花粉管通道介导转cbf1基因蝴蝶兰遗传转化体系构建

发布时间：2018-04-09 04:18

本文选题：蝴蝶兰　切入点：花粉管通道法　出处：《西南科技大学》2017年硕士论文

【摘要】：蝴蝶兰(Phalaenopsis ssp.)素有“兰花王后”的美誉,为热带兰珍品,具有极高的观赏价值和经济价值,在世界花卉产业中占有重要的地位。现阶段蝴蝶兰的育种主要以杂交为主,但育种周期漫长,世代选择复杂。因此进行花粉管通道法介导的蝴蝶兰转基因研究,为创造特异性优异新种质开辟途径。本文较系统地开展了花粉管通道法介导转cbf 1抗寒性基因蝴蝶兰研究,主要研究结果如下:(1)对蝴蝶兰花粉活力与柱头可授性研究表明,花粉母细胞减数分裂进程与其它植物有一定差别,其生殖发育过程较为特殊,雄配子发育完成于花蕾期,开花后的蝴蝶兰花粉母细胞已进入明显的四分体时期,生殖细胞与营养核均已形成,花粉活力率(染色率)大小为:完全开放1d花蕾期花蕾展开期,在花蕾展开期的花粉粒活性最强,之后随时间的增加,活性逐渐降低;柱头可授性测定显示,蝴蝶兰完全开花10d~30d内柱头保持较高的活性,在10d~15d内具有较高的可授性。取花蕾展开期的花粉对完全开花10d~15d的柱头授粉,可获得较高的杂交成功率。(2)对蝴蝶兰花粉管通道介导的遗传转化受体系统进行了研究,蝴蝶兰种胚非共生萌发形成原球茎的最适培养基为Hyponex(3g/L)+琼脂7.5g/L+香蕉泥100g/L+蔗糖25g/L+蛋白胨1.5g/L+活性碳2.0g/L;原球茎增殖培养基为Hyponex(3g/L)+琼脂7.5g/L+香蕉泥100g/L+蔗糖25g/L+蛋白胨2.0g/L+NAA1.0mg/L+6-BA10.0mg/L;由原球茎实现植株再生的最适培养基为1/2MS+琼脂7.0g/L+香蕉泥100g/L+蛋白胨2.0g/L+活性碳2.0g/L+蔗糖25g/L。抗生素敏感性试验表明,蝴蝶兰原球茎及再生植株在卡那霉素选择压为0~200mg/L时,抑制生长不明显,在300~400mg/L处理组中,生长明显受到抑制,当达到500mg/L及更高浓度时,存活率为0%。可确定500mg/L的Kan为转化株阳性植株筛选的选择压力。(3)以携带目的基因cbf 1的p BI121质粒和转化了cbf 1表达载体的农杆菌液,分别采用花粉粒携带法和子房注射法进行遗传转化。结果表明,花粉携带法比子房注射法有更高的结实率,但子房注射法的转化率更高;质粒直接侵染比农杆菌转化侵染,有更高的结实率,质粒侵染的最适浓度为100ng/μL,农杆菌为OD600=1.0。对Kan筛选的阳性植株进行PCR检测,证明外源cbf 1基因已整合到Kan抗性植株的基因组中。
[Abstract]:Phalaenopsis sp.)Known as "the queen of orchids", it is a tropical orchid treasure with high ornamental value and economic value, and plays an important role in the world flower industry.At present, the breeding of Phalaenopsis is dominated by cross, but the breeding cycle is long and the selection of generations is complicated.Therefore, the transgene research of Phalaenopsis mediated by pollen tube channel method will open the way for the creation of new germplasm with excellent specificity.In this paper, pollen-tube pathway mediated transformation of cbf _ 1 cold resistance gene Phalaenopsis was carried out systematically. The main results were as follows: 1) the pollen viability and stigma viability of Phalaenopsis were studied.The process of meiosis of pollen mother cells is different from that of other plants, and its reproductive development process is special. The male gametes develop at the bud stage, and the pollen mother cells of Phalaenopsis after anthesis have entered the obvious tetrad stage.Both germ cells and vegetative nuclei had been formed, and the pollen vigor rate (staining rate) was as follows: at the stage of full opening for 1 day, the activity of pollen grains was the highest at the stage of flower bud development, and then decreased with the increase of time.The determination of stigma affordability showed that Phalaenopsis had higher activity in 10d~30d and higher viability in 10d~15d.Pollen pollination on the stigma of fully blooming 10d~15d was used to obtain a high success rate of hybridization. The transgenetic transformation receptor system mediated by pollen tube channel of Phalaenopsis was studied.The optimum medium for plant regeneration was 1/2MS Agar 7.0g/L banana mud 100g/L peptone 2.0g/L active carbon 2.0g/L sucrose 25 g / L.The antibiotic sensitivity test showed that the growth inhibition of Phalaenopsis protocorms and regenerated plants was not obvious when kanamycin was selected as 0~200mg/L. In 300~400mg/L treatment group, the growth was obviously inhibited, and the survival rate of Phalaenopsis was 0 when 500mg/L and higher concentration were reached.It was confirmed that the Kan of 500mg/L was the selection pressure for the selection of the positive plants of the transformed strain. The p BI121 plasmid carrying the target gene cbf 1 and Agrobacterium tumefaciens transformed into the cbf 1 expression vector were used to carry out the genetic transformation by pollen grain carrying method and ovary injection method, respectively.The results showed that pollen carrying method had higher seed setting rate than ovary injection method, but the transformation rate of ovary injection method was higher than that of Agrobacterium tumefaciens, and plasmid direct infection had higher seed setting rate than Agrobacterium tumefaciens. The optimum concentration of plasmid infection was 100ng/ 渭 L and Agrobacterium tumefaciens OD600N 1.0.PCR analysis of Kan positive plants showed that exogenous cbf 1 gene had been integrated into the genome of Kan resistant plants.
【学位授予单位】：西南科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S682.31

【相似文献】