盐生草HgNHX1对大麦的遗传转化及转基因大麦抗逆性分析

发布时间：2019-05-14 04:05

【摘要】：大麦(Hordeum vulgare L.)属禾本科植物,主要用于制造啤酒及饲用。其具有抗旱、耐盐碱等特性。随着全球气候与生态环境的日益恶化,土壤盐碱化和干旱仍然是限制大麦生长的两个主要环境因素。采用基因工程手段,培育高抗旱耐盐性大麦新品种已成为大麦栽培的迫切要求。而受体材料是遗传转化的基础,寻求一种来源广泛、取材方便、受基因型和季节限制小、高频再生的体系对于有效地开展大麦转基因研究具有重要意义。因此,本实验通过建立大麦茎尖再生体系,利用农杆菌介导法将盐生草(Halogeton glomeratus)Na~~+/H~~+逆向转运蛋白(NHX)HgNHX1基因导入大麦,获得转基因阳性植株并对转基因大麦T1代植株进行抗逆性分析。主要研究结果如下:1.以栽培中常用品种甘啤3号、甘啤4号和甘啤6号为材料,研究了大麦茎尖高频再生体系影响因素,其中种子浸泡在30%的硫酸中,30℃、180 r/min摇动1 h为去除颖壳最佳方法;75%的乙醇处理1.5 min~~+2%的NaClO处理15 min为较好的灭菌方法;3 d苗龄、茎基部2 mm处切段效果最好;3 mg/L 2,4-D有利于愈伤组织诱导,参试品种中甘啤4号再生能力较强。2.利用盐生草HgNHX1对大麦进行遗传转化,采用农杆菌介导法,真空渗透处理5 min,超声波处理30 s,100 r/min摇动侵染30 min的瞬时转化率较高,获得了抗性再生植株23株,PCR检测阳性植株数7株,并获得了T1代种子。3.对T1代转基因大麦植株抗逆性分析发现,在干旱、盐胁迫下,HgNHX1基因在大麦中表达丰度增高,转HgNHX1基因大麦植株的耐盐性不明显,而抗旱性增强。
[Abstract]:Barley (Hordeum vulgare L.) Gramineae, mainly used in beer and feed. It has the characteristics of drought resistance, salt and alkali tolerance and so on. With the deterioration of global climate and ecological environment, soil salinization and drought are still the two main environmental factors that limit the growth of barley. It has become an urgent requirement for barley cultivation to cultivate new barley varieties with high drought resistance and salt tolerance by means of genetic engineering. However, the receptor material is the basis of genetic transformation, so it is of great significance to find a system with wide source, convenient collection, small genotypic and seasonal restriction and high frequency regeneration for the effective research of barley transgene. Therefore, by establishing the stem tip regeneration system of barley, the (Halogeton glomeratus) Na~~ / H / H reverse transporter (NHX) HgNHX1 gene of halophytic grass was introduced into barley by Agrobacterium tumefaciens mediated method. Transgenic positive plants were obtained and stress resistance of transgenic barley T1 plants was analyzed. The main results are as follows: 1. The influencing factors of high frequency regeneration system of barley stem tip were studied by using the commonly used varieties Ganbei 3, Ganbei 4 and Ganbei 6 as materials. The seeds were soaked in 30% sulfuric acid at 30 鈩，

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