毡毛忍冬蕾期延长相关基因AGL15的克隆与表达

发布时间：2018-08-08 21:41

【摘要】：灰毡毛忍冬Lonicera macranthoides Hand.-Mazz.来源于忍冬科,为中药材山银花来源之一,其药材为干燥花蕾或初开的花。具有清热解毒,疏散风热之功效。用于痈肿疗疮,喉痹,丹毒,热毒血痢,风热感冒,温热发病。但是长期以来,如何解决灰毡毛忍冬蕾期短,采摘不及时是灰毡毛忍冬生产和应用中一个棘手的问题。研究工作者从1999年开始以灰毡毛忍冬的自然变异株为接穗,通过多年嫁接筛选试验,初步培育出了花蕾期长,花冠不展开的无性系的灰毡毛忍冬,命名“湘蕾金银花”品种。通过前期对灰毡毛忍冬进行转录组denovo测序分析,获得了海量功能明确、具有自主知识产权的灰毡毛忍冬基因序列,为灰毡毛忍冬的深入研究提供了大量数据,其中两个品种表达量具有明显差异的Unigene基因共20709条。其中,获得具有重要调控功能的MADS-box家族基因重要基因4条,现选取其中AGL15同源基因克隆并验证其表达。本论文以灰毡毛忍冬品种为实验材料,通过同源克隆的办法,分别从其茎、叶和各不同时期花蕾中成功分离了灰毡毛忍冬LmAGL15这个花育相关基因的cDNA全长,同时借助实时定量PCR(realtime fluorescence quantitative PCR)的方法,对该基因在不同部位灰毡毛忍冬组织及花育过程中的时空表达模式进行了分析。同时,分别构建了LmAGL15基因的过表达和干扰植物表达载体,将载体转入拟南芥,经验证后,得到部分阳性植株。主要研究结果如下:1.根据已知基因的保守序列设计并引物,采用RACE的方法,第一次从灰毡毛忍冬品种的茎、叶及各不同时期花蕾中成功获得了AGL15该相关基因的cDNA全长,命名为LmAGL15(GenBank登录号:KR028478)。序列分析表明,该基因含有MADS结构域和K结构域,属于MADS-box基因家族。2.采用实时定量PCR的方法,以18SrRNA基因为内参基因,对LmAGL15基因在灰毡毛忍冬不同部位组织中的表达差异进行分析,LmAGL15基因在不同的部位组织中有不同的表达模式,具有一定的组织特异性。其中LmAGL15主要在花蕾中表达,叶和茎中几乎没有表达。对LmAGL15基因在灰毡毛忍冬花育过程中的表达差异进行分析,该基因在花育过程中有具有一定的时间特异性。其中LmAGL15在花分化前期表达最高,后期表达最低。3.成功构建了LmAGL15的过表达和干扰植物表达载体,转化拟南芥,经验证,初步得到阳性植株。4.LmAGL15基因在拟南芥中过表达,通过对转基因植株表型发现,转基因拟南芥都能够延迟开花。初步表明LmAGL15基因可能对花期调控具有重要的作用。
[Abstract]:Lonicera macranthoides Hand.-Mazz. It comes from Lonicera, which is one of the Chinese medicinal materials, and its medicinal material is dried flower bud or first blooming flower. It has the effect of clearing heat and detoxifying, dispersing wind and heat. Used for treating carbuncle, larynx, erysipelas, heat-toxic blood dysentery, wind-heat cold, warm fever. But for a long time, how to solve the problem of short bud period and untimely picking is a thorny problem in production and application of Lonicera japonica. Since 1999, the natural variant plant of Lonicera feldis was used as scion. Through many years of grafting screening experiment, the clone of Lonicera feldis with long bud stage and non-spreading flower crown was preliminarily cultivated and named "Honeysuckle in Hunan bud". Through denovo sequencing and analysis of the transcription group of Lonicera feldis in the early stage, a large number of functional and independent intellectual property rights gene sequences were obtained, which provided a large number of data for the further study of Lonicera feldis. There were 20709 Unigene genes with significant difference in expression between the two varieties. Among them, 4 important genes of MADS-box family were obtained, among which AGL15 homologous genes were cloned and their expression was verified. In this paper, LmAGL15, a floral related gene, was isolated from the stem, leaves and buds of Lonicera tenuifolia by homologous cloning. At the same time, the temporal and spatial expression patterns of the gene in different parts of Lonicera feldis were analyzed by real-time quantitative PCR (realtime fluorescence quantitative PCR). At the same time, the overexpression and interference plant expression vectors of LmAGL15 gene were constructed, and the vectors were transferred into Arabidopsis thaliana. After verification, some positive plants were obtained. The main results are as follows: 1. According to the conserved sequence of known gene and primer, the cDNA length of AGL15 gene was successfully obtained from stem, leaf and flower buds of Lonicera feldis by RACE method for the first time, and named LmAGL15 (GenBank accession number: KR028478). Sequence analysis showed that the gene contained MADS domain and K domain, belonging to the MADS-box gene family. The expression differences of LmAGL15 gene in different parts of Lonicera feldis were analyzed by using real-time quantitative PCR and 18SrRNA gene as internal reference gene. There were different expression patterns of LmAGL15 gene in different tissues. It has a certain tissue specificity. LmAGL15 was mainly expressed in buds, but hardly in leaves and stems. The difference of expression of LmAGL15 gene in flowering process of Lonicera feldis was analyzed. The expression of LmAGL15 was the highest in the early stage of flower differentiation and the lowest in the late stage. The overexpression and interfering plant expression vector of LmAGL15 was successfully constructed, and transformed into Arabidopsis thaliana. The positive plant. 4. LmAGL15 gene was overexpressed in Arabidopsis thaliana. Through the phenotype of transgenic plants, it was found that transgenic Arabidopsis could delay flowering. It is suggested that LmAGL15 gene may play an important role in the regulation of flowering.
【学位授予单位】：湖南中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S567.79

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