【作者】 乔利仙；

【导师】 戴继勋； 王斌；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2007， 博士

【摘要】 用分子标记技术进行紫菜遗传多样性分析对于紫菜种质资源的鉴定、保护和持续合理开发利用具有重要意义。本研究采用相关序列扩增多态性(sequence-related amplified polymorphism,SRAP);靶位区域扩增多态性(target region amplification polymorphism,TRAP);限制性位点扩增多态性(restriction site amplification polymorphism,RSAP)三种分子标记技术对16个紫菜系进行了遗传多样性分析,在此基础上对生产上广泛应用的条斑紫菜6-磷酸海藻糖合成酶基因(trehalose-6-phosphate synthase from Porphyra yezoensis,PyTPS )进行了转化水稻的研究。本研究首次将分子标记技术SRAP、TRAP和RSAP用于紫菜的种质资源分析。建立了适合于紫菜SRAP、TRAP和RSAP分析的PCR反应体系和反应条件,利用所建立的体系对16个紫菜系进行了遗传多样性分析,构建了这些紫菜系的聚类图和DNA指纹图。三种分子标记方法聚类分析所产生的进化树基本一致,均把这些紫菜系分成两大类,分类结果与传统分类相吻合。在所构建的DNA指纹图谱中,每个紫菜系都有其特异的指纹模式,能很容易地与其它紫菜系区分开来。根据DNA指纹图谱开发出计算机应用软件PGI-SRAP,PGI-TRAP,PGI-RSAP(Porphyra germplasm identification developed by SRAP,TRAP and RSAP method),可以辅助进行紫菜系的种质鉴定。对RSAP分析产生的17条特异性标记中的6条进行了克隆、测序,并将其中一个标记R1/R3-8119转换成紫菜系P. yezoensis Y-9101的特异序列扩增区域(sequence characterized amplified region,SCAR)标记。用本实验室克隆的条斑紫菜TPS基因(PyTPS)转化水稻品种TP309,得到的T0代转基因植株,经过连续加代、鉴定、筛选,得到5个T2代的纯合体株系,对其中3个株系(TPS155-4,TPS191-1和TPS308-1)进行了PCR和Southern杂交检测,结果都呈双重阳性,表明PyTPS基因已整合到这些转基因株系中。用0.8%NaCl模拟盐碱条件,16% PEG-6000模拟干旱条件,以非转基因水稻TP309为对照,选T2代两个纯合体株系TPS155-4和TPS191-1进行了抗盐、抗旱性分析。RT-PCR结果表明转入的基因得以表达,转基因株系的株高、单株鲜重均高于对照,在PEG处理下这种差异达到显著水平。这说明PyTPS基因的导入,明显增加了转基因水稻的抗旱性;转基因水稻的抗盐性也有所提高。更多还原

【Abstract】 It is very important to study the genetic diversity of Porphyra lines using molecular marker technique, which could promote the germplasm identification, protection and effectual exploiture of Porphyra. The genetic diversity of Porphyra lines was analysed using molecular marker techniques sequence-related amplified polymorphism （SRAP）, target region amplification polymorphism （TRAP） and restriction site amplified polymorphism （RSAP）. And the trehalose-6-phosphate synthase gene from Porphyra yezoensis （PyTPS） cloned by our laboratory was transformed to rice variety TP309, in order to study the function of the gene PyTPS.SRAP, TRAP and RSAP analytic system were set up and successfully used in germplasm identification of Porphyra lines in this study firstly. The dendrograms and the DNA fingerprints of sixteen Porphyra lines were constructed according to the result of SRAP, TRAP and RSAP analysis. The dendrograms from 3 marker techniques were accordant with the conventional taxonomy basically. In the DNA fingerprints, each of the 16 Porphyra lines has its unique fingerprinting pattern and can be easily distinguished from each other. The PGI-SRAP, PGI-TRAP and PGI-RSAP （Porphyra germplasm identification developed by SRAP, TRAP and RSAP method） developed by DNA fingerprints could be used to germplasm identification of these Porphyra lines. In addition, seventeen specific RSAP markers were identified and six of them were sequenced. One of the 17 specific markers, R1/R3-8119 from P. yezoensis Y-9101, was successfully converted into sequence characterized amplification region （SCAR） marker.Trehalose-6-phosphate synthase gene from P. yezoensis （PyTPS） cloned by our laboratory was transformed into rice variety TP309 by Agrobacterium mediated method. Three T₂ homozygous lines were obtained by the selection with kanamycin resistance, PCR amplification and Southern blotting analysis. Results indicated that the PyTPS gene had been integrated into the genome of the transgenic rice plants. The tolerance to both salt and drought was compared between two homozygous lines TPS155-4, TPS191-1 and the non-transformed rice plants TP309, imposed 0.8% NaCl and 16% PEG-6000 treatments. The plant height and fresh weight per plant in transgenic plants were better than those of control plants under water, salt and drought stress. The difference between transgenic and control plants for drought stress was obvious. The result demonstrated that the transgenic plants showed apparently increased drought stress tolerance and the slightly increased salt stress tolerance.更多还原