【作者】 潘存红；

【导师】 潘学彪；

【作者基本信息】 扬州大学 ， 植物生物技术， 2007， 博士

【摘要】 水稻适度卷叶是超高产株型育种的重要形态指标之一,其作用已在高产和超高产育种中得到充分的体现。卷叶性状对叶片光合生理、群体生态效应及经济性状的影响的研究表明,适度卷叶对塑造个体良好的株型、改善生育后期群体质量、提高产量具有重要的作用。现已发现的水稻卷叶材料在叶片的卷曲程度上存在较大差异,多数在生产上的利用价值并不高。因此,筛选和鉴定具有育种利用价值的卷叶基因是水稻育种中的一项重要研究课题。另外,水稻已成为单子叶模式作物,水稻卷叶基因的功能研究对阐明植物叶片发育规律具有重要意义。本研究发现了一批卷叶突变体材料,并确定了一份与插入的T-DNA片段表现为共分离的材料;同时,对另一卷叶材料携带的高利用价值卷叶基因rl(t)进行了精细定位,并对候选基因的功能做了推测和初步的验证,主要研究结果如下:1.T-DNA标签是一种高通量的分离和克隆植物功能基因的方法,利用T-DNA标签获得插入位点的旁邻序列,根据旁邻序列所在基因组的位置,再结合突变体的表型能快速克隆水稻功能基因。通过对4416份T1代水稻T-DNA标签系的表型筛选,获得了12份卷叶突变体材料。用可扩增T-DNA特定序列的引物对卷叶材料所在的分离小区进行PCR扩增,从中挑选出突变体都能扩增出目标产物而部分表型正常株不能扩增出产物的小区。有3个小区(小区号为267、1199、1345)符合这样的要求。种植这3个小区的突变株和表型正常株后代,进行T2代共分离验证。共分离小区的标准:突变体小区表现纯合突变表型,且PCR检测都为阳性;表型分离小区正常表型与突变表型呈3:1分离,且突变体PCR检测为阳性。筛选出1个共分离小区(小区号为1345)。初步分析:1345小区的T0代为双拷贝T-DNA插入,这2个拷贝的T-DNA插入在T1代发生了分离,并在T2代鉴定出引起卷叶突变的单拷贝T-DNA插入突变体。用TAIL-PCR的方法对单拷贝突变进行侧翼序列的分离,但得到了假阳性的测序结果(详见第四章)。将用质粒拯救的方法再次分离侧翼序列。对T-DNA侧翼序列的分离方法和共分离验证进行了讨论。2.在对水稻卷叶基因rl(t)的精细定位过程中总结了一套简单实用的分子标记设计方法。水稻图位克隆中的亲本一般都具有籼粳差异,因此可根据粳稻日本晴和籼稻93-11的基因组序列差异设计分子标记。从IRGSP下载定位区间的日本晴BAC,在NCBI网站与93-11的基因组序列进行BLAST比对,搜寻存在于这两个基因组之间的插入-缺失多态性(InDel)和单核苷酸多态性(SNP),根据定位区间大小对标记间隔的要求(区间大于100 kb时间隔15 kb左右,区间在100 kb以内时间隔5-10kb),筛选InDel和SNP来设计分子标记,优先设计InDel标记。SNP可转化为CAPS、dCAPS和AS-PCR标记(具体设计方法见第五章)。本研究共设计47个标记,其中24个InDel标记和14个SNP标记在亲本中表现多态性,设计成功率为80.9%。对如何提高标记设计成功率进行了分析和讨论。3.对卷叶基因的研究和利用是水稻株型育种的重要组成部分。本论文研究的卷叶基因rl(t)具有较高的育种价值,本实验小组已对其进行了初步定位。在此基础上,发展了2个大分离群体对卷叶基因rl(t)进行了精细定位。定位策略:选择初步定位区间两侧的标记组成双引物PCR检测群体的标记基因型,对交换株再用区间内的标记进行检测,分析各交换株发生染色体重组的最小范围,结合表型确定最小定位区间。检测到2个交换株(101-8、418-1),其染色体重组位置位于卷叶基因rl(t)的两侧且距离最近,将卷叶基因rl(t)定位于标记P95053和P113.6之间11kb的范围内,此区间只有一个候选基因。这为克隆卷叶基因和研究其调控机理奠定了基础。对提高精细定位的效率进行了讨论。4.在水稻自动释义数据库(RAD)和水稻释义计划数据库(RAP-DB)中查询到rl(t)的11 kb的定位区间内只有1个预测基因,属于HD-GL2类转录因子,经与已克隆的HD-GL2类基因进行氨基酸序列比对,发现与ZmOCL1和ANL2的相似性分别达到了85%和60%。HD-GL2类基因在分生组织的L1层、叶原基、叶片、花等侧生器官的表层表达。根据日本晴BAC AP005885设计13对测序引物,对亲本QMX和其卷叶近等基因系NIL-rl(t)进行PCR扩增和测序,结果表明:在候选基因的3’ UTR存在1个单碱基替换。推测可能是由于3’ UTR的点突变引起候选基因mRNA的稳定性或翻译的变化而导致卷叶性状的出现。5.用RNAi的手段使目标基因在转录后沉默是验证基因功能的一种快速有效的方法。本研究在候选基因的不同部位设计了3个干涉片段,用BP反应构建入RNAi载体,将载体分别转化中花11,使目标基因沉默来验证其功能。更多还原

