【作者】 李勇；

【导师】 陈国平；

【作者基本信息】 重庆大学 ， 微生物学， 2009， 硕士

【摘要】 红菜苔在我国南方具有广泛的种植面积、已有近千年的栽培历史。与其它属于冬性一年生蔬菜作物的芸苔属植物（如,白菜、甘蓝等）不同,红菜苔不需要春化就可以较早的开花。红菜苔与白菜具有很多相似的生理、遗传特性,是白菜的一个变种。但与白菜相比,红菜苔对春化需求却表现出巨大的差异。本课题的研究是以天然早花突变体生理、分子生物学特性为基础,重点对控制植物开花的关键基因FLC进行研究。FLC是一个MADS-box转录因子,受春化作用的抑制,通过抑制下游开花相关基因的表达抑制植物开花。FLC低水平表达与功能的缺失是导致拟南芥、白菜等春化需要植物早花的主要原因。我们以红菜苔为研究材料,对红菜苔早花突变的分子机理进行了深入的研究,并对一些春化作用相关基因做了相关研究。主要研究结果如下:（1）在红菜苔中克隆出BrpFLC1的两种不同的剪切产物,分别命名为BrpFLC1-1和BrpFLC1-2是。在BrpFLC1-1中,第六个内含子没有被完全剪切掉,导致BrpFLC1-1编码的氨基酸提前终止。在BrpFLC1-2中,由于第六个外显子被剪切掉,导致BrpFLC1-2编码的氨基酸缺失14个氨基酸。推测这两种剪切产物编码的氨基酸都是没有功能的。（2）从红菜苔中克隆了另外的两个FLC的同源基因,分别命名为BrpFLC2和BrpFLC3,分别包含一个591 bp和618 bp的开放阅读框。BrpFLC2、BrpFLC3的编码区分别编码196、197个氨基酸残基,分子量分别21.91和21.67kD,等电点分别为8.84和9.08。BrpFLC2、BrpFLC3与拟南芥中的AtFLC（NP₁96576）分别具有84%, 82%的同源性。从红菜苔中克隆的FLC基因与大白菜中的FLC具有很高的同源性。红菜苔FLC与大白菜的FLC基因相比,只有几个核苷酸的差异。（3）对BrpFLC2和BrpFLC3基因核苷酸序列及其编码的氨基酸残基序列进行生物信息学预测发现, BrpFLC2、BrpFLC3编码的蛋白具有MADS-box转录因子的结构特性,均有多个最有可能被磷酸化的位点,没有发现可能的O-糖基化位。BrpFLC2、BrpFLC3基因编码的蛋白与拟南芥和大白菜FLC类似都具有跨膜结构,二级结构预测显示BrpFLC具有典型的MADS-box蛋白的二级结构, BrpFLC2、BrpFLC3主要定位于细胞核内作为转录因子参与基因的表达调控。（4） BrpFLC基因的表达模式研究,BrpFLC1、BrpFLC2在红菜苔根、茎、叶、子叶中都有较高水平的表达。BrpFLC3在茎、叶、子叶具有较高水平的表达,在根中的表达较低。BrpFLC1、BrpFLC2、BrpFLC3的表达都受春化作用的抑制,15天的春化处理可以使BrpFLC表达都降到了很低的水平。春化处理之后,红菜苔中春化作用相关基因BrpSOC1和BrpVIN3的表达量升高,BrpFRI没有明显的变化。（5）对红菜苔开花时间的研究表明,红菜苔是天然早花突变体,比白菜具有较早的开花时间。红菜苔的成花转变不需要春化处理,但是红菜苔具有一定的春化响应,春化处理可以缩短其开花时间。（6） BrpFLC1剪切方式的改变是红菜苔早花的主要原因。与晚花型白菜相比,BrpFLC1剪切的两种没有功能的转录产物。推测这BrpFLC1剪切出无功能的BrpFLC1是红菜苔早花的主要原因。推测FLC1第六个内含子的第一个碱基G到A改变,可能导致FLC1的剪切紊乱,从而导致红菜苔早花。（7）对BrpFLC的启动子和内含子I的研究表明,BrpFLC2启动子与白菜BrFLC2启动子具有类似的长度,序列同源性很高。对BrpFLC2启动子进行生物信息学软件分析表明,BrpFLC2启动子包含了多个TATA-box, CAAT-box,以及其他顺式作用元件。BrpFLC1和BrpFLC2的内含子I分别与白菜BrFLC1和BrFLC2的内含子I具有类似的长度,序列同源性很高。没有发现BrpFLC基因的这些区域与红菜苔天然早花突变的原因有关。从红菜苔中克隆了FLC上游基因FRI的同源基因BrpFRI的起始密码子附近的一段区域,这一在拟南芥中经常发生突变导致早花的区域,在红菜苔中没有发生导致功能丧失的突变。（8）分析FLC在红菜苔和白菜中的表达模式发现,在红菜苔中BrpFLC具有较低水平的表达。红菜苔的早花突变与BrpFLC较低水平的表达具有一定的关系。（9）从红菜苔中克隆了VIN3的同源基因BrpVIN3的一段包括VIN3的PHD和FNIII区序列,并根据siRNA序列设计要求,构建了BrpVIN3基因的RNAi载体。为生产转基因蔬菜和研究芸苔属植物中VIN3的功能打下了基础。更多还原

