鸢尾属植物遗传多样性评价及干旱高温胁迫生理响应机制

发布时间：2018-07-14 22:01

【摘要】：鸢尾属(Iris L.)是鸢尾科(Iridaceae)所有属中涵盖种类最多的一个属,据统计目前已知的鸢尾属植物共计300余种。鸢尾属植物生长的气候带以北温带为主,分布的区域集中在亚洲、欧洲及北美洲。我国也是鸢尾属植物的分布中心之一,已知的种有64个,变种13个,此外还有1个亚种和6个变型。鸢尾属植物属多年生草本类,花色丰富(有单色也有复色),花型多变(旗瓣与垂瓣形态、大小比例等多变),是闻名于世的宿根花卉之一。随着鸢尾属植物园艺品种的增多,其遗传背景愈发复杂。本研究以鸢尾属园艺品种和种共38份材料,利用形态学标记结合ISSR和SRAP两种分子标记方法对其遗传多样性及亲缘关系进行了评价分析。另外,结合鸢尾属植物的生产应用,测定了鸢尾属部分材料高温、干旱胁迫下的生理响应与分子机制,建立了鸢尾属植物的耐热、抗旱生理评价体系,并对实验材料的耐热、抗旱能力进行了评价。本研究获得主要结果如下:1、鸢尾属参试材料观测的29个表型性状中,变异系数差异较大。对表型性状主成分分析得出,基于表型性状分类时首先应考虑花器官性状的分类,其次是根据株型、营养器官生长以及花瓣上附属物的情况分类。主成分分析的结果也反映出表型性状的主成分贡献率集中度不明显,其中的累积贡献率呈现缓慢的增长,据此推测其中主要是相关性强的变量较分散的原因,而性状分别属于多类,由此也说明鸢尾属的表型性状在演化中的多样性十分明显,这也是其存在着丰富性状变异的主要原因。2、ISSR和SRAP这两种标记方法的多态性条带比率均为100%,但SRAP较ISSR检测出的条带数高,两种方法的多态性含量PIC值均为1,说明鸢尾属种质资源遗传差异较大,在分子水平具有非常高的遗传多样性。另外,两种分子标记对鸢尾属植物的遗传多样性分析具有较高的一致性,且ISSR和SRAP相结合的方法比单一的ISSR或SRAP分子标记对鸢尾属植物遗传多样性分析的结果与表型分类结果更为接近。采用ISSR和SRAP两种方法相结合进行分子标记聚类,其遗传距离矩阵进行Mantel检验得出相关性系数r=0.8052,可见两种标记方法结合的结果较为可靠,也能更好的反映出鸢尾属植物的遗传结构,可为其他1ii相关研究提供参考。3、前人很多研究表明鸢尾属植物种间杂交存在有一定的障碍,杂交结实率很低或不结实,而本研究中反应出德国鸢尾‘Gala Madrid’和‘Nibelungen’在有髯鸢尾中遗传距离与其他德国鸢尾品种较远,且分子标记的三种方式聚类中均与无髯鸢尾聚为一类,由此推测这两种鸢尾很有可能具有无髯鸢尾的遗传背景,可以将其作为鸢尾属植物无髯与有髯鸢尾类种间杂交的优势亲本进行种质创新。4、对鸡冠状附属物亚属在分类中存在的争议,通过本研究支持其按照Rodionenko系统作为亚属的分类地位存在。另外,琴瓣鸢尾亚属的喜盐鸢尾三种方法聚类后都与无附属物亚属的种聚在了一起,可见将喜盐鸢尾作为琴瓣鸢尾单独成为一个亚属存在的意义不大,建议将其归入无附属物亚属中。另外,无附属物亚属中马蔺、黄菖蒲、路易斯安那鸢尾都单独聚为亚类群,与其它鸢尾关系较远单独成一组,结合其形态学的差异,建议可将无附属物亚属分成几个组。5、不同鸢尾品种(种)在高温和干旱胁迫下,测定部分氧化酶和非酶活性物质生理生化指标,利用主成分分析法结合隶属函数法分别对测定指标与耐热、抗旱的相关性进行评价。结果表明,电导伤害率(x1)、叶绿素(x3)、可溶性糖(x4)及丙二醛(x5)4个指标与鸢尾属植物抗旱能力强弱的相关性较高;SOD活性(x1)、POD活性(x2)、叶绿素(x3)、可溶性蛋白(x4)及丙二醛(x5)5个指标与鸢尾属植物耐热能力相关性较高。进一步采用逐步回归法结合相关性高的生理生化指标建立了鸢尾属植物抗旱、耐热评价最优回归方程。抗旱最优回归方程:D=-439.27+1.744x1+4.913x3+0.5822x4+2.6369x5,耐热最优方程:D=29.667-0.208x1-2.502 x2+0.405x3-1.742 x4-0.036 x5。依据抗旱、耐热最优回归方程供试的10个鸢尾品种(种)抗旱性由强到弱的顺序依次为‘金娃娃’、‘笛声’、‘魂断蓝桥’、德国鸢尾、扁竹兰、西伯利亚鸢尾、马蔺、‘黑骑士’、黄菖蒲和花菖蒲;耐热性由强到弱的顺序依次为:花菖蒲、黄菖蒲、马蔺、扁竹兰、‘笛声’、‘黑骑士’、‘魂断蓝桥’、‘金娃娃’、西伯利亚鸢尾和德国鸢尾。6、从观赏性较高且同为德国鸢尾系列的4个德国鸢尾中分别筛选出2个耐热性相对较差的‘金娃娃’与德国鸢尾通过Ca Cl2处理,以及2个耐热性相对好的的品种‘笛声’和‘黑骑士’通过Ca2+的拮抗剂La Cl3处理,测定Ca2+对于鸢尾高温胁迫下的耐热性的调控作用。结论是Ca Cl2较为明显的提高了高温胁迫下金娃娃与德国鸢尾叶片中的可溶性蛋白质的浓度,这可能与Ca2+信号介导的热激蛋白的产生有关,使其在较短时间内可保持对高温的抗性,但高温胁迫时间超过其耐受能力,则呈现叶片焦枯逐渐死亡;La Cl3作为Ca2+阻断剂对笛声和黑骑士叶片的耐热性影响显著;由此,推测笛声和黑骑士依赖Ca2+信号介导来产生耐热性。7、根据基因库模式植物的热激因子基因的保守区域设计引物序列,通过PCR克隆得到鸢尾热激因子基因的片段;利用荧光实时定量PCR检测不同鸢尾品种在不同温度胁迫与不同高温胁迫时间下热激因子基因转录表达水平的变化。结果显示耐热性较高的笛声和黑骑士热激因子转录水平高于耐热性差的金娃娃与德国鸢尾;同时,Ca2+信号参与了这四个鸢尾品种叶片的热激因子转录表达的调控。
[Abstract]:Iris L. is one of the most species of all genera of the family irisfamily (Iridaceae). According to the statistics, there are more than 300 species of iris. The climate zone of the genus Iris is mainly in the north temperate zone, which is distributed in Asia, Europe and North America. China is also one of the distribution centers of the Iris plants. There are 64 species, 13 varieties, and 1 subspecies and 6 variants. Iris is a perennial herb with rich color (monochromatic color) and one of the most famous perennial flowers in the world. With the increase of Iris plants, the genetic background of iris is more complex. In this study, the genetic diversity and relationship of 38 species of Iris species and species were evaluated by two molecular markers combined with morphological markers combined with ISSR and SRAP. In addition, the physiological responses and molecular machines under drought stress were measured in combination with the production and application of Iris plants. The system of heat resistance and drought resistance evaluation of Iris plants was