小花碱茅遗传转化体系的建立和PtHAK5编码基因的克隆及功能验证

发布时间：2018-06-16 00:51

本文选题：小花碱茅 + 遗传转化　；参考：《兰州大学》2017年硕士论文

【摘要】：钾是植物体内第二大矿物营养元素,它对植物生长和作物生产至关重要。盐碱化是影响农业生产和生态环境的重要因素,土地盐碱化已成为我国农业生产发展的主要限制因素之一。由于Na+和K~+在结构和化学性质方面的相似性,植物体内过多Na+的积累可干扰对K~+的吸收,抑制植物生长发育甚至导致植物死亡。自然界中的盐生植物在长期抵御外界盐碱胁迫的过程中形成了特殊的适应机制,深入系统研究这些植物的耐盐机理,筛选和培育适应盐土环境的盐生植物,对于盐碱土地生态系统的改善及土地资源的可持续发展与利用具有深远的意义。小花碱茅作为典型的拒盐型盐生植物,其主要耐盐机制为限制根系单向Na+内流,减少Na+的净积累以维持较强的K~+/Na+选择性。可见,小花碱茅具有极强的调控体内K~+、Na+平衡的能力。但是对于小花碱茅K~+、Na+吸收与转运体系的系统性研究并不多。本研究以小花碱茅为材料,建立了小花碱茅遗传转化体系;采用RT-PCR等方法克隆了KT/HAK/KUP家族高亲和性K~+转运蛋白编码基因PtHAK5,并通过酵母异源表达试验验证了该蛋白的功能,主要取得以下结果:1.优化了小花碱茅高频再生体系:(1)诱导愈伤组织的最佳激素组合为5.0 mg·L-1 2,4-D和0.5 mg·L-1 KT,继代过程中降低2,4-D的含量(2.5 mg·L-1)有利于提高愈伤分化率;0.5 mg·L-1 NAA、2.0 mg·L-1 6-BA和2.0 mg·L-1 KT能显著提高幼苗的再生率,为小花碱茅愈伤分化的最佳激素组合。(2)适量有机添加物(100 mg·L-1水解酪蛋白、500 mg·L-1脯氨酸和300 mg·L-1谷氨酰胺)和0.3%的植物凝胶能有效防止愈伤玻璃化,显著提高了胚性愈伤率。2.建立了小花碱茅高效遗传转化体系:(1)将继代培养3-4次的愈伤组织,在添加10 g·L-1葡萄糖和200μM·L-1 AS的MS培养基上预培养4 d,其细胞结构有利于外源DNA的整合,愈伤抵御农杆菌侵染伤害的能力强,侵染后转化体的恢复能力强。(2)将预培养后的愈伤组织用OD600为0.6的GV3101农杆菌菌液侵染15min,然后在添加200μM·L-1 AS的MS培养基上共培养2 d,为小花碱茅遗传转化的最优侵染组合条件。(3)将共培养2 d的愈伤投入含250 mg·L-1 Cef的溶液中浸洗20 min,然后接种于添加250 mg·L-1 Cef的MS培养基上培养,抑菌效果好;脱菌后,用含50mg·L-1 Kan的分化培养基筛选抗性愈伤。3.从小花碱茅中克隆了高亲和性K~+转运蛋白编码基因PtHAK5,ORF框长2355 bp,推测编码784个氨基酸,具有11个跨膜区,与其他高亲和性K~+转运蛋白具有较高的同源性,为KT/HAK/KUP家族第Ⅰ亚族成员。4.PtHAK5的超表达能够恢复K~+吸收功能缺失的酵母突变菌株CY162在低K~+条件下的生长,而不会增加Na+外排功能缺失的突变菌株G19的盐敏感性,表明PtHAK5只介导K~+吸收,不参与Na+转运。
[Abstract]:Potassium is the second most important mineral nutrient in plants, which is very important for plant growth and crop production. Salinization is an important factor affecting agricultural production and ecological environment. Land salinization has become one of the main limiting factors for the development of agricultural production in China. Because of the similarity between Na and K ~ in structure and chemical properties, excessive Na accumulation in plants can interfere with the absorption of K ~, inhibit the growth and development of plants and even lead to plant death. Halophytes in nature have formed special adaptation mechanism in the process of resisting external saline-alkali stress for a long time. The mechanism of salt tolerance of these plants is studied systematically and the halophytes adapted to salt soil environment are screened and cultivated. It is of great significance for the improvement of saline-alkali land ecosystem and the sustainable development and utilization of land resources. As a typical salt-repellent halophyte, the main salt tolerance mechanism of Festuca florescens is to limit the one-way Na influx of roots and reduce the net accumulation of Na to maintain strong K- / Na selectivity. It can be seen that Flos lanceolata has a strong ability to regulate K ~ + Na balance in vivo. But there are few systematic studies on K ~ + Na absorption and transport system. In this study, the genetic transformation system of Festuca flosiflora was established, and the gene PtHAK5 of KT / HAKR / KUP family was cloned by RT-PCR, and the function of PtHAK5 was verified by yeast heterologous expression test. The main results are as follows: 1. The optimal hormone combinations for callus induction were 5.0 mg L ~ (-1) 2H ~ (4-D) and 0.5 mg ~ (-1) KT, and the decrease of 2H _ 4-D content was 2.5 mg / L ~ (-1) during subculture.) the callus differentiation rate was increased by 0.5 mg 路L ~ (-1) NAA ~ (2 mg 路L ~ (-1) 6-BA and 2.0 mg ~ (-1) KT. Increase the regeneration rate of seedlings, It is the best hormone combination for callus differentiation of Flos alkaloids. 2) 100 mg L-1 hydrolysate of casein 100 mg L-1 proline and 300 mg L-1 glutamine) and 0.3% plant gel can effectively prevent vitrification of callus and significantly increase the rate of embryogenic callus. The callus was subcultured for 3-4 times and precultured on MS medium supplemented with 10 g L-1 glucose and 200 渭 M L-1 as for 4 days. The cell structure was beneficial to the integration of exogenous DNA. The ability of callus to resist the infection injury of Agrobacterium tumefaciens is strong. The precultured callus was infected with GV3101 Agrobacterium tumefaciens solution with OD600 0.6 for 15 min, and then co-cultured on MS medium supplemented with 200 渭 M L-1 as for 2 days. The callus co-cultured for 2 days was soaked in 250 mg L-1 Cef solution for 20 min and then cultured on MS medium supplemented with 250 mg L-1 Cef. The antimicrobial effect was good, and the resistant callus was screened by the differentiation medium containing 50mg L-1 Kan. The high affinity K ~ transporter encoding gene PtHAK5 ORF was cloned from Festuca chinensis with a length of 2355 BP, presumably encoding 784 amino acids, with 11 transmembrane regions, with high homology with other highly compatible K ~ transporters. The overexpression of KT / Hak / KUP family member. 4. PtHAK5 overexpression could restore the growth of yeast mutant CY162 without increasing the salt sensitivity of the mutant strain G19, which was deficient in K ~ absorption function, but could not increase the salt sensitivity of mutant strain G19, which was deficient in Na efflux function. PtHAK5 only mediates K ~ absorption and does not participate in Na transport.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2;S543.9

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