红掌遗传转化体系与抗菌肽基因表达载体的构建

发布时间：2017-12-28 11:04

本文关键词：红掌遗传转化体系与抗菌肽基因表达载体的构建　出处：《苏州大学》2015年硕士论文　论文类型：学位论文

【摘要】：红掌(Anthurium andraeanum)为天南星科花烛属多年生草本花卉,因其花叶形态奇特,花期持久,常作为切花材料;盆栽品种株型优美,是广泛应用于室内装饰的花卉。但是,红掌在栽培生产过程中,极易受到细菌性叶斑病的侵害,严重时造成毁灭性损失,这是国内外红掌生产上的一大障碍。因此,开展抗病基因工程研究、培育抗病品种具有重要理论意义及应用价值。本研究在前人研究的基础上,就红掌的再生体系、农杆菌介导的遗传转化体系、抗菌肽基因及其修饰、融合基因及植物表达载体、农杆菌转化等方面进行了研究,取得了以下实验结果。1、优化了红掌的再生体系。以盆栽红掌品种SYN-A的愈伤组织为供试材料,以1/2MS培养基为基本培养基,探讨了不同生长素(2,4-D、NAA、IBA)、不同细胞分裂素(6-BA、TDZ)及其浓度配比对红掌愈伤组织不定芽分化的影响。在本试验条件下,红掌愈伤组织的不定芽分化率都达到了76.67%以上,其中最优培养基组合为1/2MS+6-BA 0.5 mg/L+2,4-D 0.1 mg/L,愈伤组织的不定芽分化率可达100%,再分化系数高达31.06,且不定芽生长良好。2、探明了红掌对三种抗生素的敏感性差异。试验结果表明,红掌外植体对潮霉素的敏感性高于卡那霉素,前者更适用于红掌转基因组织的筛选,其使用浓度以20mg/L为宜;其次,红掌外植体对头孢霉素的敏感度较低,在添加浓度为600 mg/L、培养8周时,外植体依然能正常生长,试验时使用浓度500 mg/L即可达到良好的抑菌效果。3、初步建立了农杆菌介导的红掌遗传转化体系。以红掌愈伤组织为受体材料,以农杆菌EHA105为供试菌种,通过正交试验方法,探讨了外植体预培养时间、农杆菌侵染时间、超声波处理时间和共培养时间等对遗传转化的影响,其影响程度依次为共培养时间预培养时间侵染时间超声时间。初步建立了适用于红掌的遗传转化体系:切取1 cm×1 cm×0.5 cm大小的愈伤组织块、预培养3 d,以OD600值为0.6的菌液、侵染时间20 min,侵染期间超声波处理20 s,无需共培养,而后经抑菌培养、筛选培养,可获得抗性愈伤组织。4、构建了含抗菌肽融合基因的植物表达载体及工程农杆菌。应用生物信息学方法,以病程相关蛋白PR1a的信号肽与抗菌肽Shiva-1为基本序列,利用红掌偏爱密码子进行修饰优化后,合成了含特异酶切位点的抗菌肽融合基因(命名为Aa AMP),并连接入中间载体p CAMBIA1301,成功构建了植物表达载体p CAMBIA1301-Aa AMP,进而转化农杆菌EHA105获得了工程菌株。
[Abstract]:Anthurium (Anthurium andraeanum) for Araceae Anthurium perennial herbaceous flowers, flowers and leaves because of its peculiar shape, long flowering, often used as cut flower material; potted plant types is exquisite, is widely used in indoor decoration flower. However, in the cultivation of Anthurium production process, vulnerable to bacterial leaf spot damage, serious when a devastating loss, which is home to a large obstacle on Anthurium production. Therefore, it is of great theoretical significance and application value to carry out the research on the genetic engineering of resistance to disease and to cultivate the disease resistant varieties. This study on the basis of previous research on Anthurium regeneration system and Agrobacterium mediated genetic transformation system, antimicrobial peptide gene and its modification, fusion gene and plant expression vector and Agrobacterium transformation and other aspects of the study, obtained the following results. 1, optimize the regeneration system of anthurium. The potted anthurium callus variety SYN-A as the tested material, with 1/2MS as basic medium, effects of different auxin (2,4-D, NAA, IBA) and cytokinins (6-BA, TDZ) of different concentration ratio and its influence on Anthurium callus differentiation of adventitious buds. In this experiment, the callus differentiation rate of adventitious buds have reached more than 76.67%, the optimal medium was 1/2MS+6-BA 0.5 mg/L+2,4-D 0.1 mg/L combination, the callus differentiation rate of adventitious buds was 100%, and the differentiation coefficient was 31.06, and the growth of adventitious buds were good. 2, proved the difference in sensitivity to three antibiotics of anthurium. The test results show that Anthurium andraeanum explants of hygromycin sensitivity is higher than that of kanamycin screening, the former is more suitable for the use of transgenic Anthurium tissue, the concentration should be 20mg/L; secondly, on Anthurium andraeanum explants cephalosporin sensitivity in low concentration was 600 mg/L and cultured for 8 weeks. The explants still can grow normally, the test using concentration of 500 mg/L can achieve good antibacterial effect. 3, preliminary established Anthurium Agrobacterium mediated genetic transformation system. With Anthurium calli, Agrobacterium tumefaciens EHA105 as tested strains, through orthogonal test method, discusses the explants culture time, Agrobacterium infection time, ultrasonic treatment time and incubation time of genetic transformation, the degree of CO cultivation time of pre culture time infection time of ultrasonic time. Establishment of genetic transformation system for Anthurium: cut 1 cm * 1 cm * 0.5 cm in size of callus block, pre cultured for 3 D, the OD600 value is 0.6, the bacteria infection time 20 min infection during ultrasonic treatment for 20 s, there is no need of culture, and then by bacteriostatic culture, Shai Xuanpei raise, can obtain the resistant callus. 4. The plant expression vector and Agrobacterium tumefaciens containing the antibacterial peptide fusion gene were constructed. Using bioinformatics method, and the antibacterial peptide Shiva-1 signal peptide pathogenesis related protein PR1a as the basic sequence, modified by optimization of codon preference of Anthurium, antibacterial peptide fusion gene containing specific restriction sites were synthesized (named Aa AMP), and connected into the intermediate carrier P CAMBIA1301, successfully constructed plant the expression vector p CAMBIA1301-Aa AMP, and then transformed into Agrobacterium EHA105 strain to obtain the engineering.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S682.14

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