established, and the heat resistance and drought resistance of the experimental materials were evaluated. The main results of this study were as follows: 1, the variation coefficient of the 29 phenotypic characters observed in iris was large. The principal component analysis of phenotypic traits was based on the first classification of phenotypic traits. First, the classification of flower organs should be considered, followed by the classification of the plant type, the growth of the vegetative organs and the appendages on the petals. The results of the principal component analysis also reflect that the concentration of the principal component contribution rate of the phenotypic traits is not obvious, and the cumulative contribution rate of the phenotypic traits is slow. Because of the more scattered reasons, the characters of the characters belong to multiple categories, which also shows that the diversity of the phenotypic traits of the iris is very obvious. This is the main reason for the existence of.2, ISSR and SRAP, which are all two of the polymorphic bands of 100%, but the number of bands detected by SRAP is higher than that of ISSR, and two methods are found. The PIC value of polymorphic content is 1. It shows that the genetic diversity of iris germplasm is large and has very high genetic diversity at the molecular level. In addition, the two molecular markers have high consistency in the genetic diversity analysis of Iris plants, and the method of combining ISSR and SRAP is better than single ISSR or SRAP molecular markers. The results of genetic diversity analysis of plants are more close to the results of phenotypic classification. The combination of two methods of ISSR and SRAP is used to cluster molecular markers, and the genetic distance matrix is used to test the correlation coefficient r=0.8052 by Mantel test. It can be seen that the results of the combination of the two methods are more reliable, and can also better reflect the remains of the iris plant. The transmission structure can provide reference for other 1ii related studies. A lot of previous studies have shown that there are some obstacles in the interspecific hybridization of iris, and the hybrid seed setting rate is very low or not strong. In this study, the genetic distance of the German iris' Gala Madrid 'and' Nibelungen 'in the beard kite is far from the other German iris varieties. The three types of molecular markers are all clustered with the beard iris. Therefore, it is presumed that these two kinds of iris are likely to have the genetic background of the beard irises. They can be used as the dominant parent of the hybrid irises between the irises and the bearded irises to carry out the germplasm innovation.4, and the contention of the subgenus of the coronal appendage of the chicken in the classification. According to this study, it supports the classification status of the subgenera according to the Rodionenko system. In addition, the three methods of halophilic iris in the genus Iris are clustered together with non appendage subgenera. It is obvious that it is not of great significance to make iris iris as a subgenus as a single subgenus. In addition, the subgenus of appendages, iris iris, Huang Changpu and Louisiana irises are separated into subgroups separately, and separate from other irises. According to their morphological differences, the subgenus of non appendages can be divided into several