子。细胞学观察和扫描电镜结果表明, 突变体花药发育过程中, 花药中小孢子外壁异常、破裂, 最后没有花粉形成。遗传分析表明, 该突变体为隐性单核基因突变所致; 利用图位克隆的方法将MS1142基因定位于第1条染色体的BAC克隆F16P17上44 kb区间内, 目前尚未见该区间内有雄性不育基因的报道。以上结果结合生物信息学分析表明, MS1142是一个新的调控花药发育的关键基因。该工作为花药发育关键基因MS1142的克隆及功能分析奠定了基础。     

拟南芥bso-1突变体的基因定位及表型分析

通过EMS化学诱变在拟南芥Columbia(Col-0)野生型突变体库中筛选获得1株器官显著增大的突变体,命名为big size organ1(bso-1)。遗传分析表明,bso-1受单个隐性核基因控制。表型观察发现,突变体植株的幼苗、花、果荚及种子与野生型相比都表现出明显的增大。组织切片结果显示,突变体种子的增大主要由胚细胞个体增大导致胚体积增大而实现,因此突变体种子的重量也较野生型有明显增加。利用图位克隆方法将相关基因初步定位在4号染色体上SSLP标记T5L19与F28M11之间58kb区间内,生物信息学分析显示此区间内未见调控植物器官大小发育相关的已知基因的报道。该研究结果为进一步克隆bso-1突变体相关基因及探讨其在控制植物器官发育尤其是种子发育过程中的作用奠定了基础。     

MS1521是拟南芥花药发育的必需基因

通过对3个拟南芥（Arabidopsis thaliana）雄性不育突变体（ms1521,st350,st454）的分析,研究了MS1521基因在花药发育过程中的功能。ms1521是通过EMS诱变野生型拟南芥得到的一株突变体,遗传分析表明ms1521是隐性单核基因控制的。利用图位克隆的方法对不育基因MS1521进行了定位,结果将MS1521定位于拟南芥第一条染色体上26kb的区间内,该定位区间内有一个影响花器官形态建成的基因UFO。测序结果表明在ms1521突变体中UFO基因编码区的958bp处发生了单碱基突变,导致MS1521该位点的氨基酸由天冬酰胺变成了天冬氨酸。另外两个表型与ms1521相似的突变体st350和st454来自T-DNA插入突变体群体。测序结果表明突变体st350和st454分别在UFO基因编码区发生了提前终止突变。等位分析表明它们与MS1521基因是等位的。3个突变体营养生长期发育正常,但生殖生长发育出现异常：有的雄蕊只有花丝没有花药;或者有花药但花丝变短;或者雄蕊有正常的花丝和花药,花药中有可育的花粉,但药室不能开裂;最终导致突变体不育的表型。进一步细胞学观察发现药室不能开裂是由于药室内壁细胞纤维化和木质化增厚不明显造成的。以上这些结果表明MS1521基因在花药发育过程中起重要作用。     

调控拟南芥花粉发育突变体zy1511的基因定位及特征分析

为了进一步研究花药花粉发育过程,我们通过EMS诱变,筛选到拟南芥雄性不育突变体zy1511。遗传分析表明,zy1511为隐性单位点突变。细胞学观察表明．突变体花药中小孢子从四分体释放出后绒毡层并没有开始退化,花药发育后期绒毡层依然部分存在。说明突变体花药绒毡层退化比野生型的要迟,因此,小孢子不能发育成正常花粉粒。利用图位克隆的方法将zv1511定位于第一条染色体上分子标记F25P12和T8L23之间134．kb的区间内。本项工作为zy1511基因的克隆及对花粉发育功能分析奠定了基础。目前尚未见到该区间内雄性不育基因的报道。因此,zy1511是控制花粉发育的尚未发现的关键基因。     

拟南芥雄性不育突变体ms1502的遗传及定位分析

通过EMS诱变、背景纯化与遗传分析,从拟南芥（Arabidopsis thaliana）中筛选到了一棵隐性单基因控制的雄性不育突变体ms1502。细胞学观察发现,突变体在小孢子从四分体释放出后花药绒毡层过早衰亡,小孢子的内容物不正常地凝聚,最终无法形成正常的花粉粒。利用图位克隆的方法对该基因MSl502进行了定位,结果表明MS1502位于第4条染色体上分子标记F25124和T12H20之间105kb区间内。目前该区间内尚未见到花药发育必需基因（不育基因）的报道,因此MS1502是一个控制花粉发育的新基因。     

