【作者】 代晓霞；

【导师】 张启发； 吴昌银；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 随着水稻基因组测序的完成,水稻基因组学研究已经步入功能基因组学时代。为了在基因组或系统水平上全面分析水稻基因的功能,最直接有效的方式是构建大量的突变群体,然后利用正向遗传学或反向遗传学策略进行基因功能的研究。对于T-DNA或转座子等插入型突变体的基因克隆,无论采用正向遗传学方法或采用反向遗传学方法,都必须分离插入位点的侧翼序列。通过建立侧翼序列数据库,人们可以很容易地检索到目的基因的突变体,从而进行基因克隆。本研究以本实验室的T-DNA插入突变体库为材料,完成了12,432份T0代转基因植株的PCR阳性检测工作,利用TAIL-PCR技术分离得到T-DNA插入位点侧翼序列5,082条。COBRA-like蛋白在植物纤维素合成和细胞扩张中具有重要的作用,本研究采用正向遗传学与反向遗传学相结合的方法,研究鉴定了水稻中两个COBRA-like基因OsBC1L4和OsBC1L5的生物学功能。本研究还克隆了一个影响水稻分蘖数的基因LTWl并进行了功能分析。本研究获得的主要研究结果如下：1.完成12,432份水稻T-DNA插入突变体库TO代转基因植株的PCR阳性检测工作,其中10,770个家系为T-DNA插入阳性植株,T-DNA阳性率约为86.6%。2.对T-DNA插入阳性的家系分离侧翼序列,共计得到T-DNA插入位点侧翼序列5,082条,其中的2,644条序列与水稻基因组匹配较好(E-value<10-5),基因组定位率约为52.0%。3.通过对分蘖突变表型的正向筛选,获得2个T-DNA侧翼序列和分蘖突变性状共分离的家系04Z11EM13 (OsBC1L4)和LTWl。4.对水稻OsBC1L家族成员进行了分子特征、染色体位置、系谱关系及表达分析,并通过反向遗传学策略,搜集了5个OsBC1L基因的插入家系,发现OsBC1L5基因参与水稻花粉的发育。5.在04Z11EM13家系中,T-DNA插入到OsBC1L4基因的第4个外显子中,造成了分蘖少、矮化的突变表型。通过共分离检测和互补实验克隆了OsBC1L4基因。经光学显微镜和扫描电镜观察,发现osbc1l4突变体具有不正常的细胞扩张、降低的次生壁厚度和增加的淀粉积累。通过测定纤维素和木质素含量,发现osbcll4突变体的纤维素含量下降了24%。采用RT-PCR和组织原位杂交技术分析了OsBC1L4基因的表达模式,发现该基因在薄壁组织和厚壁组织都有表达。通过进行OsBC1L4蛋白的亚细胞定位,发现该蛋白主要位于细胞壁和细胞膜上。通过表达相关性分析,发现OsBC1L4基因与初生壁形成相关的纤维素合成酶基因共表达。用real-time方法检测了纤维素合成相关基因和OsBC1L基因在osbc1l4突变体与野生型中的表达量,发现纤维素合成相关基因的表达在osbc1l4突变体中升高了,这可能反映了纤维素合成过程中的一种反馈机制,即通过升高其它纤维素合成相关基因的表达来弥补osbc1l4突变体中的纤维素损失。6.在OsBC1L5家系中,T-DNA插入到OsBC1L5基因的唯一的外显子中。该Tos17插入家系无纯合单株。通过杂合植株与野生型植株的正反杂交,发现osbc1l5基因型的雄配子缺陷。经体外花粉萌发实验,发现OsBC1L5基因可能参与水稻花粉的萌发。通过RNAi抑制实验,发现OsBC1L5基因对水稻花粉壁的发育可能也有影响。7.在LTWl家系中,T-DNA插入到LTWl基因的唯一的外显子中,造成了分蘖少、易枯萎的突变表型。通过共分离检测和互补实验克隆了LTWl基因。另外,在韩国POSTECH突变体库找到了一个LTWl基因的等位突变体,发现它具有与LTWl家系相同的突变表型,并且这种突变表型与在LTWl基因内的T-DNA插入也是共分离的。通过对韩国等位突变体(promoter trap line)的GUS染色,研究了LTW1基因的表达模式。更多还原

【Abstract】 With the finish of the rice genome sequencing project, the study on rice genomics has stepped into the era of functional genomics. In order to analyze the function of rice genes on the genome scale, a best way is to generate lots of mutants by which people can analyze the function of genes by forward genetics or reverse genetics strategies. For the cloning of T-DNA or Tos17 inserted genes, the isolation of flanking sequence is necessary. Using the flanking sequence database, people can find easily the mutant of interested gene and clone it.In this study, I finished the PCR positive test of 12,432 T0 T-DNA inserted lines in our mutant library. By TAIL-PCR, I isolated 5,082 flanking sequences of T-DNA. COBRA-like proteins play important roles in cellulose synthesis and cell expansion, so I studied the biological function of two COBRA-like genes, OsBC1L4 and OsBC1L5, by both the forward genetics and reverse genetics methods in this research. In addition, I also cloned a gene which affects the tiller number of rice and analyze its function.The detail results in this study were summarized as follows:1. I