【作者】 李才顺；

【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， 2009， 博士

【摘要】 改变基因的表达模式是一种有效的基因功能研究策略,可以帮助研究者发现特定基因的未知功能以及时空效应。本研究采用的GAL4/UAS异位表达系统是一种成熟的改变基因表达模式的双因子系统,通过目标系与特异表达模式系的杂交,可以实现改变目的基因的时空表达谱。本室构建的基于GAL4/UAS系统的水稻Enhancer Trap系,既可以作为筛选异位表达系统模式系的原始材料,通过对其报告基因表达模式的观测,构建报告基因表达谱；又可以作为插入突变体库进行突变表型的筛选以及相应的基因功能分析。本研究所得到的模式系以及用作水稻抽穗期基因研究的rid1突变体均来源于这些Enhancer Trap系。本研究的主要结果如下：1.将吴昌银博士改造的pEGFP载体用于构建水稻Enhancer Trap突变体库,统计数据显示引入该载体后,突变体库平均分化效率为57.35%,对其中4,416份样品检测所得阳性率为91.21%,该载体用于在水稻品种中花11号中构建突变体库是可行的。2.从用pEGFP载体构建的Enhancer Trap系中选取4,416个单株,单独移栽并进行PCR阳性检测,然后对其中的阳性单株进行全生育期不同部位(不含根)报告基因表达观测,构建了T0代报告基因表达谱,T0代报告基因表达频率约为60%。3.从T0代特异表达家系中,选取了140份特异表达材料进行T1代全生育期水培,观测不同时期不同部位(包含根)GFP表达情况。观测结果显示T1报告基因表达情况相对于T0代出现丢失现象,而且不同部位丢失比例不一样。4.T0代及T1代所得的特异表达材料,经过多次复筛及选择,得到了一批模式系候选材料,通过对这些候选材料的进一步复筛得到了45个特异表达模式系,这些家系的GFP表达部位涵盖了水稻的主要器官。5.对来自45个特异表达模式系的143个株系进行了T-DNA插入拷贝数检测,检测结果显示虽然所得的特异表达模式中报告基因的表达模式特异并稳定遗传,但是没有出现明显的拷贝数分离现象,大多数株系仍然存在多拷贝,没有按照预期分离出大量单拷贝。6.筛选所得的部分特异表达模式系材料,与目标系植株(携带UAS控制下的GUS报告基因和目标基因的pGOC17载体转化水稻品种中花11号)进行杂交,对杂交后代的报告基因表达观测显示,本研究所的模式系可以成功应用于GAL4/UAS异位表达系统。7.从所得的特异表达模式系中,选取了6个在花器官特异表达的家系,进行幼穗发育过程的GFP表达观测。观测结果显示,在不同模式系的穗发育过程中,GFP报告基因表现出不同的表达模式,说明了被标记基因在水稻穗发育过程中的呈现不同的表达谱。8.针对68个家系的表达谱材料,分离了106条T-DNA插入位点左、右端的侧翼序列,并对这些侧翼序列进行了分析,从中获得13个与报告基因表达模式一致的基因序列。9.从构建模式系的材料中,选取了578个家系观测其正常田间种植条件下突变表型,从中筛选得到了177份有明显突变表型的材料,并找到了31个报告基因表达与突变性状相关的家系。10.对水稻“不抽穗”突变体rid1进行了详尽的表型描述。在正常植株抽穗成熟以后,rid1突变体继续保持营养生长状态,甚至到了播种后600 d还是无法抽穗。叶片数统计结果显示在长、短日照条件下,野生型植抽穗以后,突变体植株在观测期间平均每10 d产生1片新的叶片。rid1突变体可以拔节,节数也比野生型植株多,但是电镜观测结果显示其SAM一直处于营养生长状态,无法向生殖生长状态转换。rid1突变体的“不抽穗”突变性状与SAM由营养生长向生殖生长的转换受阻直接相关。11.65%以上的rid1互补阳性植株可以恢复抽穗表型,但是它们的抽穗期存在极大的差异,单株间差异甚至达到了111d。推测原因是由于互补片段插入水稻基因组中的位置不同,导致RID1基因的表达模式发生了改变,从而引起了抽穗期的改变。12. RID1::RID1:GFP材料报告基因表达观测结果显示RID1:GFP蛋白在幼叶及SAM中存在,而前期数据显示RID1 mRNA仅存在于幼叶中,结合已发表的文献推测原因为该基因表达量太低导致不同检测手段出现不同结果,需要进一步的实验来加以验证。13.通过杂交方式将rid1突变引入3个典型的籼稻和3个典型粳稻品种后,6个组合的杂交后代中都出现了“不抽穗”的突变性状,F2代植株有表型的分离,说明RID1基因在籼稻和粳稻品种中都起到了重要作用。14.超表达Hd3a和RFT1 (FTL3)的结果显示,单独超量表达Hd3a或者RFT1均可以使rid1突变体恢复抽穗表型并且表现出极早抽穗的现象,说明RID1基因的突变抑制了Hd3a和RFT1的表达与积累。更多还原