拟南芥雄性不育突变体fne的遗传与定位分析

在T-DNA插入突变体Salk_118481株系的群体中,筛选到一株雄性不育突变体,用T-DNA序列上的一对引物进行PCR鉴定表明其基因组中没有T DNA插入。通过背景纯化与遗传分析发现该雄性不育突变体是由单个隐性基因控制的,引起不育的主要原因是在花药发育的第13~14期,花丝不能伸长以完成授粉,故该突变体命名为fne （filament no elongation）。利用图位克隆的方法对FNE基因进行了定位,结果表明FNE基因位于第五条染色体上分子标记MBD2和MMG4之间的97kb区间内。目前该区间内尚未见到控制花丝伸长基因的报道,因此,FNE基因是一个控制花丝伸长的新基因。     

拟南芥白化突变体心口的基因定位与分析

EMS30是拟南芥经甲基磺酸乙酯（EMS）诱变得到的白化突变体。该突变体的叶绿体结构存在严重缺陷,同时伴随叶绿素缺失。遗传分析显示EMS30突变体的突变表型受隐性单基因控制。采用图位克隆的方法对EMS30突变基因进行定位的结果显示,该基因位于拟南芥第一条染色体的分子标记F21M12和F14N23之间的96kb区间内,该区间包含25个基因。通过生物信息学分析发现,该区间内有3个基因定位在叶绿体或与叶绿体发育相关。这些结果有助于该基因的克隆,为阐释叶绿体发育提供线索。     

一个控制拟南芥小孢子发育基因的定位

通过EMS诱变、背景纯化与遗传分析,从拟南芥突变群体中分离到一株单隐性核位点控制的雄性部分不育突变体pms15-16-2-3.细胞学观察表明,突变体在花药发育的过程中,中层细胞延迟降解,绒毡层细胞形态分化异常,出现异常的四分体,导致最终只能形成少量的花粉.利用图位克隆的方法对该基因进行了定位,结果表明PMSl5-16-2.3基因位于拟南芥第3条染色体BAC克隆T24C20 上的28 kb区间内.目前该区间内尚未见到控制小孢子发育基因的报道,因此该基因是一个控制小孢子发育的新基因.本研究结果对同的基因的克隆及其在化粉发育中的功能研究奠定了基础.     

MS1516是拟南芥四分体形成和小孢子发育的必需基因

以前报道了雄性育性下降突变体ms1516,而且图位克隆的方法已将突变基因MS1516定位到拟南芥基因组第3条染色体上28kh的区间内。本文通过进一步的生物信息学分析,发现该定位区间内有一个与减数分裂有关的基因AtATM,而且等位实验结果表明rns1516和nfm0是等位突变体。细胞学分析结果表明,ms1516突变体在花药发育过程中产生多个不均等的小孢子,而且大多数的小孢子不能发育成成熟的花粉。DAPI染色的结果显示小孢子母细胞减数分裂过程中,染色体不能正常分离,对成熟花粉的扫描电镜观察结果发现突变体多数花粉形态异常。以上结果说明MS1516基因在小孢子形成和发育过程中具有重要作用。     

ST273是拟南芥绒毡层和小孢子发育的必需基因

绒毡层在拟南芥花药花粉发育过程中具有重要作用,包括分泌降解胼胝质的胼胝质酶、为花粉壁的形成提供原料以及为小孢子发育提供营养物质.本文通过对拟南芥雄性不育突变体st273的分析,研究了ST273基因在花药花粉发育过程中的功能.st273是通过T-DNA插入诱变野生型拟南芥得到的一株突变体,遗传分析表明st273是单隐性核基因控制的.利用图位克隆的方法对不育基因ST273进行了定位,结果表明ST273基因与拟南芥第三条染色体上分子标记CIW11连锁.生物信息学分析发现该分子标记附近有一个调控花粉发育的基因TDF1.测序分析结果表明在st273突变体中,TDF1基因第三个外显子上459位的碱基发生了由G459变成了A459的单碱基变化,导致ST273基因该位点提前终止突变.等位分析结果表明st273与tdf1是等位突变体.st273突变体营养生长期发育正常,但生殖生长发育出现异常.亚历山大染色结果显示st273突变体花药中没有花粉.组织切片观察结果表明,突变体花药绒毡层异常肥大且空泡化,四分体不能正常释放小孢子,最终无法形成花粉.这些结果揭示了ST273蛋白质参与调控了绒毡层和小孢子发育过程.     

