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棉花植株再生困难、基因型依赖性强和转化周期长等因素一直制约着棉花遗传转化的发展。本研究利用1种不依赖组织培养的转化方法 ,即子房滴注转化方法将獐茅高亲和性钾离子转运蛋白基因(Al HAK1)导入棉花基因组中。结果表明在1006株转化幼苗中有44株为卡那抗性植株,其中35株经PCR检测为阳性(T0代),转化率为3.5%。Southern与Northern杂交结果进一步表明外源基因已整合至棉花基因组中并在转录水平上表达。在提供外源0.05 mmol·L-1 K+水平下,T1转基因棉花叶片中K+含量约为对照植株的2倍,在根中约为野生型植株的1.5倍;而在2.5 mmol·L-1 K+的正常水平下,转基因棉花与野生型植株K+含量差异不明显。在50~200 mmol·L-1 Na Cl胁迫条件下,转基因棉花的种子发芽率明显高于野生型植株,尤其是在150mmol·L-1Na Cl胁迫条件下,转基因植株种子的发芽率是野生型的2.8倍左右。本研究为子房滴注转化体系在生产实践中的广泛应用提供了理论依据,并为培育适应土壤钾素匮乏及盐渍化环境下生长的棉花新品种提供了新的种质资源。
Cotton plant regeneration difficulties, genotype-dependent and long transformation cycle and other factors have been restricting the development of genetic transformation of cotton. In this study, a tissue-free transformation method was used to introduce the Alhumna pelamis high affinity potassium transporter gene (Al HAK1) into cotton genome. The results showed that 44 of 1006 transformed seedlings were kanamycin resistant plants, of which 35 were positive by PCR (T0 generation) with a conversion rate of 3.5%. Southern and Northern hybridization results further indicate that foreign genes have been integrated into the cotton genome and expressed at the transcriptional level. At the level of exogenous 0.05 mmol·L-1 K +, the content of K + in T1 transgenic cotton leaves was about 2 times of that in the control plants and about 1.5 times that of the wild type plants in the roots. However, in the 2.5 mmol·L-1 K + The normal level of transgenic cotton and wild-type plants K + content was not significantly different. Under the condition of 50 ~ 200 mmol·L-1 NaCl stress, the seed germination rate of transgenic cotton was significantly higher than that of wild-type plants, especially under the condition of 150 mmol·L-1 NaCl stress, the germination rate of transgenic plants was wild type About 2.8 times. This study provides a theoretical basis for the widespread application of ovarian infusion transformation system in production practice and provides new germplasm resources for breeding new varieties of cotton adapted to the conditions of potassium deficiency and salinization in soil.