铁皮石斛试管苗开花及相关机理研究

发布时间：2018-03-14 19:31

本文选题：铁皮石斛　切入点：试管开花　出处：《西南交通大学》2017年硕士论文　论文类型：学位论文

【摘要】：通过组培以及一些分子生物学方面的技术,优化铁皮石斛开花条件。其主要目的是通过试管苗开花授粉得到更多的果荚,为后续胚胎及相关的实验提供物质基础。通过5种不同激素的单因子实验,得到更高的成花植株,并确定花与苗的质量优异。通过对铁皮石斛进行植物组织培养,优化铁皮石斛的开花条件,促进铁皮石斛提前成花;通过石蜡切片技术,观察铁皮石斛在由营养生长到生殖生长的转变过程中,最具代表性的茎尖的细胞切片图,观察细胞发育变化:通过RACE技术克隆出与开花相关的基因序列;采用生物信息学的方法初步分析了这些序列的一些基本性质,包括长度、亚细胞定位、等电点、二级结构等;采用实时荧光定量PCR(Quantitative real-time reverse transcription-PCR,RT-qPCR)对这些基因在不同组织,不同激素条件下的表达模式进行分析,与优化条件相结合并进行分析,可以进一步得到哪些基因与激素促进条件有关。ABA(Abscisic Acid)促进铁皮石斛的试管开花,当在栽培在ABA开花培养基中15-20天,再转移到含0.2mg·L-1 NAA和0.2mg·L-1 6-BA的诱导后培养基中,发芽数量和发芽率显著增加。当Spermidine HCL浓度为0.1 mg·L-1时,其最适宜培养的天数为20天左右,在Spermidine HCL浓度为1.5 mg·L-1时,其最适宜培养的天数为15天左右。花芽诱导率在PP333(Paclobtrazo1)0.6mg·L-1时最高,为24%。花芽诱导率在乙烯利1 mg·L1时最高,为13.3%,其致死浓度一般在2.5 mg·Ll1左右,在此时刻,铁皮石斛在诱导30d后会伴随着茎变黄,叶子枯萎凋零,并最终死亡。TDZ(Thidiazuron)可以缩短营养生长期2-3年,乙烯处理会加速营养生长到生殖生长的转变。根对铁皮石斛的花芽分化具有抑制作用。采用RACE技术,基于转录组数据,得到了 16条开花相关基因的完整cds序列。通过生物信息学的方法,得到了基因的一些基本性质。氨基酸序列长度从237bp到2958bp,分子量从19304.06ku到245735.14ku,所有序列的G+C含量都在0.44-0.54之间。通过实时荧光定量PCR技术,得到基因在不同组织、不同激素处理下的表达情况。基因在激素ABA、spermidine HCL、TDZ等处理下,与组培优化的实验结果相吻合。在用ABA预处理15天左右,铁皮石解的开花率会达到比较高的峰值;基因在激素spermidine HCL处理下,大部分的基因都在21d左右的表达量最高;基因在激素TDZ处理下,大部分的基因都在18d左右的表达量最高。4号基因Flowering Locus T、5 号基因 Flowering time control protein FCA、12 号 Flowering time control protein FPA、14号基因Flowering time control protein FC4和16号基因LEAFY表达模式比较特殊,可以筛选作为花特异性基因。这些实验结果为后续的分子机理的研究打下了基础。
[Abstract]:The flowering conditions of Dendrobium candidum were optimized by tissue culture and some molecular biological techniques. To provide the material basis for the subsequent embryo and related experiments. Through the single factor experiment of five different hormones, the higher flowering plants were obtained, and the quality of the flowers and seedlings was determined to be excellent. The plant tissue culture was carried out on Dendrobium candidum. To optimize the flowering conditions of Dendrobium candidum, to promote the early flowering of Dendrobium candidum; to observe the most representative cell sections of the stem tip of Dendrobium candidum during the transition from vegetative growth to reproductive growth through paraffin section technology. Observe the changes of cell development: clone the gene sequence related to flowering by RACE, analyze the basic properties of these sequences by bioinformatics, including length, subcellular localization, isoelectric point, secondary structure, etc. The expression patterns of these genes in different tissues and different hormone conditions were analyzed by real-time fluorescence quantitative PCR(Quantitative real-time reverse transcription-PCRR-RT-qPCR. Which genes can be further obtained to promote the flowering of Dendrobium candidum in vitro, which are related to hormone promoting conditions. When cultured in ABA flowering medium for 15 to 20 days, it was transferred to the induced medium containing 0.2 mg 路L -1 NAA and 0.2 mg 路L -1 6-BA. When the concentration of Spermidine HCL was 0.1 mg 路L -1, the most suitable culture days were about 20 days, and when the concentration of Spermidine HCL was 1.5 mg 路L -1, the most suitable culture days were about 15 days. The flower bud induction rate was the highest at PP333(Paclobtrazo1)0.6mg 路L -1. When ethephon was 1 mg 路L ~ (-1), the highest rate of flower bud induction was 13.33.The lethal concentration was about 2.5 mg 路Ll1. At this time, Dendrobium candidum would become yellow with the stem after 30 days of induction, and the leaves would wither and wither. And finally death. TDZ Thidiazuron can shorten the vegetative growth period of 2-3 years, ethylene treatment can accelerate the transformation of vegetative growth to reproductive growth. Root has inhibitory effect on flower bud differentiation of Dendrobium candidum. Using RACE technique, based on transcriptional data, The complete cds sequence of 16 flowering related genes was obtained. Some basic properties of the gene were obtained. The length of amino acid sequence ranged from 237bp to 2958bp, the molecular weight ranged from 19304.06ku to 245735.14ku. the G C content of all sequences ranged from 0.44-0.54. By real-time fluorescence quantitative PCR, the gene was obtained in different tissues. The expression of the gene under different hormone treatments was consistent with the results of tissue culture optimization under the treatment of the hormone ABA Spermidine HCLmidine TDZ etc. The flowering rate of ferrite pyrolysis reached a relatively high peak after 15 days of pretreatment with ABA. Under the treatment of hormone spermidine HCL, the expression of most genes was the highest at 21 days, and the gene was treated with hormone TDZ. Most of the genes were expressed at about 18 days. The expression patterns of Flowering time control protein Flowering time control protein FPA12, Flowering time control protein FC4 and LEAFY 16 of 4 gene Flowering Locus TG5 and 14 gene Flowering time protein FC4 and 16 gene were very special. These results provide a basis for the further study of molecular mechanism.
【学位授予单位】：西南交通大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S567.239

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