水稻卷叶基因RL14和叶绿体发育基因TLC1的图位克隆与功能分析
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摘要
1.RL14基因的图位克隆与功能分析
     叶片是植物接受和转化太阳能的主要器官。水稻等高密度栽培的农作物,适度卷叶对改善中下部叶片受光条件、延缓叶片衰老、延长叶片功能期,从而提高产量具有一定的作用。袁隆平等育种家也提出,叶片适度卷曲是水稻理想株型要素之一。在长期的育种实践和基础研究过程中,也积累一部分性状优良的卷叶材料和突变体,但是对其卷叶形成的生理和分子遗传机理的研究还相对滞后。本研究在EMS(甲基磺酸乙酯)诱变缙恢10号,所得突变体库中筛选获得2个卷叶等位突变体,命名为rolling leaf14-1(rll4-1)、rolling leaf14-2(rl14-2)。因其卷叶形态相似,所以本研究以缙恢10号为野生型对照,对突变体rl14-1农艺性状、株型特征进行了分析。利用扫描电镜、组织切片等仪器和技术,对rl14-1突变体进行了细胞学、生理学特征研究。以分子标记为基础完成了RL14基因的图位克隆,并结合qRTPCR技术、mRNA组织原位杂交技术对RL14基因的时空表达特征进行了分析。主要结果如下:
     1.1 rl14突变体特征
     rl14-1突变体全生育期表现叶片卷曲性状。抽穗期功能叶卷曲度介于40%-50%之间,突变体功能叶叶基角显著小于野生型对照,表现叶片卷曲、挺直。突变体株高低于野生型,而千粒重显著高于野生型对照。突变体叶片蒸腾速率、气孔导度均显著低于野生型对照,而水分利用效率显著高于野生型对照。突变体叶片气孔复合体面积显著低于野生型。对不同时期突变体叶片石蜡切片分析表明,突变体叶片泡状细胞形态异常呈萎缩失水状。
     1.2图位克隆
     rl14突变体突变性状受隐性单基因控制,初步定位将RL14定位在第10染色体SSR标记RM3123和RM1162之间遗传距离分别为7.5cM和9.6cM。精细定位将RL14定位在标记SID2与SW24之间,物理距离24.5kb区域内。对定位区间内的注释基因测序分析发现,突变体r114-1为启动子序列突变,突变导致基因表达量显著下降。突变体r114-2为基因编码序列突变,突变导致RL14蛋白保守区域氨基酸序列发生异亮氨酸(I)到苏氨酸(T)的替换。转基因功能互补验证试验表明转基因植株叶片平展,泡状细胞形态正常。蛋白质序列分析发现RL14基因编码2OG-Fe(II)氧化酶蛋白,属2OG-Fe(II)氧化酶基因家族。RL14蛋白在细胞核、细胞质中均有分布。组织特异性分析表明RL14基因在根、全展叶、叶鞘中表达量较高,在未抽叶、茎及幼穗中微量表达。mRNA组织原位杂交分析表明在尚未抽出的幼叶中RL14基因仅在维管束两侧厚壁细胞中表达,而在全展叶片中RL14基因的表达扩展到整个叶肉细胞中,表达时期与细胞次生壁的形成时期相符。
     1.3相关基因表达分析及纤维素木质素含量测定
     细胞次生壁形成相关转录因子、纤维素和木质素合成关键基因表达量在rl14-1叶片中均显著变化;突变体叶片纤维素含量显著上升,木质素含量显著下降,进一步证明RL14基因影响厚壁细胞及叶肉细胞次生壁的形成。结合rl14-1突变体所表现的生理和细胞学特征,我们推测细胞次生壁组成和结构的变化影响从叶片维管束到泡状细胞的水分运输导致叶片卷曲。
     2温敏性黄绿叶突变体tlc1的基因克隆和功能分析
     叶绿体是植物特有的、称为质体的细胞器家族中的一员。叶绿体也像线粒体,有自己的DNA、RNA和核糖体,表现出相当程度的自主性。但是许多关键的叶绿体组分蛋白仍由细胞核基因编码,所以叶绿体的发育和功能发挥,需要核基因和叶绿体编码基因相协调。叶色突变常直接或间接与叶绿体发育相关,因此是研究叶绿体发育的重要材料。本研究所在水稻EMS突变体库中,发现一个温度敏感型黄叶突变体,命名为temperature-impressible leaf color1(tlcl)。在不同栽培条件下,对其表型特征进行了跟踪观察,对突变体光合色素含量,叶绿体特征等进行了观测分析。同时对突变基因进行了遗传分析和图位克隆。主要结果如下
     2.1突变体tlc1表型及生理特征
     大田栽培条件下tlc1突变体叶色表现为黄、绿相间。20℃、30℃恒温条件下,突变体叶片分别表现为黄色和淡绿色。20℃突变体叶绿素含量急剧下降,H202大量累积,叶绿体分化受阻,呈质体状态。PSⅡ(光系统11)各项参数,类囊体膜蛋白含量均显著下降。
     2.2基因精细定位及候选基因分析
     精细定位将TLC1基因定位于第3染色体SSR标记RM15330与Y10之间,物理距离68.4Kb范围内,与SSR标记Y7共分离。对定位区间内所包含的13个编码基因测序分析,未在突变体与野生型间发现编码序列差异。对13个基因在不同温度下表达量进行qRTPCR分析,发现突变体中TLC1基因的表达量在低温下显著低于野生型。结合TLC1基因表达模式和突变体叶绿体发育特征将候选基因确定为TLC1。
     2.3叶绿体发育、叶绿素合成、PSⅠ、PSⅡ相关基因表达分析
     对不同栽培条件下叶绿体发育、叶绿素合成、PSⅠ、PSⅡ相关基因表达分析发现,TLC1基因突变显著影响叶绿体编码基因的表达。特别是在20℃条件下,叶绿体编码基因表达量不足野生型50%。综上所述,我们认为TLC1基因具有调控叶绿体编码基因表达,促进叶绿体分化的功能。突变导致TLC1基因自身表达量降低,是造成tlc1突变体,温度敏感表型的主要原因。但是TLC1基因突变的遗传机理,和对叶绿体编码基因的调控途径,有待更深入的研究。
1. RL14 gene map base clone and functional research
     As an important agronomic trait, leaf rolling in rice(Oryza sativa L.) has attracted much attention from plant biologists and breeders. However, the relevant molecular mechanism for some kind of leaf rolling mutant remains unclear. Here, we use histological analysis, SEM and other basic method to research rl14-1 mutants various characteristic and map-based cloning of the gene RL14, and through qRT-PCR, in situ hybridization analysis RL14 genes expression pattern.