【Abstract】 Purple Flowering Stalk （Brassica campestris ssp. chinensis L.var. utilis Tsen et Lee） is one of the most popular vegetable crops in the south of China, and has been cultivated for thousands of years. Unlike other Brassica species, such as Chinese cabbage which is a vernalization-responsive plant, Purple Flowering Stalk can flower early without vernalization. Purple Flowering Stalk which is a unique variety of Chinese cabbage has similar physiology character and genetic background with Chinese cabbage. But they greatly differ from requirement of vernalization and flowering time. Purple Flowering Stalk has a weak requirement of vernalization and flowering early. After reviewing the advances and achievements in research on physiology character and molecular biology of Purple Flowering Stalk which is a natural early flowering mutant, this research addressed the FLC gene which regulates flowering of plant. FLOWERING LOCUS C （FLC）, which encodes a MADS-box transcription factor acts as a repressor of the floral transition. Weak expression of FLC and its functional deletion results in early flowering of Arabidopsis and Chinese cabbage et al. which have vernalization responsive. We investigated the molecular mechanism of Purple Flowering Stalk flowering earlier and FLC homologous gene, BrpFLC, and other vernalization-related genes from Purple Flowering Stalk and obtained the main results as follows:（1） Two alternative splicing patterns, one with different parts of intron 6 retained and one with the entire exon 6 excluded from the transcript, were identified in Purple Flowering Stalk. These two alternative splicing patterns named BrpFLC1-1 and BrpFLC1-2 were confered having no function（2） Another FLC homologous genes cloned from Purple Flowering Stalk were named as BrpFLC2, BrpFLC3 respectively. They contain 91 bp, 618 bp ORF which encode 196, 197 amino-acid sequence with molecular mass of 21.91, 21.67kD and isoelectric point of 8.84, 9.08, respectively. According to the blast results in GenBank database ,the cDNA nucleotide sequence shares 84%, 82% identity with AtFLC（NP₁96576）of Arabidopsis . BrpFLCs of Purple Flowering Stalk have very high identity with that of Chinese cabbage.（3） Bioinformatics prediction on BrpFLC genes nucleotide and encoded amino acid residue sequence indicates that the encoded proteins are MADS-box transcription factor and possibly have multi phosphorylation sites, but no 3 O-glycosylation sites. These proteins have trans-membrane structures which were predicted in FLC of Arabidopsis and Chinese cabbage. BrpFLCs have similar secondary structure with most of MADS-box transcription factor. They may regulate expression of genes as transcription factor locate in a nucleolus.（4）Study of expression pattern of BrpFLC indicated that BrpFLC1、BrpFLC2 were found to be ubiquitously expressed in roots, stems, leaves and cotyledon of purple stem mustard. BrpFLC3 was found to be expressed highly in leaves and stems, leaves and cotyledon but low in roots. Reduced BrpFLC1、BrpFLC2 and BrpFLC3 expression were observed in Purple Flowering Stalk which was vernalized. The expression of BrpSOC1and BrpVIN3 increased after being vernalized, but there was no obviously change of expression of BrpFRI.（5） Study of flowering time of Purple Flowering Stalk indicated that Purple Flowering Stalk which flowers much earlier than Chinese cabbage was a natural early flowering mutant. Its transfer from vegetative growth to reproductive growth did not need vernalization, but it has vernalization responsive. Vernalization decreased the flowering time of Purple Flowering Stalk.（6）Three BrFLC1 alleles with alternative splicing patterns, including two with different parts of intron 6 retained and one with the entire exon 6 excluded from the transcript, were identified in addition to alleles with normal splicing. The analysis conferred that a G/A polymorphism at the 5’splice site in intron 6 of BrFLC1 is associated with splicing mutation in the BrFLC1 gene and flowering-time variation in Purple Flowering Stalk.（7） Upstream flanking region of 5’UTR of BrpFLC2 is similar with that of BrFLC2 in Chinese cabbage. It contains several TATA-box, CAAT-box and other cis-acting elements. Intron 1 of BrpFLC1 and BrpFLC2 are similar with that of BrFLC1 and BrFLC2 in Chinese cabbage. We also detected the sequence around initiation codon of BrpFRI which is a most common mutation site inducing early flowering in Arabidopsis. None of these regions we detected caused Purple Flowering Stalk flowering early.（8） Comparing with Chinese cabbage, BrpFLC1、BrpFLC2 and BrpFLC3 have weak expression in Purple Flowering Stalk. The weak expression of BrpFLC may cause Purple Flowering Stalk flowering early to some extent.（9）A fragment of VIN3 homologous gene which contains the PHD and FNIII region cloned from Purple Flowering Stalk was named as BrpVIN3. According to siRNA sequence design principles, a RNAi expression vector of targeting gene BrpVIN3 was constructed and will be transformed into Chinese cabbage. This research provides the foundation for further study on the function of VIN3 in Brassica species.更多还原