【作者】 焦锋；

【导师】 楼程富；

【作者基本信息】 浙江大学 ， 特种经济动物饲养， 2003， 博士

【摘要】 桑树是多年生叶用木本植物,桑叶是蚕丝业的物质基础,桑叶的大小形状与经济产量密切相关,对桑树叶片的生长发育和形态建成机理进行研究,具有特别重要的意义。为探讨桑树叶片发育的分子机制,以桑树叶形变异株“凤尾一之濑”和野生型“新一之濑”的顶芽和叶片为试材,进行了组织学和分子机理的研究。桑树变异株系凤尾一之濑是由新一之濑冬芽经60Coγ射线诱变处理后,形成的叶片形态变异株系,叶片细长,横向皱缩。本研究利用组织切片技术观察了桑树变异株系的叶片组织形态的变化。采用cDNA-AFLP(cDNA扩增片段长度多态性)技术进行叶片形态变异株系和野生型的mRNA差异表达分析。得到了如下几条主要结论：1.桑树品种新一之濑和其变异株系凤尾一之濑在叶片指数上存在显著差异,新一之濑为1.17,凤尾一之濑为3.05。两者的幼叶厚度基本没有差异,但凤尾一之濑的成熟叶比新一之濑稍厚。两者在叶片组织结构,细胞层数和表皮结构等方面沿叶片的纵横轴没有明显的差异。2.应用Trizol,可以获得高质量的桑树RNA样品,可应用于cDNA反转录、Northern杂交等。应用PolyATtract(?) mRNA Isolation Systems (Promega)提取桑树顶芽总RNA,利用SMARTTM cDNA library construction kit (Clontech)中的LD-PCR方法合成cDNA,以及利用聚丙烯酰胺凝胶电泳和银染法,都得到良好的实验结果,这些技术可以满足桑树cDNA-AFLP的要求。3.利用AseⅠ和TaqⅠ对cDNA进行双酶切后,连接寡聚核苷酸接头,利用有两个选择性碱基的引物进行PCR扩增,结果显示,在所用材料相同的情况下,不同的引物组合有不同的电泳图谱,同一引物组合可以得到相同的条带。而且在克隆得到的DNA片段序列中含有特定的接头和选择性碱基序列(选择性引物序列),表明cDNA-AFLP方法具有较高的可靠性和重复性。4.本研究共选用8个TaqⅠ和8个AseⅠ引物,共64对引物组合进行选择性扩增,每个引物组合扩增的条带数可达50条以上,条带清晰,共扩增得到大约3200个条带,在新一之濑和凤尾一之濑之间,大约有30个条带只在其中一个品种的扩增图谱中出现,呈现明显的差异表达,这些条带代表了在叶片形态突变体与野生型之间差异表达的基因。5. cDNA-AFLP电泳图谱表明,在桑树叶形突变体和野生型之间,大部分条带是相同的,反映了大部分生理过程是相同的。少数条带存在差异,反映了造成二者之间性状差异的遗传改变。结果表明用cDNA-AFLP对桑树叶形变异株系进行差异显示分析的实用性。6.对聚丙烯酰胺凝胶电泳后得到的差异条带进行了克隆和测序分析,在所得到的15个基因片段中,有2个与已知基因有同源性,一个为脯氨酰4-羟化酶a亚基基因,该片段来自新一之濑。另一个是羟甲基转移酶基因,该基因片段来自凤尾一之濑。其他片段与已知的功能基因尚没有发现同源性。用5个克隆片段作为探针用于Northern杂交,没有发现明显的差异,说明这些突变的基因位点可能是点突变。7.本研究建立了一套应用于桑树变异株系表达差异显示的技术体系,从现有的资料看,本研究的内容,在桑树研究中应属首次报道。从得到的结果看,该方法费用少,有较强的可行性。8.从桑树现有的大量诱变株系入手,可以得到一些特殊基因序列。本研究为利用我国丰富的桑树种质资源,分离有特殊应用前景的目的基因应用于桑树分子育种领域,开创了新的研究途径和方法。9.本研究利用桑树叶片形态变异株和野生型进行mRNA表达差异显示分析,所得到的基因片段可能与桑树的叶形发育有某些相关性。对所得到的基因片段可以进一步克隆全长,研究基因功能。本研究为进一步研究叶片性状变化和发育分化的分子机理研究奠定了基础。更多还原

