【作者】 刘燕；

【导师】 郭光沁；

【作者基本信息】 兰州大学 ， 细胞生物学， 2010， 博士

【摘要】 雄配子体在被子植物有性生殖过程中起着重要作用,然而目前已知的参与调节雄配子体发育和功能的基因还很少。本文通过统计拟南芥幼苗卡那霉素(选择性标记)抗性与敏感性分离比,从Ds插入突变体库中筛选到四个偏离孟德尔分离比的突变体：KD360、KD361、KD362、KD375。正反交实验表明KD360、KD362和KD375仅影响雄配子体的传递,而KD361既影响雄配子体传递又影响雌配子体传递；KD362完全不能通过雄配子体传递,而KD360、KD361和KD375仅部分不能通过雄配子体传递。形态学和细胞学研究表明KD360、KD361和KD375雄配子体败育发生在单核时期,而KD362雄配子体败育发生在两核之后三核之前。基因克隆显示KD360 Ds插入到Atlg44224唯一的外显子内,预测Atlg44224编码ECA1配子体发生相关蛋白。KD361Ds插入到At4g00380 ATG上游1bp处。预测At4g00380编码含XH/XS元件或XS锌指元件蛋白。KD362有两个Ds插入,一个插入到At2g01560第一个外显子内,另一个插入到At2g01910和At2g01913之间,距At2g01910终止密码子TGA下游460bp,距At2g01913终止密码子TTA下游1284bp。KD375 Ds插入到Atlg26815 ATG上游134bp处。预测At1g26815编码的蛋白含有一个F-box结合区域和一个跨膜元件。通过Ds插入位点纯杂合鉴定以及等位突变体分析我们推测：Atlg44224和At4g00380突变可能是KD360和KD361雄配子体部分致死的原因,即Atlg44224和At4g00380可能参与调控拟南芥雄配子体发育：KD362雄配子致死表型可能是Ds插入导致At2g01913突变造成;KD375雄配子体部分致死表型与Atlg26815上游的Ds插入紧密连锁。Pro::GUS转基因植株Gus染色结果表明：Atlg44224在小孢子、成熟花粉粒、萌发的花粉粒、柱头、以及早期胚胎中表达;At4g00380在四分体、小孢子、成熟花粉粒、萌发的花粉粒、柱头以及果荚中表达。此外我们得到一个新的雄性不育突变体ms360。刚从四分体释放出来的ms360小孢子外壁呈颗粒状,小孢子不能正常发育成花粉粒。此外ms360绒毡层的发育也不正常。遗传分析确定ms360是由单基因控制的隐性突变体。采用图位克隆技术,将MS360定位在拟南芥第三号染色体分子标记nga162和nga172附近。更多还原

【Abstract】 The male gametophyte plays a critical role in sexual reproduction of angiosperms. Currently, the genes and pathways involved in male gametophytic development and function in flowering plants remain largely unknown. A large-scale mutant screen of Ds transposon insertion lines was employed to identify four mutants (KD360, KD361, KD362, and KD375) of Arabidopsis thaliana with defect in male gametophyte development and function. Genetic and morphologic analyses indicated that KD362 mutant was a completely male gametophytic lethal, but KD360, KD361 and KD375 mutants were partly male gametophytic lethal. In addition, KD360, KD362 and KD375 mutants affect only the male gametophyte, while KD361 mutation affects both the female and male gametophyte. We analyzed male gametophyte development of four mutants and found that the KD360, KD361 and KD375 mutants degenerated before the PMI, while the KD362 microspores arrested during the binucleate stage to trinucleate stage. Complete cosegregation of the phenotype with the antibiotic resistance marker and consistent segregation ratios suggested that Ds was tightly linked to or responsible for the reduced genetic transmission. The sequences flanking the Ds element were isolated with TAIL-PCR. The flanking sequences obtained were run against BLASTN to identify the genomic location of the Ds element. In KD360, Ds inserts into the exon of At1g44224, which encodes an ECA1 gametogenesis related family protein. In KD361, Ds inserts in the upstream of At4g00380, which encodes XH/XS domain-containing protein or XS zinc finger domain-containing protein. In KD362, Ds inserts not only in the first exon of At1g44224 but also between At2g01910 and At2g01913. In KD375, Ds inserts in the upstream of Atlg26815, which is similar to F-box family protein. Analyses the additional insertion lines of these genes suggested that At1g44224 and At4g00380 may be responsible for the reduced genetic transmission and pollen abortion. Pro::GUS transgenic Arabidopsis showed that At1g44224 and At4g00380 driven GUS activity during pollen maturation and also in germinating pollen.Besides four male gametophytic mutants, we obtained a male sterile mutant fortuitously. In ms360 mutant, after released from the tetrad, the microspores’exine and tapetum are abnormal. ms360 is a recessive male sterile mutant controlled by single gene. Using map-based cloning technique, we mapped MS360 gene close to the molecular marker nga162 and nga172 of Chromosome 3.更多还原