利用子房滴注法获得转高亲和性钾离子转运蛋白基因(AlHAK1)棉花植株  

Generation of Transgenic Cotton (Gossypium hirsutum L.) with High-affinity K+ Transporter (AlHAK1) via Ovary-drip Method

作　　者：刘灵娣[1,2] 唐宏亮[1] 董丽君[1] 张书玲[1] 吴立柱 杜环[1] 高佳佳[1] 赵晓雅 刘晗[1] 蒋肖[1] 刘建凤[1] 

机构地区：[1]河北大学生命科学学院,河北保定071002 [2]河北省农林科学院经济作物研究所,石家庄050051 [3]河北农业大学生命科学学院,河北保定071001

出　　处：《棉花学报》2015年第1期9-15,共7页Cotton Science

基　　金：河北省自然科学基金(C2013201219);河北大学人才引进科研启动项目(2014-277);生物学强势特色学科(植物学)

摘　　要：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（AIHAK1）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％。Southem与Northem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0．05mmol·L-1K＋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2．5mmol·L-1K＋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50～200mmol·L-1NaC1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-1NaC1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。The progress for development of transgenic engineering of cotton is restricted by plant regeneration difficulty, geno- type-dependent response and a prolonged culture period and so on. In this study, a high-affinity K＋ transporter（AIHAK1）, isolat- ed from a Aeluropus littoralis Pad., was introduced into cotton （Gossypium hirsutum L.） by a ovary-drip transformation method that is tissue culture-independent. The results showed that 44 out of 1006 transformed plants were kan-resistance and 35 were still positive by PCR detection, corresponding to a positive rate of 3.5%. Southern and Northern blot analysis confirmed that the A/HAK1 gene was correctly integrated into the genome and expressed in the transgenic plants. Under 0.05 mmol· L-1 K＋ con- centrations stress, potassium contents in leaves and roots of T1 generation transgenic plants were respectively 2-fold, 1.5-fold of that in the wild-type plants. While under 2.5 mmol· L 1 K＋ concentrations, there was no significant difference in potassium con- tent between the transgenie plants and the wild-type plants. Under NaC1 treatment, the germination rates of the transgenic cotton were remarkably higher than that of the wild-type plants within the NaC1 concentration scope of 50- 200 mmol. L- 1, especially under 150 mmol·L-1 NaC1 the germination rate of the transgenic lines was approximately 2.8-fold of that of wild-type plants.＂File research findings may provide theoretical evidence lbr the extensive application of ovary-drip translbrnlation system in pro- duction practice and important germplasm resources for breeding of new cotton variety adapting to K＋ deficiency and salinity soil condittion.

关 键 词：棉花 子房滴注转化 钾离子转运蛋白 转基因植株 钾营养 盐胁迫 

分 类 号：S562[农业科学—作物学]