【Abstract】 Semi-rolled leaf is one of the most important morphological characters in plant breeding. Some high yield varieties or their hybrids were semi-rolled leaf cultivars. Studies on effects of leaf rolling on photosynthetic physiology, population ecology and economical traits showed that semi-rolled leaf had some upstanding effects, for instance making leaf erect, optimizing canopy light transmission, increasing effective leaf area per unit land and improving the quality of population in late growth stage. However, most of rolled leaf materials found in past research have different leaf rolling degrees and most of them could not be applied in rice breeding. Therefore, identifying more useful rolled-leaf genes is still an important goal for practical rice breeding. On the other hand, rice has become a model plant of monocots, so the function research for it on rolled leaf genes will provide good chances for elucidating leaf development mechanism in rice.Some rolled leaf mutants were obtained via screening the T-DNA inserted mutant pool and one of them was confirmed to be co-segregated with T-DNA. A rolled gene, rl(t), with high value for breeding, was fine mapped and its putative function was preliminarily analysed and identified.1. T-DNA tagging is a high throughput method for identifying and cloning novel genes. The sequence flanking the T-DNA insertion site could be obtained according to T-DNA tagging, then the gene tagged by T-DNA could be discovered based on the flanking sequence position in the rice genome. Twelve rolled leaf mutants were obtained via screening 4416 rice T1 tagged lines. These lines were tested by PCR using primers amplifying special T-DNA segment and 3 co-segregated lines were selected. Mutants were PCR positive and several plants with natural phenotype were PCR negative in the co-segregated lines. Seeds from natural plants and mutants grew out T2 generation. The co-segregation criterion of T2 generation was as follows, in the progeny blocks of mutants all plants had mutant phenotype and were PCR positive, moreover, in the progeny blocks of some narural plants, the ratio of narural plants to mutants fit 3:1 and all mutants of them were PCR positive. Line 1345 was co-segregaed according to this criterion. The results indicated line 1345 in T1 generation had two copy T-DNA insertion and one resulted in mutant phenotype. The mutant lines with single copy T-DNA were obtained. The flanking sequence from the line with targeted copy was amplied by TAIL-PCR, but its sequencing result was false positive (detail in chapter 4). We would separate the flanking sequence by plasmid rescue. The measure of separating flanking sequence and the co-segregation test were discussed.2. A applied method of designing molecular markers was brought forward in the process of fine mapping the rolled leaf rl(t) in rice. The parents of map-based cloning have japonica-indica diversity, so markers could be designed based on the sequence diversity between the genome of Nipponbare and that of 93-11. Nipponbare BAC in the mapping region were downloaded from IRGSP, and then these BAC were aligned with the 93-11 genome in NCBI. InDel and SNP could be discovered according to the alignment result. Some InDel and SNP were screened out to design markers (about 15 kb interval when region was longer than 100 kb, and 5 to 10 kb when shorter than 100 kb). SNP could be transferred into CAPS, dCAPS and AS-PCR markers (detail in chapter 5). Forty seven markers were designed in this research. Twenty four InDel markers and 14 SNP markers have polymorphism. How to improve the efficiency of marker design was discussed.3. Research and application for rolled leaf genes were important for plant type breeding in rice. The rolled gene rl(t) was valuable in rice breeding and primarily mapped in our past research. Two great segregated population were developed to fine map rl(t). The strategy of fine mapping was as follows. First, marker genotypes of population were tested by double primers PCR method. Two primers of this method should be flanked to the region covering rl(t). Then, recombinants were chosen out and analyzed by markers in the region. Finally, the co-segregation relationship between marker genotype and plant phenotype was analyzed in all recombinants, and the precise region was confirmed. Two recombinants were found, whose recombined sites were flanked to rl(t) and very close to each other. The result indicated that the rolled gene rl(t) was located between marker P95053 and P113.6. The physical distance from marker P95053 to P113.6 was 11 kb, and only one candidate gene lay in this region. All conclusion laid a foundation to clone rl(t) and research the regulation mechanism of rl(t). How to improve efficiency of fine mapping was discussed.4. Only one predicted gene in the 11 kb region was discovered from rice automated annotation database (RAD) and rice annotation project database (RAP-DB). The putative gene belonged to HD-GL2 type transcription factor. Alignment between the putative gene and HD-GL2 type genes indicated that the amino acid sequence of rl(t) showed high similarity to that of ZmOCL1 (85%) and ANL2 (60%). The HD-GL2 type genes showed L1-layer or protoderm-specifc expression in the SAM during the shoot and flower development. Thirteen pairs sequencing primers were designed according to Nipponbare BAC(AP005885) to amplify the DNA of QMX and NIL-rl(t). The PCR product were sequenced and the result indicated that a nucleotide substitution lay in the 3’ UTR of rl(t). It was suggested that the point mutant in 3’ UTR of rl(t) could result in change of mRNA stability or translation and induce rolled leaf.5. Post-transcriptional silencing of plant genes could identify the gene function effectively. The RNAi technique could efficiently silence genes in this way. Three interference segments were designed from different position of the candidate gene. Subsequently, they were constructed into RNAi vector by BP reaction. Finally, the constructed vectors were transformed into Zhonghua 11. The targeted gene would be silenced to identify function.更多还原