【作者】 严松；

【导师】 顾铭洪； 李欣； 严长杰；

【作者基本信息】 扬州大学 ， 植物生物技术， 2008， 博士

【摘要】 叶片是植物进行光合作用和呼吸作用的主要器官,对植物的生命活动起着重要的作用,叶片的发育作为植物形态建成的一个重要方面,关系到植物株型和农业产量的形成。水稻是世界上最重要的粮食作物之一,卷叶性状是超高产育种的一项重要形态指标。因此,探明卷叶形成的分子机理,不仅能使我们更多地了解水稻的叶发育机制,而且能帮助我们通过分子设计改良株型。利用⁶⁰Co-γ射线辐射水稻品种中花11,在M₂代获得了三份卷叶突变体:M425、M429和M475,在日本晴的组培后代中得到一份卷叶突变体M7。其中,M425和M7表型相似,叶片都表现筒状卷曲,且籽粒畸形;M429和M475两者表型相似,叶片半卷,籽粒增多呈密穗。本研究对这些卷叶突变体进行了形态观察、突变性状的遗传分析、基因的等位性检测及图位克隆,主要研究结果如下:一、水稻卷叶基因RL9的定位和克隆1、对叶片细胞结构进行电镜观察,发现rl9-1突变体（M425）叶片小维管束远轴面厚壁细胞缺失,叶肉细胞取而代之,近轴面特有的茸毛在远轴面也有分布,而且叶绿体发育异常,基粒片层排列紊乱。2、将rl9-1突变体与中花11配制杂交,F₁植株叶片表现平展,F₂中正常株和突变株的分离比符合3:1,表明突变性状受单隐性基因控制。3、利用rl9-1突变体与籼稻品种Dular杂交产生的F₂和F₃群体,运用SSR、STS和CAPS标记,将RL9基因定位在第9染色体PAC克隆AP005904上22kb的区段上;此外,利用图位克隆的方法分离了一个与rl9-1等位的突变基因rl9-2,来自突变体M7。4、通过基因预测发现,在RL9基因所在的22kb内,仅有一个候选基因,预测编码类似MYB的蛋白;对该基因的测序结果表明,rl9-1在第一个外显子中发生了5个碱基的缺失,引起移码:rl9-2在第一个内含子和第二个外显子的剪切位点处发生了1个碱基的替换,使内含子不能被正确剪切,引起RL9阅读框改变,导致RL9功能的丧失。5、通过RT-PCR的方法确定了RL9基因位于AP005904上103061-108515处,编码区长1134bp,编码377个氨基酸,包含6个外显子和5个内含子。6、将来自日本晴BAC克隆的全长RL9基因导入rl9-1突变体后,rl9-1恢复成野生型表型,证实RL9是控制M425卷叶性状的基因。7、半定量RT-PCR结果表明,RL9基因在植株所有器官中均有表达,在根、叶片和穗中的表达量较高,在茎和叶鞘中表达量较低。8、对RL9所编码的氨基酸序列进行分析发现,RL9含有一个GARP结构域。9、将RL9蛋白序列与数据库中已命名的GARP超家族成员进行多序列比对,以邻位相连法构建进化树,发现RL9与拟南芥KANADI同源,根据KANADI在拟南芥中的功能,结合RL9功能丧失突变在水稻中的表现,推测RL9控制叶片的远轴发育。10、将RL9与GFP融合在洋葱表皮瞬时表达,融合蛋白定位在细胞核内,表明RL9是一个转录因子。二、卷叶基因RL10的定位和克隆1、对叶片细胞结构进行电镜观察,发现rl10-1突变体（M429）叶片部分小维管束远轴面的厚壁细胞缺失,并有少量类似C4植物的“花环”结构产生,而且近轴面所属的茸毛在远轴面也有分布。2、将rl10-1突变体与中花11配制杂交,F₁植株叶片表现平展,F₂中正常株和突变株的分离比符合3:1,表明突变性状受单隐性基因控制。3、利用rl10-1突变体与籼稻品种Dular杂交产生的F₂和F₃群体,将RL10基因定位在STS标记f70和f87之间约38kb的区段内;同样,利用图位克隆的方法分离了一个与rl10-1等位的突变基因rl10-2,来自突变体M475。4、通过基因预测发现,在RL10基因定位的38kb内,存在5个基因,对它们进行测序分析发现,第二个基因在两个突变体中发生了序列变化,rl10-1在外显子中发生了单碱基替换（T→C）,导致一个丝氨酸突变成脯氨酸,rl10-2在终止密码子前发生了两个碱基的缺失,导致翻译终止滞后。5、对野生型品种的候选基因2进行RNA干涉,产生类似突变体的表型,表明该基因是控制rl10突变性状的基因。6、半定量RT-PCR结果表明,RL10基因在植株所有器官中均有表达,在叶片、叶鞘和穗中的表达量较高,在根和茎中的表达量稍低。更多还原

