AtCKX3基因转化东方百合‘索邦’的研究

发布时间：2018-06-24 09:30

本文选题：百合 + 遗传转化　；参考：《山西农业大学》2016年硕士论文

【摘要】：百合(Lilium spp.),百合科百合属多年生草本球根植物,是一类我国资源丰富,兼具营养药用价值,观赏价值和商业价值的栽培花卉品种。传统的百合育种方式受到育种周期长,后代分离复杂等因素限制,使得百合定向选育困难。而分子育种方式可以弥补其不足,在短时间内实现新品种的定向选育。但是由于百合基因组庞大,外源基因与受体基因间互作方式不清楚,常导致外源基因沉默不表达,转化效率低。本研究以东方百合‘索邦’(Lilium Oriental'Sorbonne')鳞片为试材,通过三种不同的转化方式,即以喷雾式植物反应器(开放式培养)为基础的遗传转化方式,以组织培养为基础及以花粉管通道法为基础的遗传转化方式,分别建立了相应的高效无抗生素标记的百合遗传转化体系,对百合鳞片进行外源基因AtCKX3的转化,并成功获得了转基因植株,以期为提高百合抗逆性研究提供参考,对寻求可提高百合基因转化效率的新的转化系统做出尝试,获得的研究结果如下：1.基于喷雾式植物反应器的AtCKX3基因转化索邦的研究(1)对‘索邦’幼苗进行不同除草剂草丁膦(PPT)浓度的喷施,通过喷施后百合幼苗的植株叶色、形态变化等表型特征,筛选出PPT喷施的临界浓度为8.5 mg·L-1。(2)首次建立了将喷雾式植物反应器与农杆菌介导的遗传转化手段相结合的新的遗传转化体系。研究结果表明喷雾式反应器具有操作简便,成本低廉,对操作要求低等优点,更易于实现外源基因转化工作的大量操作。并且通过分子检测,初步证明了转基因植株的获得,转化率为0.29%左右。此外,对转化植株进行移栽后发现,植株移栽的成活率也有很大提高,可达75%,证明了该遗传转化体系的可行性。2.基于组织培养的4tCKX3基母转化索邦的研究(1)将以两种不同培养基(MS+0.01 mg·L-1 TDZ+0.2 mg·L-1NAA; MS+2.0 mg·L-16-BA+0.2ng·L-1NAA)培养的百合鳞片的分化效率和生长状态进行比较,结果表明培养基激素配比：0.01 mg·L-1 TDZ+0.2 mg·L-1 NAA较后者可更高效直接地诱导出鳞茎苗,更利于百合‘索邦’外源基因转化受体系统的建立。(2)对‘索邦’离体再生鳞片分别进行了相应的抑菌剂羧苄(Cb)浓度以及除草剂草丁膦(PPT)浓度筛选。结果表明,‘索邦’组培鳞片对抗菌剂羧苄(Cb)的耐受浓度为500 mg·L-1,草丁膦筛选的临界浓度为2.5 mg·L-1。(3)通过农杆菌介导法对‘索邦’无菌苗鳞片进行了转化,并对抗性植株进行了PCR检测,初步证明目的基因AtCKX3已整合到百合基因组中,转化效率为0.17%。3.基于花粉管通道法的AtCKX3基因转化索邦的研究对以花粉管通道法为基础的遗传转化方式进行了初探。通过对东方百合杂交系的4个栽培品种和铁炮百合(雷山1号)开花植株为材料,进行最优杂交组合和合适的DNA穿膜肽溶液导入方式的筛选。结果表明,杂交组合(铁炮×索邦)结实率,种子的有胚率均为最高,分别为94.74%,18.31%。而直接对柱头进行涂抹,注射与涂抹相结合的两种方式对百合植株造成的伤害较小,适宜DNA穿膜肽溶液的导入。
[Abstract]:Lilium (Lilium spp.), Lilium Lilium perennial herb, is a kind of cultivated flower species, which is rich in resources and has both medicinal and medicinal value, ornamental and commercial value in our country. The traditional breeding method of lily is limited by long breeding cycle and complicated separation of offspring, which makes it difficult to select Lilium orientated breeding. The method can make up for the deficiency and realize the directional breeding of new varieties in a short time. However, because of the huge genome of Lilium, the interaction mode between the foreign gene and the receptor gene is not clear, which often leads to the silence of the foreign gene and the low conversion efficiency. The three different transformation methods, namely, the genetic transformation based on the spray plant reactor (open culture), based on the tissue culture and the pollen tube pathway based genetic transformation, established the corresponding efficient and non antibiotic labelled Lilium relic transformation system, and the exogenous gene AtCKX was carried out on the lily scales. 3 transformation, and successfully obtained transgenic plants, in order to provide reference for improving the resistance research of Lilium, and try to find a new transformation system which can improve the transformation efficiency of Lilium. The results are as follows: 1. the study on the AtCKX3 gene conversion of the spray plant reactor (1) to the seedlings of Sorbonne By spraying PPT concentration of different herbicides, the critical concentration of PPT spraying was selected as 8.5 mg. L-1. (2) by spraying the leaf color and morphological characteristics of Lilium seedlings after spraying, and the new genetic transformation system combining the spray plant reactor with Agrobacterium mediated genetic transformation was established for the first time. It shows that the spray reactor has the advantages of simple operation, low cost and low operating requirements. It is easier to achieve a large number of operation of exogenous gene transformation. And through molecular detection, it has been proved that the conversion rate of transgenic plants is about 0.29%. In addition, the survival rate of transplanting is also found after transplanting. A great increase, up to 75%, proves the feasibility of the genetic transformation system.2. based on the tissue culture 4tCKX3 base mother transformation Sorbonne (1) will compare the differentiation efficiency and growth state of the lily scales in two different cultures (MS+0.01 mg. L-1, TDZ+0.2 mg. L-1NAA; MS+2.0 mg. L-16-BA+0.2ng L-1NAA). 0.01 mg. L-1 TDZ+0.2 mg. L-1 NAA can be more efficient and direct induction of bulb seedlings than the latter, which is more beneficial to the establishment of exogenous gene transformation receptor system in Lilium. (2) the concentration of bacteriostat carboxylic benzyl (Cb) and herbicide butylphosphine (PPT) concentration sieves are carried out respectively. The results showed that the tolerance concentration of "Sorbonne" tissue culture scale against bacterial agent carboxybenzyl (Cb) was 500 mg. L-1, and the critical concentration of chlorinyl phosphine screening was 2.5 mg. L-1. (3) through agrobacterium mediated transformation of "Sorbonne" aseptic seedlings, and the antagonistic plants were detected by PCR. It was preliminarily proved that the target gene AtCKX3 had been integrated into the lily base. In the group, the conversion efficiency was 0.17%.3. based on the pollen tube channel method of AtCKX3 gene transformation Sorbonne. The genetic transformation method based on the pollen tube pathway was studied. 4 varieties of oriental lily hybrid and the flowering plant of Lilium Lilium (Lilium 1) were used as the material, and the optimal hybrid combination and appropriate DN were carried out. The results showed that the seed setting rate of the hybrid combination (iron gun, SAC) was the highest, and the seed embryo rate was the highest, which was 94.74%, 18.31%. respectively, which was applied directly to the stigma, and the injections of the two methods combined with the injection and smearing were less harmful to the lily plants and suitable for the introduction of DNA membrane peptide solution.
【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S682.29

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