Characterization and genetic mapping of a mutation (ms35) which prevents anther dehiscence in Arabidopsis thaliana by affecting secondary wall thickening in the endothecium

The male sterile mutant, ms35 , of Arabidopsis thaliana was produced by X-irradiation of seeds. The mutant produces fertile pollen, but is male sterile because the anthers do not dehisce. Anther development in ms35 plants occurs as in wild-type Arabidopsis until shortly after microspores are released from meiotic tetrads. Thereafter, in the wild type, bands of lignified, cellulosic secondary wall thickenings are laid down around the cells of the anther endothecium. In contrast, wall thickenings are not formed in the endothecium of the ms35 mutant. Development of other lignified tissues, for example the vascular tissue of the stamen, occurs normally in ms35 plants. In mutant anthers, as pollen maturation is completed, the stomium is cleaved but the anther wall does not retract to release pollen. The block in anther dehiscence in ms35 plants is specifically correlated with the absence of endothecial wall thickenings. The ms35 mutation represents the first genetic evidence in support of the proposed role of the endothecium in anther dehiscence. The ms35 gene was mapped to the top arm of chromosome 3 ( hy2 -(4.17±2.31 cM)- ms35 -(32.14±5.45 cM)- gl1 ).     

ST273 Is Essential for Tapetum and Microspore Development in Arabidopsis thaliana*

In Arabidopsis, the tapetum plays important roles in anther and pollen development by providing enzymes for callose dissolution, materials for pollen wall formation, and nutrients for microspore development. This paper describes the functional analyses of the ST273 gene in anther and pollen development by using Arabidopsis male sterile mutant st273. Mutant st273 was identified from a T DNA insertion mutant population, and genetic analysis showed that st273 mutant was controlled by a single recessive nuclear gene. A map based cloning approach was used, and ST273 gene was mapped to be linked to a molecular marker CIW11 on chromosome 3. Bioinformatics analysis revealed that there is a TDF1 gene near the marker CIW11. Sequencing analysis indicated that st273 mutant had a G459 to A459 base pair change in the third exon of TDF1 gene, which resulted in premature termination mutation in this region. Allelism test indicated that ST273 and TDF1 belong to the same locus. The mutant plant grows normally during the vegetative growth stage, but show developmental defects at the reproductive growth stage. Alexander staining showed that there was no pollen in the mature anther locule. Cytology observation indicated that the mutant tapetum was enlarged and vacuolated, the tetrads could not release the microspores timely, and finally no pollen was formed in the anther. These results demonstrated that ST273 protein plays an important role in tapetum and microspore development.     

ses, a novel visible mutant ofArabidopsis thaliana related to anther development

Analysis of mutants and clone of genes is crucial for unraveling the mechanism of anther development. The lack of elongation of siliques in a novel shortened early-stage siliques (ses) mutant was caused by unsuccessful pollination because of aborted pollens. At stage 11, microspores of mutantses appeared weakly stained and degenerated. At stage 14, mutantses produced abnormal pollens, and the anther wall was collapsed and crumpled; furthermore, some cavities were also found under the epidermis. Mutantses showed that the gene responsible for the defects plays a role in anther development. By mapping, mutantses was narrowed into a 67-kb interval on chromosome 1 between CER448792 (2,000,541 bp) and CER464544 (2,067,844 bp). By using the sequence-based map ofArabidopsis genes with mutant phenotypes,ses was localized on the right of phenotype marker AT1g06150 and on the left of phenotype marker AT1g08060. The phenotyping and mapping data both pointed to the conclusion thatses was a novel mutant related to anther development. Sequencing showed that there was a point mutation in gene AT1G06710.1; thus, gene AT1G06710.1 is the mist likely candidate gene responsible for the mutation.     

The transformation of anthers in the <Emphasis Type="Italic">msca1</Emphasis> mutant of maize

In normal anther development in maize (Zea mays L), large hypodermal cells in anther primordia undergo a series of proscribed cell divisions to form an anther containing microsporogenous cells and three distinctive anther wall layers: the tapetum, the middle layer and the endothecium. In homozygous msca1 mutants of maize, stamen primordia are initiated normally and large hypodermal cells can be detected in developing anthers. However, the normal series of cell division and differentiation events does not occur in msca1 mutant plants. Rather, structures containing parenchymal cells and ectopic, nonfunctional vascular strands are formed. The epidermal surfaces of these structures contain stomata, which are normally absent in maize anthers. Thus, all of the cell layers of the "anther" have been transformed in mutant plants. The filaments of the mutant structures are normal, and all other flower parts are normal. The msca1 mutation does not affect female fertility, but transformed "stamen" structures remain associated with mature ovules rather than aborting as in normal ear development. The msca1 mutation is distinctive in that only one part of a single (male) reproductive organ is transformed. The resulting structure has general vegetative features, but cannot be conclusively identified as a particular vegetative organ.     