【Abstract】 Leaves play a very important role in plant development for their function of photosynthesis and transpiration.Leaf morphogenesis,as an important part of plant morphogenesis,determines plant type and agricultural yields.Rice is one of the most important crops in the world,and semi-rolled leaf is one of the most important morphological characters in rice breeding.Therefore,to elucidate the mechanisms of leaf development would be critical not only for us to get a deep understanding of leaf morphogenesis but also for plant type improvement via biodesign.In this study,3 rolled leaf mutants:M425,M429,M475 were isolated from Zhonghua-11（O.sativa ssp.japonica） induced by ⁶⁰Co-y ray.The forth rolled leaf mutant,M7,was derived from japonica variety Nipponbare during the tissue culture process.Of the four mutants,M425 and M7 have the similar phenotype,displaying rolled leaves severely like a cylinder and malformed seeds;the other two mutants, M429 and M475,which look similar to each other too,have a performance of semi-rolled leaf and dense panicle.Anatomical analysis,genetic analysis,gene allelic test and map-based cloning were conducted.The main results were as follows:Part 1:The mapping and cloning of the rice RL9 gene1.Electron microscopic observation revealed that in rl9-1 mutant（M425）,the mesophyll cells covered the vascular bundles on the abaxial side of the leaf,where filled with sclerenchyma cells in wild-type;triehoms,which restricted to adaxial domain in wild-type,distributed on both sides in rl9-1;and the arrangement of chloroplast grana lamellae were disordered and irregular in rl9-1. 2.rl9-1 mutant was crossed to the wild-type Zhonghua-11,the F₁ plants were of flat leaves and the ratio of normal plants to mutant plants in F₂ population fitted 3:1, suggesting the phenotype of the rolled-leaf mutant was controlled by single recessive gene.3.Based on the progenies from F₂ and F₃ populations of rl9-1/Dular（O.sativa ssp. indica）,the RL9 gene was restricted to a 22-kb region on AP005904 of the long arm of chromosome 9 by using SSR（Simple Sequence Repeat）,STS（Sequenced Tagged Site） and CAPS（Cleaved Amplified Polymorphisms） markers.Moreover,rl9-2,the allelic mutant gene of rl9-1,was isolated from M7 by map-based cloning.4.There’s only one MYB-like gene located in RL9 anchored region.Sequencing results showed that a 5-bp deletion in exon 1 occurred in rl9-1,which resulted in frame shift;a substitution of A（wild-type） by G at the splice site of intronl/exon2 occurred in rl9-2,which made the intron can not be cleaved accurately and lead to loss-of-function of RL9.5.By means of RT-PCR,coding sequences（CDS） of RL9 gene were redefined.The RL9 gene was located at 103061-108515 of AP005904,consisting of 6 exons and 5 introns,totally 1134-bp CDS,encoding 377 amino acids.6.RL9,the true gene controlled the rolled-leaf phenotype of M425,was confermed by the result of complementation test,which showed that the rl9-1 phenotype was recovered by introduction of the RL9 gene from Nipponbare.7.The results of semi-quantitative RT-PCR showed that the RL9 is expressed in all organs of the wild-type plants,higher in roots,leaves and panicles,and lower in stems and leaf sheathes.8.Sequence analysis revealed that RL9 encodes a GARP domain.9.Alignment of amino acid sequences of RL9 and GARP super-family members was carried out,and a neighbor-joining tree was generated.The results showed that RL9 is homologous to Arabidopsis KANADIs.Based on the role of KANs in Arabidopsis and the performances of loss-fuction of RL9 in rice,it is suggested that RL9 has a function in specifying the abaxial fate of leaves.10.Transient expression analysis using an RL9-GFP fusion protein in onion epidermal cells indicated that the RL9 protein was localized to the nucleus,confirming that the RL9 acts as a transcription factor.Part 2:The mapping and cloning of the rice RL10 gene1.Electron microscopic observation revealed that in rl10-1 mutant（M429）,the mesophyll cells covered some vascular bundles on the abaxial side of the leaf,and interestingly,few garland-structures resembling C4 plants existed,in addition, trichoms distributed on both sides.2.rl10-1 mutant was crossed to the wild-type Zhonghua-11,the F₁ plants were of flat leaves and the ratio of normal plants to mutant plants in F₂ population fitted 3:1, suggesting the phenotype of rl10-1 mutant was controlled by single recessive gene.3.Using the progenies from F₂ and F₃ populations of rl10-1/Dular,the RL10 gene was restricted to a 38-kb region between markers f70 and f87.Moreover,rl10-2,the allelic mutant gene of rl10-1,was isolated from M475 by map-based cloning.4.Five genes located within the RL10 anchored region.Sequencing results of the five genes showed that gene 2 mutated in both two rl10 mutants,i.e.a substitution of T （wild-type） by C occurred in rl10-1,which resulted in a Ser transformed to Pro;a 2-bp deletion occurred ahead of the stop codon in rl10-2,which lead to post-termination of translation.5.RNAi aimed at the candidate gene 2 in wild type plants could result in the mutation phenotype of rl10 mutants.6.The results of semi-quantitative RT-PCR showed that the RL10 is expressed in all organs of the wild-type plants,higher in leaves,leaf sheathes and panicles,and lower in roots and stems.更多还原