【作者】 位芳；

【导师】 张改生；

【作者基本信息】 西北农林科技大学 ， 遗传学， 2010， 博士

【摘要】 在植物界,大约70%的被子植物都曾发生过多倍化现象。多倍化在植物界物种进化过程中有着非常重要的意义,是植物进化和变异的重要推动力量,也是保持物种多样性和稳定性的重要途径。因此,在多倍体植物中,深入研究和探索多倍化效应对植物生殖和发育的影响,对揭示多倍体植物的进化和变异有着重要的意义。正是基于此,本研究充分利用模式植物拟南芥生殖和发育优越性,采用人工同源或异源多倍化技术,成功合成出同源和异源四倍体拟南芥,并结合与植物有性生殖紧密相关的减数分裂过程,通过分子及细胞遗传学手段,深入研究了多倍化效应对基因突变、基因重组、配子传递等的影响,获得以下重要结论:1.对拟南芥二倍体中3种减数分裂突变体spo11-1、dmc1和asy1的细胞遗传学研究和相应表达蛋白质产物序列比对分析发现,spo11-1功能是在减数分裂间期启动DNA双链产生缺口,dmc1与其他修复蛋白构成修复复合体,对产生缺口的DNA单链进行同源依赖性修复,asy1对减数分裂前期I同源染色质(体)联会和中期I同源染色体配对有重要作用。虽然3个基因功能不同和表达时期不同,但在细胞遗传学上具有相似性,3个基因突变都能导致同源染色体联会和配对失败,最终减数分裂期间以单价体形式出现。但是,3种基因突变导致的细胞遗传变化程度不同,spo11-1和dmc1导致的突变效应较为强烈,减数分裂期同源染色体完全以单价体出现,而asy1突变体中,平均每个减数分裂细胞能产生1.26个二价体。此外,序列比对发现,3种基因在植物界较为保守,对保证植物正常的有性生殖有重要意义。2.通过人工多倍化,本研究成功合成了同源四倍体拟南芥A.thaliana。对多倍化后的突变体dmc1和asy1减数分裂过程研究发现,在同源四倍体中,dmc1基因导致的突变效应较为强烈,减数分裂过程未发现二价体或四价体的形成;而同源四倍体突变体asy1中,减数分裂过程中,二价体数量增多,并能检测到部分四价体现象。因此,本实验认为,多倍化可以缓冲基因突变带来的不良影响,为多倍体通过有性生殖延续后代提供条件。3.对同源四倍体突变体asy1有性生殖过程中非整倍体配子传递规律的研究发现,在细胞遗传学上,减数分裂后配子染色体数目决定了配子传递的方式,非整倍性亚倍体配子传递主要通过雄性配子,而超倍体配子传递可以通过雌雄配子,雌配子体主要传递超倍体。此外,双受精后,来自不同亲本的染色体数目和父母本间基因组含量相对平衡性对植物发育有重要影响4.在同源多倍体中,以一对紧密连锁基因GFP和RFP(G-R,物理距离约为5Mb)为模型,估计了同源多倍体减数分裂过程中多价体联会和配对对基因重组频率影响,并在实际研究中得到了证实。对于一对物理距离较近的连锁基因G-R,减数分裂期同源染色体间的多价体,如三价体和四价体的形成并不影响基因重组的频率。5.在二倍体拟南芥中,对连锁基因G-R的重组频率的研究发现,位于拟南芥3号染色体上的平均重组率为16.8%,父本重组率为20.2%,母本重组率为7.4%;位于5号染色体上的G-R连锁基因,平均重组率为14.9%,父本重组率为18.1%,母本重组率为14.0%,因此,本研究认为,同一对基因G-R重组率受染色体、染色体上位置的影响。此外,基因重组也受性别的影响,一般父本重组率较高。6.利用连锁基因G-R,通过同源和异源多倍化手段,分别对拟南芥同源四倍体A.thaliana和异源四倍体A.suecica基因重组频率进行了研究,研究结果表明,与二倍体相比较,同源和异源多倍化都可以导致基因重组频率的显著提高。同源四倍体A.thaliana中,连锁基因G-R的平均重组率为23.2%,父本重组率为28.0%,母本重组率为15.0%。异源四倍体A.suecica中,连锁基因G-R的平均重组率为28.2%,父本重组率为29.8%,母本重组率为13.0%。因此,多倍化后,性别特异性基因重组频率有所变化,都受到多倍化效应的影响。7.结合荧光原位杂交技术,针对不同来源拟南芥染色体组,通过利用特异性探针,研究结果表明,在新合成异源四倍体拟南芥A.suecica中,由于基因组冲击(genomic shock)的影响,尽管基因表达受到某种程度的影响,但是异源四倍体形成后,可能由于类似Ph1基因的存在,在减数分裂过程中,能进行正常的减数分裂,同源染色体联会和配对的机制不受干扰,保证了同源染色体间的等位基因交叉互换,为产生功能性配子提供细胞学物质基础。8.细胞遗传学研究表明,在异源多倍体中,减数分裂中同源染色体多价体的联会和配对并不能导致基因重组频率提高,因此,关于多倍化效应导致基因重组频率改变的内在原因有待于进一步研究。更多还原

