农杆菌介导的黑麦草丛生芽芽尖的遗传转化及转基因植株再生

【作者】 姜素云；

【导师】 杨爱芳；

【作者基本信息】 山东大学 ， 细胞生物学， 2005， 硕士

【摘要】 黑麦草是具有世界栽培意义的禾本科牧草和草坪草,其遗传转化常用的方法是基因枪法,此外有硅碳纤维介导法以及农杆菌介导法,受体体系一般是经离体培养获得的胚性愈伤组织、悬浮细胞系和原生质体。目前黑麦草遗传转化方法尚存在一些缺点,如组织培养时间长、受基因型限制、愈伤组织经长期继代培养易出现体细胞无性系变异、转化周期长等,只从少数品种获得了少量的转基因植株。因此,建立一套高效、快速、不受基因型限制的黑麦草遗传转化体系是加快黑麦草基因工程发展的关键。 本工作以优良黑麦草栽培品种海湾（一年生）、泰垂莱特（多年生）的黄化丛生芽的裸露芽尖分生组织作为遗传转化的受体,通过农杆菌介导法进行遗传转化,将来自大肠杆菌编码胆碱脱氢酶的betA基因转入两种基因型黑麦草细胞中,获得了可育的转基因植株。研究了影响黑麦草芽尖转化效率的因素,优化了转化体系。具体内容如下: 黑麦草离体丛生芽培养体系的建立:以黑麦草无菌种子苗的茎尖生长点部分为外植体,在附加2.0mg/L 6-BA和0.5mg/L 2,4-D的诱导培养基上诱导丛生芽发生,在附加2.0mg/L 6-BA的增殖培养基上继代培养,建立起高效的黑麦草丛生芽离体培养体系。 黑麦草丛生芽芽尖的遗传转化:取在增殖培养基上培养8-10天的丛生芽块剥成1mm大小的单芽或小芽块,尽可能地裸露出生长点,在携带质粒pCAMBIA1300-betA-als的农杆菌LBA4404浸染液中（加入0.1mmol/L乙酰丁香酮）浸染,同时在0.5×10⁵Pa负压下处理5min,无菌滤纸略吸小芽块上的残留菌液后,于丛生芽增殖培养基上共培养3天,然后转移到加有100mg/L头孢霉素的抑菌培养基上培养8天,再转移到加有3-5mg/L绿磺隆的筛选培养基上连续筛选3代,每代15天。存活的小芽在无除草剂的培养基上恢复培养后,转入生根培养基,20天后,移栽入上层蛭石、下层壤土的花盆中。2个月后对移栽成活植株进行PCR检测,确定转基因植株。 研究了影响黑麦草遗传转化效率的因素,优化了转化条件,以继代培养8-10天的丛生芽为受体,浸染液浓度OD₆₀₀为0.5,加入0.1mmol/L乙酰丁香酮,在0.5x10⁵Pa负压下浸染5min,抗性芽比例较高,最高达到32.7%。该转化体系以黄化丛生芽裸露芽尖为转化受体,基因型依赖性小,仅需3-4个月即可获得转基因植株,且转化效率较高,这为黑麦草基因工程育种打下了良好的基础。更多还原

【Abstract】 Ryegrass (Lolium multiflorum Lam. and Lolium perenne L.) is one of the important grass species in the world. Genetic engineering may contribute to the development of improved ryegrass cultivars for forage and turf purpose as conventional selection procedure is slow. Transgenic plants of ryegrass have been reported using microprojectile bombardment, silicon-carbide fibre-mediated and Agrobacterium-mediated transformation with embryogenic calli, suspension cells and protoplasts as recipient However, these ryegrass transformation systems were basically influenced by the regeneration potential and genotype of donor plant, long in vitro culture period, resulted in only a few transgenic ryegrass plants have been obtained successfully. Hence it is prerequisite to establish an efficient transformation system for ryegrass genetic breeding. Here we reported an efficient procedure for ryegrass genetic transformation involving bud tips of multiple bud clumps derived from etiolated shoot tips of sterile seedlings from two elite ryegrass cultivars Gulf (annual ryegrass) and Tetrilite (perennial ryegrass) and many fertile transgenic plantlets were obtained.The first step to establish the transformation system is to establish an in vitro system with higher regeneration frequency. Bud clumps were successfully induced from shoot tips of ryegrass in order to develop an efficient propagation system. The percentage of bud clumps was significantly improved on induction medium containing 2.0mg/L 6-BA and 0.5mg/L 2,4-D, and multiple bud clumps were obtained on proliferation medium containing 2.0mg/L 6-BA.The single bud (about 1mm in height) and bud clumps derived from the multiple bud clumps subcultured for 8-10 days were infected with Agrobacterium LBA4404 harboring plasmid pCAMBIA1300-betA-als with 0.5×10~5Pa pressure for 5 minutes and co-cultured for 3 days on induction medium after blotted on sterile filter paper. Then, the inoculated explants were transferred to medium supplemented with 100mg/L cefotaxime to inhibit the growth of Agrobacterium for 8 days and further 45 days subcultures on medium containing 3-5mg/L lvhuanglong (chlorsulfuron) at a 15-day interval. Survival buds were transferred to medium without herbicide for recovery growth and 20 days later these recovery buds were transferred onto rooting medium to induce root formation. Rooted plantlets were transferred to plastic pots containing medium composed of autoclaved vermiculite-soil (1/1, v/v) and transgenic plants were obtained after PCR assay to the transplants 2 months later.Factors related with the genetic transformation were investigated respectively. Higher percentage of resistant buds (chlorsulfuron-resistant buds/infected buds) was obtained under the following conditions: explants from multiple bud clumps subcultured for 8-10 days were inoculated in Agrobacterium suspension of OD6oo=0.5 supplemented with O.lmmol/L acetosyrigone for 5 min under O-SxK^Pa pressure, and the highest percentage of resistant-buds reached 32.7%. This transformation procedure used shoot tips of etiolated multiple bud clumps as target, avoiding genotype-dependence and produced transgenic plants within a short period (3-4 months) with higher transformation efficiency.PCR assay results of the "I*! plants after open pollination showed that out of 21 transgenic lines, 3 were PCR positive in Ti generation, indicating that the transformation procedure using bud tips of ryegrass as explants mediated with Agrobacterium is feasible in ryegrass genetic breeding. The seeds (Tj) and non-transgenic controls were sown in pots full of sand and irrigated with 1.0%NaCl solution for 10 days and further 20 days with 2.0%NaCl solution. The salt-tolerance of T] was improved at least 0.5%NaCl than the controls based on the results of the survival rate of the seedlings.更多还原