     1.1. Phenotypic characterization of rl14-1 mutants
     Histological analysis found that rl14-1 mutant plants had incurved leaves due to the shrinkage of bulliform cells on the adaxial side. Moreover, rl14-1 mutant plants displayed smaller stomatal complexes and decreased transpiration rates, compared with the wild type.
     1.2. Map-based cloning and expression pattern analysis of RL14
     The RL14 locus was localized further to a 24.5-kb region between markers SID2 and SW24. Amplification of relevant DNA fragments and sequence comparison revealed that rl14-1 and rl14-2 resulted from a single base deletion in the 517 bp upstream of the start codon and a single nucleotide mutation at codon 190 (T/C) in the second exon, respectively. The identity of rl14 was subsequently confirmed by genetic complementation experiments. The shape of leaves and bulliform were all restored to levels of wild-type plants upon transformation with the RL14 gene.Our results reveal RL14 was transcribed in sclerenchymatous cells in leaves that remained wrapped inside the sheath. In mature leaves, RL14 accumulated mainly in the mesophyll cells that surround the vasculature.
     1.3. RL14 affected formation of the secondary cell wall
     Expression of genes related to secondary cell wall formation was affected in rl14-1 mutants, cellulose and lignin content was altered in rl14-1 leaves. These results reveal that the RL14 gene affects water transport in leaves by affecting the composition of the secondary cell wall. This change in water transport results to water deficiency, which is the major reason for the abnormal shape of the bulliform cells.
     2. TLC1 gene map base clone and functional research
     Chloroplasts are essential for the unique photoautotrophic and sessile existence of higher plants. Chloroplasts account for 50% of the total soluble protein in leaves, and these proteins are encoded by both nuclear and chloroplast genomes. Plastid development from proplastids to photosynthetically active chloroplasts is one of the most important metabolic processes during plant growth and is coordinately regulated by both plastid and nuclear genes. A number of chlorophyll (Ch1) and chloroplast associated mutations that affect leaf coloration have been identified and are referred to as virescent (v), stripe (st), albino, chlorina, zebra, and yellow variegated depending on their diverse phenotypes. In this study, we describe a leaf color mutant temperature-impressible leaf color1 (tlcl)(tlc1) from the EMS mutant library. The mutant plant exhibits a yellow-green leaf phenotype, decreased level of Chl, and delayed chloroplast development at 20℃. Fine mapped tlcl locus and identified tlcl gene by its expression pattern.
     2.1. Phenotypic characterization of tlcl mutants
     under paddy field conditions, tlcl plants largely exhibit a yellow and pale green leaves, while it displayed yellow at 20℃and pale at 30℃.The chlorophyll content decreased but the H2O2 content increased in tlcl mutant leaves. TEM observation found the development of chloroplast in mutant leaves was delayed. Photosynthetic capacity was significantly lower in tlcl mutant leaves; assembly of photosynthetic complexes was impaired in tlcl mutant.
     2.2. Fine map of tlcl locus and tlcl mutation analysis.
     Fine mapped the tlcl locus between RM1533O and Y10 from a segregating population of 560 recessive individuals. The tlcl gene was narrowed down to a 68.4kb genomic DNA region between the SSR markers RM15330 and Y10, and cosegregated with Y7. There were 13 hypothetical and expressed genes in this region that were already annotated and we identified tlcl candidate gene by its expression trait.
     2.3. Transcription analysis of chloroplast development-related genes in mutant plants.
     We analyze the expression of genes associated with Chl biosynthesis, photosynthesis, or chloroplast development by qRTPCR. The result show transcript levels of chloroplast code gene were down regulated in mutant.Together we conclude TLC1 gene functioned for chloroplast code gene expression and chloroplast differentiation and development.
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