【Abstract】 The diversity of leaf morphological characteristics shows a wide adaptive feature in the environment. This distinct characteristic of leaves is not only important for plant production, but also aids the plants classification, such as that of in mulberry (Morus spp.). However, the expression and regulation of leaf morphological characteristics remains unclear. In the present study, the mulberry leaf shape mutant (Fengwei-Ichinose), and its wild type (Shin-Ichinose), were used to evaluate the regularity of leaf mutations and to identify the differentially expressed genes that respond for the changes of leaf shape. Fengwei-Ichinose was induced byγray of Co60 from’Shin-Ichinose’ Histological observation was performed to analysize the leaf morphological characters, and the cDNA-AFLP was consducted to fish out the differentially expressed genes. The results are summarized as follows:1. The leaf index was distinctly different between the mutant and its wild type. That of Fengwei-Ichinose was 3.05, while that of the wild type "Shin-Ichinose" was 1.17, The leaves of Fengwei-Ichinose were much narrower and with transversal crimples, and thicker at leaf fully expanded stage when compared to those of the wild type. The constructure of leaf tissue, cell layers and epidermis, however, had no differences along the leaf length and leaf width direction between the mutant and its wild type.2. Total RNA was extracted from buds of mulberry using Trizol method with minor modifications. mRNA was isolated by PolyATtract(?) mRNA Isolation Systems (Promage). The cDNA was synthesized by SMART cDNA library construction kit (CLONTECH.). cDNA-AFLP was performed by polyacrylamide gel electrophoresis and silver staining. The results indicated that the quality of the RNA obtained by Trizol was very high. These set of techniques were applicable in mulberry.3. cDNA was digested by AseⅠand TaqⅠ, ligated by two annealed anchors, and then, amplified by primers complementary to the anchors with two selective bases at 3’ end. The result of electrophoresis showed different primer combinations produced different banding pattern, while same banding patterns could be repeatedly obtained by a same primer combination Sequence of the specific between the mutant and its wild type showed the cloned fragments had the primer sequence and the two selective bases. These suggested cDNA-AFLP was of high reliability and repeatability.4. Totally 64 of combinations of eight AseⅠand eight TaqⅠprimers were used. More than 50 bands were detected from each primers combination, and approximately 3200 expression bands were obtained in sum. Of which,30 bands only appeared in the mutant or the wild type, were suggested may corresponsable to the leaf morphogenesis.5. Most of the bands between the mutant and its wild type were identicall, which demonstrated there was similarity between them. A few bands only appeared in one of the two materials, mutant or its wild type. This result suggested cDNA-AFLP is practiceal for gene different expression display.6. Fifteen of the different expressed gene fragments were cloned and sequenced. Two of them showed homological with known gene after BLAST on NCBI GeneBank. One fragment, obtained from Shin-Ichinose showed similarity to the UniGene of putative prolyl 4-hydroxylase, alpha subunit in Arabidopsis. Another fragment obtained from Fengwei-Ichinose showed homologous with hydroxymethyltransferase in Arabidopsis. The other 13 fragments did not show any homology with any known genes in GeneBank, suggesting they might be are new gene fragments responsible to mulberry leaf development, relating to the change of the leaf morphology in mulberry.7. It was the first time for cDNA-AFLP to be used to analyze mulberry. In the present study, the cDNA-AFLP technique system was established for screening the differentially expressional genes between mutants and their original type.Summarilly, From the above results we can conclud that the work of analyzing the mulberry mutants using cDNA-AFLP technique has developed a new strategy in the study of leaf development mechanism and cloning new genes in woody plant, which have been rarely reported up to now. The study established a solid basic for analysing molecular mechanism of leaf morphology development.更多还原