甜菜红素合成关键基因的克隆及不同条件下的表达研究

发布时间：2019-01-08 11:31

【摘要】：甜菜素是存在于食用红甜菜中的天然植物色素,对食品具有良好的着色性,具有重要的食用和药用价值。甜菜素包括甜菜红素和甜菜黄素,其中甜菜红素的生物合成较为复杂,目前国内外对于甜菜红素生理方面活性的研究已经较为透彻,而从分子角度水平去解释甜菜红素的代谢过程途径,仍处于研究阶段。多年来学术界一直推测糖基转移酶、酪氨酸酶参与了甜菜红素合成代谢途径,但尚缺乏分子生物学证据。为研究在甜菜红素代谢过程中糖基转移酶基因UDP及酪氨酸酶基因TYR的功能表达调控情况,本实验首先克隆了以上两种基因,对其进行了生物信息学分析,得到了其编码蛋白质的一系列信息。以克隆得到的基因为模板设计引物,采用染色体步移技术得到了UDP及TYR基因的启动子序列,通过在线软件plant CARE对其进行了分析,根据启动子序列分析结果推测出甜菜UDP及TYR基因的表达对光照及各种有机酸类物质较为敏感。为进一步明确化学物质及光对甜菜生长及UDP、TYR基因表达的影响效果,本实验在不同环境下对甜菜苗进行了培育,探讨各生长时期不同处理条件下甜菜生长状况、甜菜红素积累量及UDP、TYR基因相对表达量之间的关系。与实践结合,考虑到甜菜的实际种植及生产,在化学物质的处理下,分别对甜菜苗进行了大豆、玉米、菊苣、秋葵、甜菜的化感物质浸提液处理,并使用不同的化感物质浸提液对甜菜进行了发芽实验;在光处理下,分别在红光、蓝光、绿光下对甜菜苗进行了培育。发芽实验中,5种化感物质对甜菜种子萌发的影响不同。甜菜和菊苣化感物质对甜菜种子萌发表现出抑制作用,大豆,秋葵,玉米化感物质为促进作用。秋葵、玉米、大豆化感物质均对幼苗的鲜重、株高的增加有促进作用,菊苣、甜菜化感物质则表现为抑制作用。此外,大豆化感物质中的对羟基肉桂酸通过抑制酪氨酸酶活性降低了甜菜色素的合成,其余四种化感物质均促进了幼苗中甜菜红素的含量。进一步证实了酪氨酸酶在甜菜色素合成中的关键作用。不同农作物由于所含具体化感物质的种类不同,对甜菜基因表达的影响各不相同。在不同的光处理下,绿光、蓝光、红光均会对幼苗的生长产生抑制作用,红光的抑制作用最为明显。绿光和蓝光的抑制作用差别不大。此外,在不同的光处理下,色素含量的总体变化趋势均为先上升后下降。蓝光促进色素的积累,说明短波长有利于色素合成。在三种光的照射条件下,TYR基因的表达量几乎一直高于对照组,说明单色光可以有效的激发TYR基因的表达。通过在不同化感物质及光下培育甜菜苗,得到基因表达水平与甜菜中甜菜红素代谢累积的关系,为甜菜红素的合成研究提供分子生物学理论基础和依据。
[Abstract]:Betaine is a natural plant pigment in edible red beet. It has good coloring property and important edible and medicinal value. The biosynthesis of beberelin is very complex, and the physiological activity of beberelin has been studied thoroughly at home and abroad. However, it is still in the research stage to explain the metabolic pathway of beet from the molecular level. For many years, it has been speculated that glycosyltransferase and tyrosinase are involved in the biosynthesis pathway of beet, but there is still no molecular biological evidence. In order to study the functional regulation of glycosyltransferase gene (UDP) and tyrosinase gene (TYR) during the metabolism of beet, the two genes were cloned and analyzed by bioinformatics. A series of information about the protein is obtained. The promoter sequences of UDP and TYR genes were obtained by chromosome step technique using the cloned genes as template design primers. The promoter sequences were analyzed by online software plant CARE. According to the results of promoter sequence analysis, it was inferred that the expression of UDP and TYR genes in beet was sensitive to light and various organic acids. In order to further clarify the effect of chemical substances and light on the growth of sugarbeet and the expression of UDP,TYR gene, sugarbeet seedlings were cultivated in different environments, and the growth status of sugarbeet was studied under different treatment conditions. The relationship between the accumulation of beet and the relative expression of UDP,TYR gene. Considering the actual planting and production of sugarbeet, soybean, corn, chicory, okra and sugar beet were treated with allelochemicals extraction solution under the treatment of chemical substances. The germination experiment of sugarbeet was carried out with different allelochemicals. The sugarbeet seedlings were cultured in red light, blue light and green light respectively under light treatment. The effects of five allelochemicals on the germination of sugarbeet seeds were different. The allelopathic substances of sugar beet and chicory showed inhibitory effect on the germination of sugarbeet seeds, soybean, okra and maize alleles were promoted. Allelopathic substances such as okra, maize and soybean allelopathy could promote the growth of seedling fresh weight and plant height, while chicory and beet allelopathy were inhibited. In addition, p-hydroxycinnamic acid in soybean allelopathy decreased the synthesis of beet pigment by inhibiting the activity of tyrosinase, and the other four alleles promoted the content of beet. The key role of tyrosinase in the biosynthesis of beet pigment was further confirmed. Different crops have different effects on sugarbeet gene expression due to the different species of specific sensitive substances. Under different light treatments, green light, blue light and red light could inhibit the growth of seedlings, and the inhibition of red light was the most obvious. The inhibitory effects of green and blue light were not different. In addition, under different light treatments, the overall change trend of pigment content was first increased and then decreased. Blue light promoted the accumulation of pigment, which indicated that short wavelength was beneficial to pigment synthesis. Under three light irradiation conditions, the expression of TYR gene was almost higher than that of the control group, indicating that monochromatic light could effectively stimulate the expression of TYR gene. The relationship between the gene expression level and the metabolic accumulation of beet was obtained by cultivating sugarbeet seedlings under different allelopathic substances and light, which provided the theoretical basis and basis of molecular biology for the biosynthesis of beberelin.
【学位授予单位】：哈尔滨工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2

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