中间锦鸡儿组织培养体系的建立及其两个PP2C基因的克隆与表达分析

发布时间：2018-02-15 08:41

本文关键词： 中间锦鸡儿组织培养 CiPP2C8-like基因 CiPP2C27-like基因　出处：《内蒙古农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：中间锦鸡儿抗旱、耐寒,对沙地环境有很强的适应能力,具有防风固沙,保持水土,改善局部小环境的作用,对保护和恢复生态平衡有着重要的意义。近二十年来,我国西北地区生态环境日趋恶化,锦鸡儿属在沙地植被恢复进程中常作为主要物种。因此,尽快培育出具有优良遗传特性的中间锦鸡儿品种迫在眉睫。植物在生长过程中,很难逃避各种变化不定的灾害性环境因素,如低温、干旱和高盐等,为适应环境的胁迫而形成了不同的生理生化机制。其中蛋白磷酸酶PP2C参与植物的生长发育、细胞周期、信号转导和渗透胁迫等各种生物学过程。本研究以中间锦鸡儿茎段为外植体,经器官再生途径长出植株,探索主要影响因素与培养效果的关系,初步建立了中间锦鸡儿组织培养体系,为中间锦鸡儿品种的改良和遗传转化奠定基础;对CiPP2C8-like和CiPP2C2 7-like基因进行了克隆和生物信息学分析,通过qRT-PCR技术发现CiPP2C8-like基因受盐和脱水胁迫诱导。主要研究结果如下:1.以中间锦鸡儿茎尖和茎段分别为外植体进行了愈伤诱导,结果显示茎段比茎尖的愈伤诱导率高。2.利用SPSS19软件设计GA3、6-BA、NAA和活性炭AC的四因素三水平正交试验。以中间锦鸡儿的茎段为外植体,在GA3 0.15mg/L+6-BA 0.2mg/L+NAA 0.4mg/L+AC 0.1g/L的MS培养基中,愈伤诱导率为33.33%;影响愈伤诱导的因素顺序依次为GA3、NAA、AC和6-BA;最佳激素配比为GA3 0.2mg/L+6-BA 0.2mg/L+NAA 0.4mg/L+AC 0.3g/L。3.在6-BA1.0mg/L+NAA0.2mg/LMS培养基中,丛生芽诱导率为13.33%,长势良好,有 3-5 个丛生芽;在 GA30.05mg/L+6-BA0.05mg/L+KT 1.0mg/L MS 培养基中,丛生芽诱导率为20.00%,但只有1-2个丛生芽,部分褐化。4.丛生芽长至2-3cm时,用手术刀剥离,在GA30.15mg/L+IAA0.5mg/L的MS培养基中生根率最高,生根率为60.00%。将经过生根培养后的小植株从培养瓶中取出,移栽成活率为100%。5.克隆得到了CiPP2C8-lik 和CiPP2C27-like基因cDNA全长序列。CiPP2C8-like基因的cDNA长为999 bp,共编码333个氨基酸,起始密码子为ATG,终止密码子为TAG;CiPP2C27-like基因的cDNA长为1149 bp,共编码383个氨基酸,起始密码子为ATG,终止密码子为TAG。以上两个基因所编码的蛋白均属于亲水蛋白。6.利用实时荧光定量PCR技术检测胁迫处理后CiPP2C8-like和CiPP2C27-like基因表达量的变化。实验结果显示,盐胁迫处理后,CiPP2C8-like基因表达量上调,在48h最高,达到未处理的33倍;脱水胁迫处理后,CiPP2C8-like基因表达量上调,在12h最高,达到未处理的252倍。7.构建了表达载体 CiPP2C8-like-HA。
[Abstract]:Caragana intermedium has the functions of drought resistance, cold tolerance, strong adaptability to sandy land environment, wind and sand prevention, soil and water conservation, and improvement of local environment, which is of great significance to the protection and restoration of ecological balance. The ecological environment of northwest China is deteriorating day by day. Caragana is often used as the main species in the process of vegetation restoration in sandy land. Therefore, it is urgent to cultivate the species of Caragana intermedium with excellent genetic characteristics as soon as possible. It is difficult to escape from various environmental factors, such as low temperature, drought and high salt, which have formed different physiological and biochemical mechanisms to adapt to environmental stress. Protein phosphatase (PP2C) is involved in plant growth and development, cell cycle, etc. In this study, the stem segments of Caragana intermedia were used as explants to grow plants through organ regeneration pathway, and to explore the relationship between the main factors and the culture effect, and other biological processes such as signal transduction and osmotic stress. The tissue culture system of Caragana intermedia was established preliminarily, which laid the foundation for the improvement and genetic transformation of Caragana intermedia, cloning and bioinformatics analysis of CiPP2C8-like and CiPP2C2 7-like genes. CiPP2C8-like gene was induced by salt and dehydration stress by qRT-PCR technique. The main results were as follows: 1. Callus induction was carried out with stem tip and stem segment of Caragana intermedia, respectively. The results showed that the callus induction rate of stem segment was higher than that of stem tip. Using SPSS19 software to design four factors and three levels orthogonal experiment of GA _ 3N _ 6-BANAA and activated carbon AC. The stem segment of Caragana intermedia was used as explant in MS medium of GA3 0.15mg / L 6-BA 0.2mg / L NAA 0.4mg / L AC 0.1 g / L. The rate of callus induction was 33.33, the order of factors affecting callus induction was GA _ 3N _ NAA _ (AC) and 6-BA, the best hormone ratio was GA3 0.2mg / L 6-BA 0.2mg / L NAA 0.4mg / L AC 0.3g / L. 3.In 6-BA 1.0mg / L NAA0.2mg/LMS medium, the induction rate of cluster buds was 13.3333, the growth rate was good, and there were 3-5 cluster buds. In GA30.05mg/L 6-BA 0.05 mg / L KT 1.0 mg / L MS medium, the induction rate of cluster buds was 20.00%, but there were only 1 or 2 clusters of buds, and partial browning. 4. When the buds grew to 2-3 cm long, the rooting rate was the highest in MS medium of GA30.15mg/L IAA0.5mg/L when the buds grew to 2-3 cm, and the rooting rate was the highest in MS medium of GA30.15mg/L IAA0.5mg/L. The rooting rate was 60. 00. the rooting rate of the small plant was extracted from the culture bottle, and the survival rate of transplanting was 100. 5. The length of cDNA of the cDNA sequence of CiPP2C8-lik and CiPP2C27-like gene was 999bp. encoding 333 amino acids. The initial codon is ATG, and the termination codon is TAGN CiPP2C27-like gene. The cDNA length of the gene is 1149bp. it encodes 383 amino acids. The initial codon is ATG and the termination codon is tag. The proteins encoded by the above two genes belong to hydrophilic protein .6.Real-time fluorescence quantitative PCR technique is used to detect the changes of CiPP2C8-like and CiPP2C27-like gene expression after stress. After salt stress, the expression of CiPP2C8-like gene was up to 33 times at 48h, and the expression of CiPP2C8-like gene was up to 252 times at 12h after dehydration stress. The expression vector CiPP2C8-like-HA was constructed.
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2;S793.3

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