【作者】 郭冬；

【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， 2008， 博士

【摘要】 突变体在水稻功能基因组研究中具有重要的作用,创造基因功能缺失突变体是研究目的基因功能的最直观和有效的途径之一。T-DNA侧翼序列的分离对水稻T-DNA插入突变体库的应用至关重要。一方面,我们可以根据大量的T-DNA侧翼序列来分析T-DNA在水稻基因组中的分布规律,进而明晰农杆菌介导的T-DNA转化机制;另一方面,根据侧翼序列提供的基因信息,我们可以广泛地开展反向遗传学的研究。本研究中,以本室T-DNA插入突变体库为材料,采用PCR扩增对突变体库阳性情况进行了检测。利用TAIL-PCR技术成功分离T-DNA侧翼序列7,333。本研究共收集来自全世界水稻T-DNA侧翼序列144,427条,Tos17侧翼序列22,432条,比较了它们在水稻基因组中的分布规律。通过对T-DNA插入位点序列的分析,揭示了插入位点侧翼序列DNA的物理特性和碱基组成特征。采用正向和反向遗传学的方法,利用已有的突变体库资源,鉴定了2个白化苗突变体,并具体分析由T-DNA插入而产生白化苗性状的基因OsRNase Z的功能。主要研究结果如下:1.以T-DNA上GAL4/VP16基因区段为标记,对15,535个转基因植株DNA进行PCR扩增阳性检测,扩增结果表明13,080个为PCR阳性,阳性率约88.9%。2.利用TAIL-PCR的方法单独分离侧翼序列73,33条,其中T-DNA或载体骨架序列3,343条,水稻基因组序列3,215条,能够在水稻基因组上精确定位序列(E-value＜10^-5)2361条。同时我们还收集全世界其他研究机构分离的T-DNA和Tos17侧翼序列,用于分析的T-DNA侧翼序列共144,427条,Tos17侧翼序列22,432条。3.对所有收集的侧翼序列分析显示:无论T-DNA和Tos17在染色体上的分布数量还是分布密度都偏向插入到水稻中较大的染色体上,且插入密度和染色体大小高度相关。在同一染色体上,T-DNA倾向于插入到远离着丝粒区域,且分布数量与着丝粒的距离明显正相关;Tos17在染色体两端分布较多,在着丝粒附近和染色体中部分布较少。T-DNA在染色体上的分布和cDNA的分布显著相关,Tos17中不存在这一现象。4.T-DNA和Tos17在水稻基因区间的分布显示,无论T-DNA或Tos17都极度偏向插入基因区,而极度的不偏向插入转座子相关基因中;T-DNA在基因间隔区的插入比较均一,基本不存在偏爱,而Tos17极度不偏向插入基因间隔区。在基因区域中,T-DNA和Tos17偏爱插入基因编码区而偏爱不插入基因调控区;在编码序列中,T-DNA和Tos17偏爱插入外显子区域,而不偏爱插入内含子区域。相对基因间隔区而言,T-DNA和Tos17更偏爱插入到基因编码区。5.我们还对T-DNA和Tos17插入的功能基因进行了分析。在转座子相关基因中,Tos17在在转座子相关基因的插入要相对集中,可能存在偏爱;而T-DNA的插入比较均一,不存在插入热点基因。在转座子和反转座子相关基因中,Tos17更偏爱插入反转座子相关基因,T-DNA对两者没有偏爱。对T-DNA和Tos17插入的具体基因分析,它们可能存在相同的插入热点基因。在不同功能基因中,T-DNA和Tos17共同偏向于插入“Motor”,“Signal transducer”,“Transporter”和“Transcription regulator”等4类功能基因,而不偏向插入“Binding”和“Nutrientreservoir”等2类基因,除此之外,T-DNA还偏向插入“Structural molecule”和“Translation regulator”两类功能基因,不偏向插入“Catalytic”类基因中,而Tos17在这3类功能基因中呈随机分布状态。6.对T-DNA插入位点前后各1 kb的序列（ISNS）弯曲度平均值分析结果显示:在插入位点-200 bp和200 bp的序列区间弯曲度值出现了一个明显的波形变化,在—200和200位置为波谷。最大值波峰分别出现在约-10和10的位置,且在0,即T-DNA插入位点出现陡然的下降出现一个相对低值。其他研究单位提取的ISNS中我们同样观察到了同样的现象,而随机提取的序列的弯曲度值表现为杂乱的曲线。7.对ISNS和随机提取序列的GC skew和TA skew分析发现:两组ISNS的GC与TA skews之间呈显著的负相关。GC与TA skew曲线在插入位点位置出现交叉,且值在插入位点都约为0,表明在插入位点G与C,T与A的含量均等;在-800到0的这段序列里,GC skew值表现为正值,且在-200到-100位置达到最大值,表现为G与C在单链上的极度不均衡分布,与之对应的是TA skew值为负值,但也是在同样位置达到最小值,表现为T与A在单链上的极度不均衡分布;在0-800区域内我们也发现了对应的结果,GC skew值表现为负值,TA skew值表现为正值,且在200到100位置碱基的分布出现极度的不均衡情况。在对照序列的结果中并没有发现这些特征。8.通过对1994个T-DNA插入突变体家系的T₁代苗期筛选鉴定,获得2个T-DNA侧翼序列和白化苗性状共分离白化苗突变家系03211EV57和03211DH29。9.在03211EV57家系中,T-DNA插入位点在水稻第5染色体上编码含蛋白激酶结构域的表达蛋白基因的第1个内含子里,在TIGR中的编号为LOC_Os05g47770,预测基因全长6,339 bp,CDS总长2,853 bp,由6个外显子和5个内含子组成,编码一个包含951个氨基酸的蛋白,无全长cDNA信息支持。10.在03211DH29家系中,T-DNA插入位点在水稻第9染色体上编码RNase Z基因的第Ⅶ外显子的3′端。在TIGR中的编号为LOC_Os09g30466,基因全长3,066bp。KOME提供了该基因的全长cDNA信息（AK070747）:cDNA的总长度为1,352bp,由8个外显子和7个内含子组成,编码一个包含365个氨基酸的蛋白,编码蛋白的氨基酸序列与拟南芥RNase Z有97%的相似性,因此将该蛋白命名为OsRNase Z。11.对03211DH29白化苗突变体叶片透射电镜观察发现,叶绿体数目明显减少,并且叶绿体变形,内囊体膜消失,白化苗突变表型的叶绿体中出现前片层体,但不能形成内囊体。叶绿素含量测定表明,在白化苗中叶绿素的合成系统完全丧失。12.全生育期表达谱芯片和RT-PCR结果显示OsRNase Z基因与叶绿素的合成密切相关。OsRNase Z基因的表达和组织的叶绿素含量存在十分明显的正相关,叶绿素含量高的组织基因表达水平就高,叶绿素含量低的组织基因的表达水平就低或者RT-PCR方法未检测到基因表达。13.对中花11中的OsRNase Z基因利用双链RNA进行抑制表达,30%的转化苗出现白化表型,RT-PCR结果显示,在白化苗中OsRNase Z基因的表达基本被抑制。14.P_Ubi:⊿OsRNase Z-GFP融合表达载体转化拟南芥原生质体,瞬时表达结果表明OsRNase Z基因编码的蛋白定位在叶绿体中。更多还原

【Abstract】 Mutants play an important role in rice functional genomic research.Generating loss-of-function mutant is one of the most straightforward and efficient way to study gene function.The isolation of the flanking sequence tags（FSTs） is crucial in the application of rice T-DNA insertional mutant library.First,based on the vast FST data,we can have a deep insight into the T-DNA distribution pattern in rice genome and then elucidate the Agrobacterium-mediated T-DNA transformation mechanism;on the other hand,we can carry out reverse genetics