BIOLOGICAL AND MOLECULAR ANALYSIS OF INDUCTION MECHANISM FOR SOMACLONAL VARIAION IN TOMATO

番茄体细胞克隆变异诱导机制的生物学和分子分析

• 批准号: 12660050
• 负责人: TOYODA Hideyoshi
• 金额: $ 2.24万
• 依托单位: KINKI UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12660050/
• 关键词: Retrotransposon; Tomato; Detection of gene expression; Micromanipulation; トリコーム; RT-PCR; 組織培養; うどんこ病; 変異; 抵抗性; 青枯病

When leaf segments of a tomato cultivar 'Ponderosa' were inoculated with Agrobacterium rhizogenes MAFF07-20001 carrying the binary vectors pRi and pBI121/sGFP, adventitious roots were developed from calli formed at the edges of the segments. Primordial roots that showed green fluorescence under blue light and elongated vigorously on hormone-free medium without loss of the green fluorescence were obtained. They were easily distinguishable from the non-fluorescing roots on the same segments. Successful integration of the sGFP and rol C genes into the chromosome of tomato roots was confirmed by polymerase chain reaction and Sourhem hybridization. The present method enables us to evaluate the hairy root formation without subculture, isolation and DNA analysis. This method was tested on all of commercial cultivars available in Japan(42 cultivars) and 14 breeding lines of tomato. All but two breeding lines produced the hairy roots. Thus, the present method is useful for hairy root production … More in tomato. RT-PCR was used to detect gene expression in situ in single selected cells from tomato callus aggregates. The cytoplasm from one cell was removed with a micropipette viewed under a light microscope and used directly for RT-PCR, followed by nested PCR. This method of removing cytosolic contents prevented the introduction of genomic DNA into the RT-PCR, and only intron-spliced products were amplified when intron-containing genes were used as PCR targets. In addition, transcription of the intron-free gene was possibly detected by simultaneously tracing the intron-containing and intron-free genes using mixed primers for the targeted genes. The present study indicated that some stimuli-activated genes, such as CHI3 and TLC1-retrotransposon long terminal repeat, were constitutively transcribed in tomato callus cells. A single-cell RT-PCR was conducted to detect gene expression in situ in pinpointed trichome cells of tomato leaves. The cytoplasm was removed with the micropipette using a light microscope and directly used for RT-PCR, followed by nested PCR. Two intron-containing genes, glyceraldehydes 3-phosphate dehydrogenase gene and plasma membrane H^+-ATPas gene were constantly expressed in this tissues and therefore used as the indicator, because of easy detection of shorter-size PCR-products produced by splicing. In addition, the sucking of nucleus-free cellular contents was effective to prevent contamination of genomic DNA led to miss-amplification of corresponding genomic DNA sequences of the intron-less genes in the process of RT-PCR and subsequent nested PCR. Thus, the present technique could be applicable to single trichome cells of tomato leaves for directly detecting their gene expression in response to chemical and physical stimulation. Less