【Abstract】 In plant kingdom, approximately 70% of flowering plants have experienced polyploidization. Polyploidization has a meaningful role in plant evolutionary history, and is a driving force for evolution and variation in plants, and is a pathway for plants to maintain genetic diversification and genomic stability. Therefore, to explore the effects of polyploidy on plant sexual reproduction and development can undoubtedly contribute to our understandings of tremendous events of polyploidization in plants. In the present study, the model plant Arabidopsis was used for its fast growth and development to artificially synthesize polyploid pants including autotetraplod A.thaliana and allotetraploid A.suecica (by crossing between A.thaliana and A.arenosa). The meiotic progression of newly synthesized allotetraploid A.suecica was analysed. In combination with 3 meiosis-related genes spo11-1, dmc1 and asy1, the effects of polyploidy on meiosis and transmission of sexually reproductive gametes were also exploited. Besides, aided with a pair of cis-linked transgenic fluorescent marks G-R, the frequency of meiotic recombination was investigated as well. The results were presented as follows:1. In diploid A.thaliana, 3 mutant genes were investigated during meiosis. Spo11-1 was involved in initiation of double-strand breaks (DSBs), which further affected the meiotic recombination. Dmc1 played a key role in repairing DSBs by contributing to fixing the protruded 3’single DNA strands. Asy1 was a key component controlling synapsis and pairing of homologous chromatins (chromosomes). The results showed that, although the 3 genes examined here have distinct functions during meiosis respectively, the cytological observations of meiotic chromosomes were very similar. Mutation of the 3 genes can result in the failure of synapsis and pairing of homologous chromosomes, and chromosomes displayed as a unique formation of univalents in spo11-1 and dmc1, whereas in asy1 the rare formation of bivalents was infrequently observed at 1.26 per cell. Besides, the alignment of amino acid sequences of spo11-1, dmc1 and asy1 from other systems such as human, yeast, wheat and rice showed that 3 genes are highly conserved in sequence similarity which indicated an important role performed by these 3 genes in plant sexual life.2. After polyploidization, the autotetraploid Arabidopsis was obtained in the mutants dmc1 and asy1. The effects of polyploidization on meiotic progression in tetraploid dmc1 and asy1 were observed. The results demonstrated that the defects caused by mutation of dmc1 was more severe than that of asy1, and no improved meiosis was observed in tetraploid dmc1, but an increasing number of bivalents, even quadrivalents were frequently observed. These results indicated that polyploidy of plants can buffer the defectiveness brought about by specific mutations.3. The transmission of aneuploids was investigated in autotetraploid asy1, and the results showed that chromosome number in the produced gametes determines the pathway of aneuploid transmission. Transmission of hypoploids was mainly dependent on male gametes, whereas hyperploids have a dual pathway of transmission, through male or female. And chromosome number and relative ratio balance of genomic contents between male and female determine on the fate of development after fertilization.4. The statistical model on estimation of frequency of meiotic recombination based on a pair of cis-linked markers GFP and RFP was established on condition that the linked markers were closely located on a chromosme (5 Mb). The model indicated that the formation of multivalents in autotetraploid contributed little to the frequency change of meiotic recombination, which was proved further with our solid data.5. In diploid Arabidopsis, the recombination frequency of the cis-linked markers was estimated on chromosome 3 and chromosome 5 respectively. The results showed that on chromosome 3, the averaged frequency of homologous recombination was 16.8%, and the frequency in male was 20.2% and 7.4% in female. However, on chromosome 5, the averaged frequency of homologous recombination was 14.9%, and the frequency in male was 18.1% and 14.0% in female. These data indicated that recombination frequency was affected by a series of factors including chromosome of genes, locus of gene on chromosome. Besides, sex-related difference of recombination was generally observed with a higher frequency in male than female.6. After polyploidization including autopolyploidization and allopolyploidization, the recombination frequency of the same cis-linked markers was investigated in autotetraploid and allotetraploid Arabidopsis. The data indicated that in contrast with diploid Arabidopsis, autotetraploid has averaged 23.2% of recombination frequency, 28.0% in male and 15.0% in female. Comparatively, in allotetraploid, the averaged frequency of recombination was 28.2%, and 29.8% in male, and 13.0% in female. These results indicate that polyploidization contributes much to the increased frequency of meiotic recombination, and sex-related difference of recombination existed as well in polyploid plants.7. Aided with fluorescence in situ hybridization (FISH), the newly formed allotetraploid Arabidopsis was analyzed during meiosis. The observations showed that normal meiotic process was guaranteed with possible presence of wheat Ph1-like gene albeit the genomic shock of dirrerent chromosome resources. The synapsis and pairing of homologous chromosomes progress without interference of homoeologous chromosomes, which provided the opportunity for recombination and a basis for production of functional gametes after meiosis.8. Cytogenetic analysis demonstrated that formation of multivalents in polyploid plants can not account for the increased frequency of recombination, thus the further work was needed for unraveling the mechanism of increased recombination frequency brought about by polyploidy associated effects.更多还原