The Post-meiotic Deficicent Anther1 （PDA1） gene is required for post-meiotic anther development in rice

To understand the molecular mechanism of male reproductive development in the model crop rice,we isolated a complete male sterile mutant post-meiotic deficient anther1 (pda1) from a γ-ray-treated rice mutant library.Genetic analysis revealed that the pda1 mutant was controlled by a recessive nucleus gene.The pda1 mutant anther seemed smaller with white appearance.Histological analysis demonstrated that the pda1 mutant anther undergoes normal early tapetum development without obvious altered meiosis.However,the pda1 mutant displayed obvious defects in postmeiotic tapetal development,abnormal degeneration occurred in the tapetal cells at stage 9 of anther development.Also we observed abnormal lipidic Ubisch bodies from the tapetal layer of the pda1 mutant,causing no obvious pollen exine formation.RT-PCR analysis indicated that the expression of genes involved in anther development including GAMYB,OsC4 and Wax-deficient anther1 (WDA1) was greatly reduced in the pda1 mutant anther.Using map-based cloning approach,the PDA1 gene was finely mapped between two markers HLF610 and HLF627 on chromosome 6 using 3,883 individuals of F2 population.The physical distance between HLF610 and HLF627 was about 194 kb.This work suggests that PDA1 is required for post-meiotic tapetal development and pollen/microspore formation in rice.     

Androgenesis in vitro in anther cultures of chlorophyll mutants ofNicotiana tabacum

The course of androgenesisin vitro was investigated with anther cultures of two chlorophyll mutants of Nicotiana tabacum. A different sensitivity to the hormonal composition of the medium was revealed between the cultures White Seedling and Sulfur; the stimulatory effect of kinetin on the frequency of androgenesis was observed only in White Seedling cultures. In addition to green plants, “aurea” (mutation Sulfur) or “albino” (mutation White Seedling) phenotypes also differentiated in both cultures. The possible causes of variability in the participation of green and mutant forms are discussed.     

Rice SIZ1, a SUMO E3 ligase, controls spikelet fertility through regulation of anther dehiscence

? Sumoylation, a post-translational modification, has important functions in both animals and plants. However, the biological function of the SUMO E3 ligase, SIZ1, in rice (Oryza sativa) is still under investigation. ? In this study, we employed two different genetic approaches, the use of siz1 T-DNA mutant and SIZ1-RNAi transgenic plants, to characterize the function of rice SIZ1. ? Genetic results revealed the co-segregation of single T-DNA insertional recessive mutation with the observed phenotypes in siz1. In addition to showing reduced plant height, tiller number and seed set percentage, both the siz1 mutant and SIZ1-RNAi transgenic plants showed obvious defects in anther dehiscence, but not pollen viability. The anther indehiscence in siz1 was probably a result of defects in endothecium development before anthesis. Interestingly, rice orthologs of AtIRX and ZmMADS2, which are essential for endothecium development during anther dehiscence, were significantly down-regulated in siz1. Compared with the wild-type, the sumoylation profile of high-molecular-weight proteins in mature spikelets was reduced significantly in siz1 and the SIZ1-RNAi line with notably reduced SIZ1 expression. The nuclear localization signal located in the SIZ1 C-terminus was sufficient for its nuclear targeting in bombarded onion epidermis. ? The results suggest the functional role of SIZ1, a SUMO E3 ligase, in regulating rice anther dehiscence.     

The Arabidopsis male-sterile mutant dde2-2 is defective in the ALLENE OXIDE SYNTHASE gene encoding one of the key enzymes of the jasmonic acid biosynthesis pathway

The Arabidopsis thaliana (L.) Heynh. mutant delayed-dehiscence2-2 (dde2-2) was identified in an En1/Spm1 transposon-induced mutant population screened for plants showing defects in fertility. The dde2-2 mutant allele is defective in the anther dehiscence process and filament elongation and thus exhibits a male-sterile phenotype. The dde2-2 phenotype can be rescued by application of methyl jasmonate, indicating that the mutant is affected in jasmonic acid biosynthesis. The combination of genetic mapping and a candidate-gene approach identified a frameshift mutation in the ALLENE OXIDE SYNTHASE (AOS) gene, encoding one of the key enzymes of jasmonic acid biosynthesis. Expression analysis and genetic complementation of the dde2-2 phenotype by overexpression of the AOS coding sequence confirmed that the male-sterile phenotype is indeed caused by the mutation in the AOS gene.     

一种新型矮牵牛花发育突变体的研究

报道了新发现的一种矮牵牛(Petunia hybrida L.)花发育突变体,命名为efficient(eff),并对这一突变体进行了形态学和遗传学分析。eff突变体主要表现为雌蕊心皮数目的增加和雄蕊上长出花瓣状结构,同时,雄蕊、花瓣和萼片数亦有增多,但营养器官无变化。心皮数目的增加导致雌蕊柱头和子房体积的显著增大,并形成较大的果实。雄蕊上花瓣的形成对花粉的产生无明显影响。扫描电镜观察发现,eff突变体在花器官原基形成时发生了相应数目的增加及特征的变化。遗传学分析表明,突变的表现型符合孟德尔单基因遗传规律。