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首页> 外文学位 >Analysis of transgenic plants of Vitis vinifera L. expressing genes encoding the pear PGIP and GFPs fused to secretory sequences.
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Analysis of transgenic plants of Vitis vinifera L. expressing genes encoding the pear PGIP and GFPs fused to secretory sequences.

机译：分析表达编码编码与分泌序列融合的梨PGIP和GFP的葡萄的葡萄的转基因植物。

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The major objective of this research was to develop an efficient method by which to transform Vitis vinifera L. cultivars with the pear PGIP gene in order to analyze its effect on tolerance to Pierce's disease (PD) and botrytis in transgenic plants. A second goal was the transformation of grape with several gfp constructs carrying sequences expected to enhance secretion from the cell in order to evaluate the effect of signal sequences on the targeting of transgene products to xylem tissue. Transgenic plants of cvs. ‘Chardonnay’ and ‘Thompson Seedless’ expressing the pear PGIP or the GFP genes were produced via somatic embryogenesis. Putative PGIP transgenic lines growing in the greenhouse were analyzed by PCR, Western blotting and PGIP activity assays. Western blot analysis demonstrated the presence of the protein in roots, leaves and young stems of the transgenic plants but not in untransformed controls. High levels of enzyme activity were found in crude extracts from leaves and in xylem sap of transgenic lines obtained from independent transformation events but not in untransformed controls. Enzyme activity was also detected in xylem sap of untransformed scions grafted on transgenic rootstocks. Lesion expansion was retarded in leaves of PGIP transgenic plants infected with Botrytis cinerea. The development of PD was delayed in some transgenic lines with high PGIP activity, which exhibited reduced leaf scorching, lower Xylella titers and better re-growth after pruning than the untransformed controls. GFP transgenic plants were transformed with four gene constructs that included the coding sequences for a synthetic GFP and GFP fused with the amino-terminal of the secreted protein trichosanthin (TCS) or the xylem specific protein XSP30, all under the control of the CaMV 35S promoter. In addition, a fourth construct contained gfp under the control of the potato Ubi3 promoter. Strong fluorescence was found in embryos, roots, stems and leaves of plants transformed with gfp and xsp30-gfp but the levels of fluorescence observed in tcs-gfp transformants were very low. In all cases fluorescence was detected only inside the cells. More analysis is necessary to determine whether or not the signal peptides were recognized by the grape secretory machinery. Plants transformed with gfp controlled by Ubi3 promoter exhibited green fluorescence only in the root apex and in meristematic leaves. No fluorescence was detected in these plants after wounding or ethylene treatment.
机译：这项研究的主要目的是开发一种有效的方法，通过该方法用梨PGIP基因转化 L.品种，以分析其对皮尔斯氏病（PD）和葡萄孢的耐受性的影响。转基因植物。第二个目标是用几种 gfp 构建体转化葡萄，这些构建体带有预期增强细胞分泌的序列，以评估信号序列对转基因产物靶向木质部组织的影响。转基因植物的简历。表达梨PGIP或GFP基因的“霞多丽”和“汤普森无核”是通过体细胞胚发生而产生的。通过PCR，蛋白质印迹和PGIP活性测定法分析了温室中推定的PGIP转基因品系。蛋白质印迹分析表明该蛋白存在于转基因植物的根，叶和幼茎中，但未存在于未转化的对照中。在从独立转化事件获得的转基因品系的叶片粗提取物中和木质部汁液中发现了高水平的酶活性，但在未转化的对照中未发现。在移植到转基因砧木上的未转化接穗的木质部汁液中也检测到酶活性。感染了 cinetry cinerea 的PGIP转基因植物叶片的病灶扩展受到抑制。与未转化的对照相比，在具有高PGIP活性的某些转基因品系中，PD的发育被延迟，与未转化的对照相比，它们表现出减少的叶子焦焦，更低的滴度和更好的重新生长。用四个基因构建体转化GFP转基因植物，其中包括合成GFP的编码序列以及与分泌的蛋白天花粉蛋白（TCS）或木质部特异蛋白XSP30的氨基末端融合的GFP编码序列，所有这些都在CaMV 35S启动子的控制下。另外，在马铃薯Ubi3启动子的控制下，第四个构建体包含 gfp 。在 gfp 和 xsp30-gfp 转化的植物的胚，根，茎和叶中发现强烈的荧光，而在 tcs-gfp 中观察到的荧光水平斜体>转化子非常低。在所有情况下，仅在细胞内部检测到荧光。为了确定信号肽是否被葡萄分泌机制识别，需要进行更多分析。受Ubi3启动子控制的 gfp 转化的植物仅在根尖和分生叶中显示绿色荧光。受伤或乙烯处理后在这些植物中未检测到荧光。

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