当番茄品种“黄铜”的叶片片段被根状腺根源菌群接种时，携带二进制载体PRI和PBI121/SGFP的根状腺杆菌MAFF07-20001是由在段边缘形成的呼唤开发的。原始根在蓝光下显示出绿色的荧光，并在无刺培养基上剧烈伸长而不会损失绿色荧光。它们很容易与同一细分市场的非荧光根区分开。通过聚合酶链反应和酸性杂交证实了SGFP和ROL C基因成功地整合到番茄根染色体中。目前的方法使我们能够在没有亚培养，隔离和DNA分析的情况下评估毛茸茸的根部形成。对日本可用的所有商业品种（42种品种）和14条番茄繁殖线进行了测试。除两条繁殖线外，还产生了毛茸茸的根。这是当前的方法对毛根生产很有用……在番茄中更多。 RT-PCR用于从番茄愈伤组织聚集体中单个选定细胞中检测基因表达。用一个微型夹子在光学显微镜下观察并直接用于RT-PCR，然后用嵌套的PCR去除去一个细胞的细胞质。这种去除胞质含量的方法阻止了基因组DNA引入RT-PCR，并且当使用含有内含子的基因作为PCR靶标时，仅内含子插入的产物是放大器。此外，很可能通过简单地使用混合引物作为靶向基因追踪含内含子的基因和无内含子基因的转录。本研究表明，在番茄愈伤组织细胞中，将一些刺激性激活的基因（例如CHI3和TLC1-返回跨跨跨末期重复重复）组成二次转录。进行了单细胞RT-PCR，以检测番茄叶的精确的毛状细胞中的原位基因表达。使用光学显微镜用微型移液器除去细胞质，并直接用于RT-PCR，然后使用嵌套的PCR。两个含有内含子的基因，即3-磷酸盐脱氢酶基因和质膜H^+ - ATPAS基因的两个含甘油醛基因在该组织中经常表达，因此用作指示剂，因为很容易检测到通过拼接产生的较短尺寸的PCR产品。另外，无核细胞含量的吮吸有效防止基因组DNA污染导致在RT-PCR和随后的嵌套PCR过程中对相应的无插入基因的相应基因组DNA序列的放大。这是当前的技术可用于番茄叶的单个毛状细胞，以直接检测其基因表达，以响应化学和物理刺激。较少的

项目成果

Matsuda Y, Kashimoto K, Takikawa Y, Aikawa R, Nonomura T, Toyoda H: "Occurrence of new powdery mildew on greenhouse tomato cultivars"Journal of General Plant Pathology. 67. 294-298 (2001)

Matsuda Y、Kashimoto K、Takikawa Y、Aikawa R、Nonomura T、Toyoda H：“温室番茄品种上新白粉病的发生”普通植物病理学杂志。

H.Toyoda, Y.matsuda, T.Nonomura, Y.Takikawa, Y.Otsu, H.Mori, K.Kakutani: "Chitin Enzymology"Atec Edizioni. 614 (2001)

H.Toyoda、Y.matsuda、T.Nonomura、Y.Takikawa、Y.Otsu、H.Mori、K.Kakutani：“几丁质酶学”Atec Edizioni。

Kashimoto K, Matsuda Y, Matsutani K, Sameshima T, Kakutani K, Nonomura T, Okada K, Kusakari S, Nakata K, Takamatsu S, Toyoda H: "Morphological and molecular characterization for a Japanese isolate of tomato powdery mildew Oidium neolycopersici and its hos

Kashimoto K、Matsuda Y、Matsutani K、Sameshima T、Kakutani K、Nonomura T、Okada K、Kusakari S、Nakata K、Takamatsu S、Toyoda H：“番茄白粉病 Oidium neolycopersici 及其日本分离株的形态学和分子特征

Matsuda Y, Sameshima T, Inoue K, Nonomura T, Kakutani K, Takamatsu S, Toyoda H: "Molecular Discrimination of Different Powdery Mildew Fungi Simultaneously Attacking Plant Leaves by the Phylogenetic Analysis of rDNA Internal Transcribed Spacer Sequences Am

Matsuda Y、Sameshima T、Inoue K、Nonomura T、Kakutani K、Takamatsu S、Toyoda H：“通过 rDNA 内转录间隔序列的系统发育分析对同时攻击植物叶片的不同白粉病真菌进行分子识别

Wada M, Matsuda Y, Fujita K, Nishimura M, Nonomura T, Kakutani K, Toyoda H: "Possible amplification of mature mRNAs in single cells of tomato callus aggregates by direct RT-PCR of cytosolic contents suctioned out with a micropipette"Plant Cell Tissue Orga

Wada M、Matsuda Y、Fujita K、Nishimura M、Nonomura T、Kakutani K、Toyoda H：“通过对用微量吸管吸出的胞质内容物进行直接 RT-PCR，可能扩增番茄愈伤组织聚集体单细胞中的成熟 mRNA”Plant Cell