【作者】 朱东姿；

【导师】 张宪省； 高新起；

【作者基本信息】 山东农业大学 ， 发育生物学， 2012， 博士

【摘要】 雌配子体的发育是植物有性生殖过程的重要阶段,是植物受精和种子形成的前提,直接影响到作物的产量和品质。研究雌配子体发育调控的分子机制,具有重要的理论意义和应用价值。RNA解旋酶参与基因转录、各种前体RNA的加工、成熟、转运、翻译起始及核糖体装配等诸多细胞学过程,在生物发育过程中发挥重要作用。拟南芥基因组中包含100多个RNA解旋酶基因,但目前只有少数基因的功能得到验证。相关研究发现,RNA解旋酶参与植物的多个生理过程,包括生长发育调控和逆境胁迫响应等。本研究中鉴定到一个拟南芥T-DNA插入突变体,杂合突变体植株自交后代中野生型植株数和杂合体的比例约为1：1,没有鉴定到纯合体。杂合突变体植株的角果中种子数是野生型的一半。将杂合突变体与野生型植株进行互交后,对后代基因型进行鉴定,结果发现,突变的基因不能通过雌配子传递。该突变体中的T-DNA插入到一个DEAH-BOX RNA解旋酶基因中,该基因编码的蛋白含有四个保守的结构域,DEAH、 HELICc、HA2和OB-fold。对拟南芥中DEAH-BOX家族进行聚类分析,并将该基因命名为AtDEAHb3。atdeahb3突变通过影响雌配子体发育过程中的有丝分裂进程,使雌配子体从FG2期开始出现发育延迟,导致同一雌蕊中不同雌配子体发育的同步性被破坏。野生型植株的成熟(flower stage12)雌蕊中,雌配子体处于FG6或FG7期；而杂合突变体植株相同时期的雌蕊中,约有50%的雌配子体处于FG2、FG3、FG4或者FG5期。对突变体进行延迟授粉实验发现,通过雌配子的传递效率上升,表明部分突变的雌配子体最终可以发育成熟并能完成受精。这些结果表明AtDEAHb3在拟南芥雌配子体的发育过程中起重要作用。qReal Time-PCR、GUS活性分析显示,AtDEAHb3在拟南芥分生组织、叶片、未成熟花药和胚珠中表达,表明AtDEAHb3可能参与植物多个器官的生长和发育过程。原位杂交结果表明,AtDEAHb3在整个雌配子体发育过程中均有表达,在成熟胚囊珠孔端杂交信号较强。利用35S::AtDEAHb3-GFP载体转化烟草叶片进行亚细胞定位研究,发现荧光信号分布在细胞质和细胞核中；在拟南芥中稳定表达pAtDEAHb3::AtDEAHb3-GUS融合蛋白,发现GUS信号集中在细胞质和细胞核中。AtDEAHb3与酵母Prp22p、人类DHX8同源,它们作为U5snRNP的组分,参与mRNA前体的剪接。酵母遗传互补实验发现,拟南芥AtDEAHb3能够互补酵母prp22突变体的热敏感表型,推测拟南芥AtDEAHb3参与植物mRNA前体的剪接。拟南芥GFA1在酵母中的同源蛋白Snu114p是剪接复合体U5snRNP的关键组分。利用拟南芥AtGenExpress Visualization Tool分析发现GFA1和AtDEAHb3的表达模式相似。酵母双杂交实验、BiFC实验和Pull-Down实验证实AtDEAHb3可以和GFA1相互作用。进一步通过AtDEAHb3的不同片段分别与GFAl进行酵母双杂交分析,结果发现AtDEAHb3通过其DEAH和HELICc domain之间的序列与GFA1(?)目互作用。这些结果提示,AtDEAHb3可能通过与拟南芥中U5snRNP复合体的互作,参与调节mRNA前体的剪接。AtDEAHb3-amiRNAi转基因拟南芥幼苗生长缓慢,对这一转基因植株中有丝分裂相关基因的表达进行分析,结果发现CYCB类基因表达下调幅度最大,推测AtDEAHb3通过调节CYCB类基因的表达参与对有丝分裂进程的调控。综上所述,在拟南芥雌配子体发育过程中,AtDEAHb3可能通过参与前体mRNA的剪接调节CYCB类基因的表达,进而通过影响细胞有丝分裂参与对雌配子体发育进程的调节。更多还原

【Abstract】 Female gametophyte plays a critical role in the sexual reproduction of angiosperms. Successful development of female gametophytes in higher plants is the consequence of coordination of cell specification, proliferation, and differentiation. Several mutants and genes that are involved in female gametophyte development were identified.RNA helicases are involved in mRNA transcription, pre-RNA splicing, RNA maturation, transportation, translation initiation and ribosomal assembly, and many other cellular processes, which play an important role in eukaryotic organism development. Over100RNA helicase genes in Arabidopsis genome were found, but only few of them are being functional analyzed. Previous studies revealed that RNA helicases were involved in regulating plant growth, development and stress responses.In our study, two T-DNA insertion lines were identified and no homozygous insertion was found in their selfed progeny. In the self-pollinated progeny of the heterozygotic mutant plants, the segregation ratio of heterozygous to wild type plants was about1:1, instead of the typical3:1. Reciprocal cross analysis revealed that the mutant allei could not be transmitted through the female gametophyte. There are about50%seeds in the silique of the mutant compared with the wild type. These results indicate that the mutation has a vital effect on the female gametophyte development. The T-DNA was inserted in a DEAH-box RNA helicase gene. Arabidopsis genome codes22DEAH-box RNA helicases. We named this gene as AtDEAHb3.In flower stage12, the majorities of wild-type embryo sacs are mature and develop to the stage FG6or FG7. However, about50%female gametophytes from atdeahb3/+plants show delayed development, from FG2to FG7stages, indicating that the synchrony of female gametophytic development is impaired in atdeahb3pistils.GUS staining analysis in the pAtDEAHb3::GUS transgenic Arabidopsis plants revealed that AtDEAHb3express in the various tissues, especially in the apical meristem and ovule. The expression of AtDEAHb3was detected in the whole process of female gametophytic development by using RNA in situ hybridization.Arabidopsis GFA1is the core component of U5SnRNP complex involved in pre-mRNA splicing. It was predicated that GFA1and AtDEAHb3have similar expression patterns in Arahidopsis development. Furthermore, yeast two-hybrid, BiFC and Pull-Down analysis revealed that AtDEAHb3could physically interact with GFA1, indicating that AtDEAHb3may play a role in female gametophyte development by involving in pre-mRNA splicing.In the AtDEAHb3-amiRNAi transgenic Arabidopsis lines, the expressions of CYCB genes were down-regulated, which indicates that AtDEAHb3functions in the mitotic process by regulating CYCB genes expression and pre-mRNA splicing.更多还原