finished the PCR tests of 12,432 T0 T-DNA inserted lines and found 10,770 (86.6%) lines were T-DNA positive.2. Employing TAIL-PCR method, I isolated 5,082 flanking sequences of T-DNA,2,644 (52%) of which can be located onto the rice genome (E-value<10-5).3. By screening the tiller mutants in the field, we obtained two cosegregation lines, 04Z11EM13 (OsBC1L4) and LTW1.4. I analyzed the molecular characterization, chromosomal location, phylogenetic relationships and expression patterns of rice OsBC1L family members. In addition, I obtained 5 OsBC1L inserted lines and found that OsBC1L5 was involved in the development of rice pollen.5. In the 04Z11EM13 line, T-DNA was inserted into the fourth exon of OsBC1L4 gene and caused the mutant phenotype of less tiller and dwarf. We cloned this gene by cosegregation and complementation experiments. The mutation of OsBC1L4 results in abnormal cell expansion, decreased secondary cell wall thickness, and increased starch accumulation. By measuring the content of crystal cellulose, I found a 24% reduction of cellulose in osbc1l4 mutants. I analyzed the expression pattern of OsBC1L4 by RT-PCR and in situ hybridization and found that OsBC1L4 was expressed in both the sclerenchyma and parenchyma cells. OsBC1L4 protein is mainly located in the cell wall and plasma membrane. Correlation coefficient analysis indicated that the expression of OsBC1L4 was highly correlated to that of several primary wall-forming cellulose synthase genes (CESAs). Moreover, the expression level of several cellulose-related genes is increased in osbc1l4 mutants, which suggests that a feedback mechanism may exist to compensate for the reduced cellulose production in osbc1l4 mutants by increasing the expression of other cellulose-related genes during cellulose synthesis.6. In the OsBC1L5 line, T-DNA was inserted into the single exon of OsBC1L5 gene. This line has no homozygous plant of OsBC1L5 insertion. I performed reciprocal cross between OsBC1L5/osbcll5 heterozygous and the wild type plants and found the osbcll5 male gametophyte had a severe defect. By in vitro pollen germination, I found OsBC1L5 gene might be involved in the germination of rice pollen. I investigated the phenotype of OsBC1L5 RNAi plants and found OsBC1L5 gene might also affect the development of pollen wall in rice.7. In the LTW1 line, T-DNA was inserted into the single exon of LTW1 gene and caused a less tiller and wilted mutant phenotype. I cloned this gene by cosegregation and complementation experiments. In addition, I obtained an allelic mutant from POSTECH mutant library, which had the same mutant phenotype with LTW1 and also showed the cosegregation of the mutant phenotype and T-DNA insertion. By GUS staining of the POSTECH promoter trap line, I investigated the expression pattern of LTW1 gene.更多还原