Momtaz Osama A, Hussein Emad M, Fahmy Eman M, Ahmed Sherif E
Gene Expression Regulation Technology Lab, Agriculture Genetic Engineering Research Institute, Agriculture Research Center, Giza, Egypt.
GM Crops. 2010 Jul-Sep;1(4):257-66. doi: 10.4161/gmcr.1.4.13779.
Developing drought tolerance in Egyptian cotton varieties is a strategic goal considering the need to expand cotton cultivated area and water scarcity in the Nile valley. In the present study, increasing levels of polyamine accumulation via expressing S-adenosyl methionine decarboxylase (SAMDC) gene was the main goal. SAMDC cDNA isolated from Saccharomyces cerevisiae isolate was isolated and genetically engineered into Egyptian cotton varieties Giza 88 as an extra long staple and Giza 90 as a long staple by means of particle bombardment through meristem transformation.T(0) transgenic plants were screened using basta herbicide (200 mg/l). RT- PCR analysis was used to confirm gene expression while gene integration was confirmed by Southern blot analysis. Control plants from Giza 88 and Giza 90 were subjected to drought regime using different concentrations of PEG 6000 (2.5%, 5%, 7.5%, 10%, 12.5%, 15%, 17%, and 20%) for 9 hrs to record drought stress symptoms and determine the potential concentration level for inducing polyamine accumulation. 17% of PEG 6000 was considered the sublethal concentration showing drought stress symptoms and therefore was used as potential stress concentration for estimating the level for spermine accumulation in both control and transgenic Giza varieties. T(1) transgenic plants grown under induced drought stress regime were tested positive for gene integration and expression and subjected to HPLC analysis to determine levels of spermine as polyamine accumulated compound in response to drought stress regime. Elevated spermine accumulation in Egyptian cotton varieties Giza 88 and Giza 90, were compared as non transgenic plants grown under same induced drought conditions with T(1) transgenic plants using reverse-phase HPLC analysis. Elevated spermine accumulation expressing SAMDC gene reflect main cause for increasing drought tolerance in both transgenic varieties.
考虑到尼罗河流域棉花种植面积扩大的需求以及水资源短缺的现状，培育埃及棉花品种的耐旱性成为一项战略目标。在本研究中，通过表达S-腺苷甲硫氨酸脱羧酶（SAMDC）基因来提高多胺积累水平是主要目标。从酿酒酵母分离物中分离出SAMDC cDNA，并通过分生组织转化的粒子轰击法将其基因工程导入埃及超长绒棉品种吉扎88和长绒棉品种吉扎90中。使用草丁膦除草剂（200 mg/l）筛选T(0)代转基因植株。RT-PCR分析用于确认基因表达，而Southern杂交分析用于确认基因整合。对吉扎88和吉扎90的对照植株使用不同浓度的PEG 6000（2.5%、5%、7.5%、10%、12.5%、15%、17%和20%）进行9小时的干旱处理，以记录干旱胁迫症状并确定诱导多胺积累的潜在浓度水平。17%的PEG 6000被认为是显示干旱胁迫症状的亚致死浓度，因此被用作潜在胁迫浓度，以估计对照和转基因吉扎品种中精胺积累水平。在诱导干旱胁迫条件下生长的T(1)代转基因植株经检测基因整合和表达呈阳性，并进行HPLC分析，以确定作为多胺积累化合物的精胺在干旱胁迫条件下的水平。使用反相HPLC分析，将埃及棉花品种吉扎88和吉扎90中精胺积累量的增加与在相同诱导干旱条件下生长的非转基因植株和T(1)代转基因植株进行比较。表达SAMDC基因导致的精胺积累增加反映了两个转基因品种耐旱性增强的主要原因。