groups of.5, and different iris varieties (species) can determine partial oxidation under high temperature and drought stress. The physiological and biochemical indexes of enzymes and non enzyme active substances were evaluated by principal component analysis and membership function method respectively. The results showed that the 4 indexes of conductance damage rate (x1), chlorophyll (x3), soluble sugar (x4) and malondialdehyde (x5) were more related to the drought resistance of Iris Plants, SOD The 5 indexes of activity (x1), POD activity (x2), chlorophyll (x3), soluble protein (x4) and malondialdehyde (x5) were highly correlated with the heat tolerance of Iris plants. The optimum regression equation for drought resistance and heat tolerance evaluation of Iris plants was established by stepwise regression method combined with high correlation physiological and biochemical indexes. The optimal regression equation of drought resistance: D=-439.27+1.7 44x1+4.913x3+0.5822x4+2.6369x5, the optimum heat tolerance equation: the drought resistance of the 10 varieties (species) of D=29.667-0.208x1-2.502 x2+0.405x3-1.742 x4-0.036 x5., based on the optimum regression equation of drought resistance and heat resistance, is in sequence of "gold dolls", 'flute sound', 'soul blue bridge', German iris, flat bamboo orchid, and Siberia iris. Iris, 'Black Knight', Huang Changpu and flower calamus; the order of heat resistance from strong to weak: Flower calamus, Huang Changpu, iris, bamboo orchid, 'flute', 'Black Knight', 'soul blue bridge', 'gold doll', Siberia iris and German iris.6, respectively sifting from 4 German irises with a higher appreciation and German iris series. 2 relatively poor heat-resistant 'gold dolls' and German irises were treated by Ca Cl2, and 2 varieties with relatively good heat resistance were treated by Ca2+'s antagonist La Cl3. The result was that Ca2+ was used to regulate the heat resistance of iris under high temperature stress. The conclusion was that Ca Cl2 increased the high temperature obviously. The concentration of soluble protein in the gold dolls and the leaves of German iris may be related to the production of heat shock protein mediated by Ca2+ signal, which can keep the resistance to high temperature in a short time, but the time of high temperature stress exceeds its tolerance, while La Cl3 is a Ca2+ blocker to the flute and the sound of the flute. The heat resistance of the Black Knight leaves is significant. Therefore, it is speculated that the flute and the Black Knight rely on the Ca2+ signal to produce heat resistance.7. According to the conserved region of the heat shock factor gene of the gene bank model plant, the primer sequence is designed and the segments of the iris heat shock factor gene are obtained by PCR cloning, and the different iris are detected by real time fluorescence quantitative PCR The transcriptional level of heat shock factor gene in different temperature stress and high temperature stress time was observed. The results showed that the transcriptional level of the flute and the Black Knight heat shock factor was higher than that of the poor heat resistant gold dolls and German irises, and the Ca2+ signal was involved in the transcriptional expression of the heat shock factor of the leaves of the four irises Regulation and control.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S682.19

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