studies for the corresponding genes according to the tagged gene information.In this study,we examined the transformation positive ratio of our insertional population by checking the T-DNA insertional positivity.TAIL-PCR method was employed to isolate T-DNA FSTs.144,427 T-DNA FSTs and 22,432 Tosl7 FSTs were collected from worldwide databases for analyzing distribution pattern in the rice chromosomes in this study.We revealed the DNA physical property and base composition features of the insertion sites through analyzing T-DNA insertion site nearby sequences. We screened 1,994 T₁-generation T-DNA insertion lines and obtained some leaf color mutations in seeding stage.We obtained two albinos and study OsRNase Z gene function that interrupted by T-DNA.The detail results were summarized as follows:1.The transformation positive percentage of 15,535 transformants was checked by using GAL4/VP16 fragment in T-DNA as a PCR marker,13,080（88.9%） out of them showed to be PCR positive.2.Employing the TAIL-PCR method,I independently isolated total 7,333 FSTs, including 3,343 T-DNA or vector bone sequences,5,110 rice genome sequences. 2361 of 3215 FSTs can be well mapped into the rice genome(E-value＜10^-5).A total of 144,427 T-DNA FSTs and 22,432 Tosl7 FSTs in this study were collected from several major institutes in the world.3.T-DNA and Tosl7 insertions were biased towards large chromosomes,not only in the absolute number of insertions but also in the relative density,and the insertion density is highly correlated with the chromosome size.Within chromosomes,T-DNA insertions occurred more densely in the distal ends,and less densely in the centromeric regions and the insertion number is significantly correlated with the distances to centromere;Tosl7 insertions mainly distribute in the distal ends of chromosome,and a few distribute in centromeric region and the middle part of chromosome.The number of T-DNA insertions in the chromosome is significantly correlated with the cDNA number in corresponding region,which did not observed in Tosl7 insertions.4.The distribution of T-DNA and Tosl7 insertions in different regions of genome show that T-DNA and Tosl7 insertions strongly disfavored transposable element （TE）-related sequences and favored genic sequences;Tosl7 insertions strongly disfavored intergenic but T-DNA showed a random distribution in this region.In the genic region,T-DNA and Tosl7 prefer to insert in the coding sequences,but not the regulating sequences;particularly,T-DNA and Tosl 7 prefer exons,rather than introns, in the coding sequence.Compared with the intergenic region,T-DNA and Tosl7 insert favorably in the genic region.5.We also analyzed the function genes which inserted by T-DNA and Tosl7.In TE-related genes,the distributions of Tosl7 insertions were relatively concentrated, insert hot spot may be there,but T-DNA insertions were relatively uniform and no insert hot spot were observed.Tosl7 insertions prefer retrotransposon genes than transposon genes,T-DNA have no insertional bias between them.T-DNA and Tosl7 insertions bias among the various classes of functional genes were observed: preferentially occurred in "Motor","Signal transducer","Transporter" and "Transcription regulator",but preferentially not occurred in "Binding" and "Nutrient reservoir".In addition,T-DNA also biased "Structural molecule" and "Translation regulator",not biased insert "Catalytic",but Tosl7 showed a random distribution pattern in the 3 gene classes.6.In ISNS bendability analyze result,the random CK showed an irregular curve.But in our ISNS,elevated bendability was