【Abstract】 The changing of gene expression pattern is a powerful tool for studying of gene function. This strategy can help researchers to know the temporal and spatial effects of the given genes, and discover their unknown functions. The GAL4/UAS two component ectopic expression system in this research is a recognized plan to chang the expression pattern of target genes, the tissue and time specific expression of the target gene can be put into practice by the genetic cross of pattern lines and target lines.The GAL4/UAS Enhancer Trap lines of our lab can be used as an original source of the pattern lines. A reporter gene expression profiling can be established by observing of the reporter gene expression. In the other hand, the traping lines can provide us a mutant library for studying of gene function. All of the the pattern lines and the rid1 mutant in this study come from the Enhancer Trap library. The main results are as follows:1. The introducing of pEGFP vector (modified by Dr. Wu Changyin) into the rice Enhancer Trap mutant library brings the differentiation rate to 57.35%. A positive detection of 4,416 lines shows the positive rate of those lines achieve to 91.21%. The pEGFP vector is fit for construction of mutant library in Zhonghua 11.2. A collection of 4,416 independent T0 plants from the pEGFP lines have been dissected for positive test and reporter gene expression pattern observation in different stages and tissues(without roots). The reporter gene expression ratio is about 60% in T0 plants.3. One hundred and forty specific expression T1 lines from T0 expression pattern library have been collected and grow in water culture pond for repoeter gene expression observation(within roots). The lost of the reporter gene expression in T1 generation compare to the T0 generation have been observed, and the rate of the lost is different around tissues.4. More selections have been done on T0 and T1 specific expression lines, and a number of undetermined pattern lines have been chosen.45 final pattern lines have been gathered from the undetermined lines, their expression patterns cover the main tissues of rice plants.5. Souther blot has been done in 143 strains from the 45 pattern lines for detecting the T-DNA copy number. The result shows that the copies of T-DNA have no distinctly segregation though the reporter gene expression patterns have stabilized by several generation’s selection. Multiple copy lines still remain and few single copy strains arised.6. Several pattern lines from our screening have been used to cross with the target line (pGOC17 vector containing GUS reporter gene and miR156 gene controlled by UAS transformed into Zhonghua 11). Ectopic expression effect has been observed in the offspring of this target line crossed with our pattern lines. Our pattern lines can be well used in the GAL4/UAS ectopic expression system.7. Six lines with flower specific reporter gene expression pattern have been collected and the reporter gene expression of those lines in panicle development has been observed. The difference of the reporter gene expression pattern in time and location shows the differentiation of targeted genes.8. One hundred and six flanking sequences have been isolated and analyzed from 68 Enhancer trap lines.13 of those sequences show the same direction of reporter gene and targeted gene expression.9. Five hundred and seventy eight enhancer trap lines have been observed in normal cultivation conditions, and 177 mutant lines have been collected. The relativity of the mutation and reporter gene expression pattern has been discussed and 31 relativ lines have been found.10. The phenotype of a "never-flowering" mutant rid1 has been described detailedly. rid1 mutant plants stayed in vegetative growth stage long after the harvest of wild type plant, even 600 days after sowing. The statistics of leaf numbers shows that the mutant plant produces one leaf within every 10 days after the heading of wild type plant. Elongation of the internode and more nodes can be obersived in rid1 plants. SEM analysis indicated that the SAM of ridl can not chang from vegetative growth to reproductive growth state, and the "never-flowering" phenotype of rid1 have a direct correlation with this reason.11. More than 65% of the positive complementary plants can recover the "never-flowering" phenotype of rid1 mutant with a widely distributed heading date. The difference between plants even reaches to 111 days. The possible reason for this condition is the differential expression of RID1 gene led by position effect. 12. GFP observation of RID1::RID1:GFP plants shows the presence of RID1:GFP protein in young leaf and SAM, but our pre-data shows that the RID] mRNA only appears in the young leaf. The possible reason is the low expression level of RID1 gene and different detection methods, but we need more evidence.13. The "never-flowering" phenotype could be led into other rice varieties by crossing with rid] heterozygous plants since the rid1 mutant plants can not flower. The phenotype separation can be seen in F2 plant, indicated that the RID1 gene play an important role in other rice varieties.14. Over-expression of Hd3a and RFT1(FTL3) can recover the mutant phenotype of rid1 mutant separately and bring a very early flowering phenotype to it. These phenomenons indicated that the mutation of RID] gene inhibit the expression and accumulation of Hd3a and RFT1.更多还原