陆地棉李氏纤维突变体的植物形态学观察及基因定位

发布时间：2019-05-29 10:41

【摘要】：棉花是一种重要的经济作物,其纤维是纺织业的主要原材料。纤维的长度、韧性、细度等决定了纤维的品质,如何获得高产优质的纤维是提升棉花经济效益的前提。陆地棉李氏突变体(Ligon-lintless1,Li1)是一种纤维极短的突变体,其种子上的纤维只有5~6mm,发育迟缓,植株矮小,茎叶扭曲,遗传分析表明该突变体是由显性单基因Li1控制。作为一个研究纤维发育的理想材料,Li1在克隆棉花纤维相关基因及阐述纤维发育分子机理等方面发挥了重要作用。为了对Li1植株形态差异有一个更深刻的认识及定位Li1基因,本文以海岛棉H7124和Li1为亲本,配置了F2群体,应用SSR及SSCP分子标记技术对Li1基因进行遗传定位;此外通过观察Li1野生型与突变体细胞和植株的形态,对其进行了系统的差异分析。主要研究结果如下:1.根据陆地棉基因组序列及前人对Li1定位的结果设计了73对SSR和SSCP引物,通过亲本多态性筛选表明28对引物有多态性,其中21对引物扩增的条带比较稳定清晰,用于构建遗传图谱。用这些多态性引物对620个F2单株进行遗传分型,构建了一个跨度为10.5cM,标记间平均间距为0.81cM的遗传图。所构建的遗传图谱包括13个标记位点,与Li1共分离的标记有六个:P98、P216、P219、P221、P222和P234,这些标记之间的物理距离因不同的物理图谱而不同。2.Li1野生型与突变体在株高,单个棉铃棉籽数,茎杆茸毛密度等方面有显著差异:在株高方面,野生型平均达97.84cm,杂合突变体为60.88cm,纯合突变体只有27.82cm;在种子结实方面,野生型平均每个棉铃可以结25颗饱满种子,杂合突变体为19颗,纯合突变体只有12颗;在茎秆茸毛方面,野生型比较密且长,杂合突变体虽然长但是比较稀少,而纯合突变体则又短又稀。3.Li1野生型与突变体茎和叶的光学显微镜与电子透射显微镜观察表明:突变体茎中维管束的部分细胞死亡导致细胞排列疏松,甚至出现大的空腔;叶中有些叶肉细胞发生质壁分离,与野生型相比,叶绿体等细胞器质量差,数量少。而野生型细胞排列紧密,结构正常,细胞器质量好。4.显微镜下野生型与突变体之间花粉粒及花粉管的形态无明显差异:花粉粒均成圆球状,表面有许多刺突状突起;用花粉管萌发培养基体外培养时,一个花粉粒可以萌发一之数根花粉管。在花粉活力及萌发率方面野生型与突变体之间有差异:野生型花粉活力为89%,杂合突变为73%,纯合突变为62%;野生型花粉管萌发率为84%,杂合突变为79%,纯合突变为69%。5.通过酶解法获得了高活力的棉花叶片原生质体,用F-Actin特异荧光染料Alexa Fluor 488?phalloidin对原生质体微丝骨架进行标记,在激光共聚焦显微镜下可以清晰的观察到微丝的形态及排列格局:丝状的微丝纵横交错呈网格状分布于质膜下方,叶绿体比较丰富,被微丝包裹缠绕。对比野生型与突变体之间的微丝骨架,发现野生型的微丝比较多,排列紧密,而突变体的微丝数量比较少,长度比较短,排列疏松。
[Abstract]:Cotton is an important economic crop, and its fiber is the main raw material of the textile industry. The length, toughness and fineness of the fiber determine the quality of the fiber, and how to obtain high-yield and high-quality fiber is the premise to improve the economic benefit of cotton. Ligon-lintzessl, Li1, a mutant with very short fibers, was only 5-6 mm in the fiber, and the growth was slow, the plant was small and the stem and leaf were twisted, and the genetic analysis showed that the mutant was controlled by the dominant single-gene Li1. As an ideal material for studying the development of fiber, Li1 plays an important role in cloning cotton fiber-related genes and elucidating the molecular mechanism of fiber development. In order to have a more profound understanding of the morphological differences of the Li1 plants and to position the Li1 gene, the F2 population is configured by using the sea-island cotton H7124 and Li1 as the parent, and the genetic positioning of the Li1 gene is carried out by using the SSR and the SSCP molecular marker technology; In addition, by observing the morphology of the wild type and the mutant cell and the plant of Li1, the differential analysis of the system was carried out. The main results are as follows:1. 73 pairs of SSR and SSCP primers were designed according to the genome sequence of the Gossypium hirsutum L. and the results of the previous studies. The polymorphic screening of the parents showed that 28 pairs of primers were polymorphic, and 21 pairs of primers were stable and clear, and used to construct the genetic map. The genetic classification of 620 F2 single plants was carried out by using these polymorphic primers, and a genetic map with a span of 10.5 cM and an average spacing of 0.81 cM was constructed. The constructed genetic map consists of 13 marker sites, and the markers co-separated with Li1 are six: P98, P216, P219, P221, P222, and P234, the physical distance between these markers is different for different physical maps. There was a significant difference in the hairy density of the stem: in the high aspect of the plant, the wild-type average was 97.84 cm, the hybrid mutant was 60.88 cm, the homozygous mutant was only 27.82 cm, and in the seed-bearing aspect, the wild-type average per cotton boll could have 25 full seeds, and the hybrid mutant was 19, The homozygous mutant was only 12; in the aspect of the stalk, the wild-type was relatively dense and long, and the hybrid mutant was short and thin, while the homozygous mutant was short and thin.3. The observation of the optical microscope and electron-transmission microscope of the Li1 wild-type and mutant stem and leaf showed that: Some of the cell death in the vascular bundle in the stem of the mutant resulted in loose cells and even large cavities. Some of the leaf-meat cells in the leaves were separated from the mass wall, and compared with the wild-type, the organelles such as the chloroplast were poor in mass and less in number. And the wild-type cells are closely arranged, the structure is normal, and the organelles are in good quality. There was no significant difference in the morphology of the pollen tube and the pollen tube between the wild-type and the mutant under the microscope. There was a difference between the wild-type and the mutant in the pollen activity and the germination rate: the wild-type pollen activity was 89%, the hybrid mutation was 73%, the homozygous mutation was 62%, the germination rate of the wild-type pollen tube was 84%, the hybrid mutation was 79%, and the homozygous mutation was 69%. The protoplast of the high-energy cotton leaves was obtained by the enzymatic method, and the protoplast microfilament framework was marked with the F-Actin-specific fluorescent dye Alexa Fluor 488-phaloidein, and the morphology and arrangement pattern of the microfilaments can be clearly observed under the laser confocal microscope. The filament-like micro-filaments are distributed in a grid-like shape under the plasma membrane, and the chloroplast is rich and is wrapped by the micro-filament. Compared with the microfilament framework between the wild type and the mutant, the wild type microfilaments are found to be more and more arranged, and the number of microfilaments of the mutant is less, the length is short, and the arrangement is loose.
【学位授予单位】：浙江农林大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S562

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