observed at positions from -200 to 200 bp and the bendability peak is symmetric in this region,with the highest points at both -10 and 10 bp from the insertion sites.There was an abrupt drop in the bendability value at the insertion sites.A similar result was also observed in ISNS from other research groups.7.The GC skew and TA skew calculation exhibited several features in both ISNS from our lab and other groups:The GC and TA skews appeared to be inversely correlated; The two curves crossed each other at the insertion sites（point 0） at which both GC and TA skews were equal to 0,indicating complete symmetry at this point;From the insertion site to -800 bp upstream,the GC skew was positive and reached a plateau in approximately the region from -300 to -100 bp,indicating a extremely uneven distribution of G and C in the strand analyzed,whereas the TA skew was negative and formed a valley in a similar position in approximately the region from -300 to -100 bp.The reverse was the case from the insertion sites to 800 bp downstream, whereas no such features were observed in the 2000 random sequences.8.We screened 1,994 T₁-generation T-DNA insertion lines,observed some color mutations in seeding stage.We obtained two albino lines 03Z11EV57 and 03Z11DH29 which co-segregation with T-DNA insertion.9.The albino line 03Z11EV57 which harbored a T-DNA insertion in the first intron of a gene in chromosome 5.The gene codes a protein kinase domail cotaining protein, which ID is LOC_Os05g47770 in TIGR,length 6,339 bp and CDS 2,853 bp contain 6 exons and 5 introns,encoding a 951 AA protein,but no supported by FLcDNA. 10.Line 03Z11DH29 was selected for further detailed study,which harbored a T-DNA insertion in the seventh exon of OsRNase Z in chromosome 9.The ID of OsRNase Z in TIGR is LOC_Os09g30466 with a length of 3066bp.KOME provided the full-length cDNA information（AK070747）:1,352bp in length,containes 8 exons and 7 introns,encoding a 365AA protein,which has 97%similarity with RNase Z of Arabidopsis,so we named OsRNase Z.11.We investigated the albino mutant and normal green seedlings by transmission electron microscope,which showed that the chloroplast of albino mutants had changed remarkably compared with normal green seedlings:the number of chloroplast decreased,the shape distorted,the thylakoid membrane disappeared and prolamellar body formed instead of thylakoid.At the same time,we measured the chlorophyll contents,the result suggested that the pathway of chlorophyll biosynthesis have been blocked in albino.12.Microarray based expression profile and RT-PCR data showed that OsRNase Z has a close relationship with the synthesis of chlorophyll.The expression level of OsRNase Z was positively correlated with the chlorophyll content,In the tissues with high content chlorophyll,the expression of OsRNase Z was high;correspondingly,in the tissues with low content chlorophyll,the expression of OsRNase Z was low or otherwise couldn’t be detected.13.RNA interference（RNAi） technique was used to suppress OsRNase Z expression in the rice variety Zhonghua 11.About 30 percent of transgenic seedlings appear albino phenotype.A part of these mutant plants were used to analyze the expression of OsRNase Z gene.RT-PCR result revealed that the expression of OsRNase Z was suppressed significantly in the chlorophyll mutant plants.14.The construction of P_Ubi:△OsRNase Z-GFP was transformed into Arabidopsis protoplast by PEG-mediated transformation method.Observation of protoplasts under a confocal microscope showed that OsRNase Z protein is exclusively localized in chloroplast.更多还原