DTT诱导的内质网应激基因表达及其对花青素合成的影响

发布时间：2018-07-01 20:53

本文选题：不结球白菜 + 内质网应激　；参考：《南京农业大学》2016年硕士论文

【摘要】：不结球白菜(Brassica rapa ssp.chinensis),又称青菜,原产于中国。由于其口感好,营养丰富,深受广大人民喜爱,特别是在南方,更是人们的家中常备蔬菜之一。随着生活质量的提升,人们对不结球白菜的需求也日益多样化,如何在保证口感的同时提高产量以及提高其有益次级代谢产物,如花青素、维生素等,已经成为人们重点关注的问题。花青素作为植物的二级次生代谢产物,在植物生理及食物营养方面的重要性日益凸显。近年来关于它的报道越来越多,因此提高其含量及稳定性等方面的研究日益重要。而在紫色不结球白菜中花青素含量极高,此外不结球白菜的产量极高,因此提高不结球白菜中花青素的含量具有很高的经济价值。本研究以南京农业大学白菜系统生物学实验室提供的两种不同品系的不结球白菜为试验材料,即NJZX1-3和NJZX1-0。其中NJZX1-3在成长过程叶的正面是紫色,背面为绿色,受到胁迫后背面也会产生部分紫色;而NJZX1-0是NJZX1-3的绿色突变体,无论在何种条件下,其颜色变化不显著;用DTT(二硫苏糖醇)对两种材料处理后,发现其在形态和生理方面会出现不同的响应现象,于是重点分析了 DTT诱导的内质网应激(ER应激)并引起自噬与花青素之间的关系后,再通过BLAST获得了与拟南芥中与内质网应激响应相关基因的同源基因BrcbZIP60,通过生物信息学和分子生物学方法初步断定其功能。这些研究为进一步研究花青素与自噬的关系奠定了基础。具体结果如下:1.DTT处理后的形态观察与生理指标的检测本研究以NJZX1-0和NJZX1-3两种不结球白菜为材料,在5片叶龄时用DTT喷洒材料叶片,并在0h,2 h,12 h,24 h时取样。随后测定叶片花青素的含量、抗氧化酶SOD、POD和CAT活性,以及花青素合成相关的酶BrcCHS基因和相关的由ROS引起的核基因的表达情况,在处理2 h时拍照记录。结果显示:形态上可以看出,在NJZX1-3叶片背面颜色加深。抗氧化酶的活性方面,两种材料出现不同的变化趋势,虽然都有上升趋势,但是NJZX1-0的POD,SOD,CAT等酶活性明显高于NJZX1-3;在NJZX1-3中BrcCHS基因的表达量也显著升高,而在NJZX1-0中没有显著变化;此外,核内抗氧化基因BrcGPXH在两种不同的品系中均呈现不断升高后略有下降的表达模式,NJZX1-3中的变化幅度显著低于NJZX1-0,这与之前酶活性的变化趋势相同;而另一种核内抗氧化基因BrcGST的表达量在两种品系中呈现出与之前不同的变化,即在NJZX1-3中,该基因的表达量增加程度与NJZX1-0出现显著性差别。综上所述,在DTT处理的紫色不结球白菜中花青素比没有处理的不结球白菜中提高2.5倍以上,最高产量达到了 2.7mg.L-1;NJZX1-3的变种NJZX1-0基本没有变化,此外花青素在一定程度上起到了抗氧化的作用。2.DTT诱导的内质网应激生物标记基因以及自噬相关基因表达的测定利用拟南芥相关基因通过BLAST获得不结球白菜内质网应激标志基因的同源基因BrcIRE1、BrcbZIP60、BrcERO1和BrcPDI,通过qRT-PCR测定以上基因在不同时间点的表达情况;以同样的方法获得并测定了不结球白菜中自噬相关基因BrcATG8C-1、BrcATG8C-2、BrcATG8C-3、BrcATG8D 和 BrcATI1 的表达情况。结果显示,在DTT处理的不结球白菜中,内质网应激标志基因以及自噬相关基因都有表达,其中四个内质网应激标志基因的表达模式是类似的,但是在NJZX1-3品系中,自噬相关的基因表达更为明显,而在NJZX1-0中的表达情况显著性并不高。这表明,通过引起内质网应激导致细胞自噬的过程中,花青素的合成与自噬呈现出正相关的关系。3.不结球白菜BrcbZIP60基因的克隆及初步分析为了进一步明确花青素的合成与内质网应激引起的细胞自噬之间的关系,通过BLAST获得不结球白菜的BrcbZIP60基因,并通过生物信息学与分子生物学方法对其初步分析,结果显示:通过克隆技术,成功获得了BrcbZI 60的ORF序列,它包括450 bp碱基,编码149个氨基酸,并将其构建GFP标签的载体;通过软件分析预测,BrcbZIP60基因与bZIP60基因类似;再利用瞬转技术,将之前获得的质粒转入拟南芥原生质体内,衣霉素处理后,观察该基因有大量表达。
[Abstract]:Brassica rapa ssp.chinensis, also known as green cabbage, is native to China. Because of its good taste and rich nutrition, it is very popular with the people. Especially in the south, it is one of the most common vegetables in the people's home. With the improvement of the quality of life, the demand for non heading Chinese cabbage is also increasingly diversified. Increasing production and improving its beneficial secondary metabolites, such as anthocyanin and vitamins, have become a major concern. As the two secondary metabolites of plants, anthocyanins are becoming more and more important in plant physiology and food nutrition. In recent years, more and more reports about it have been reported, thus increasing its content and stability. In the purple non heading Chinese cabbage, the content of anthocyanin is very high and the yield of the non heading Chinese cabbage is very high. Therefore, it is of high economic value to improve the anthocyanin content in the non heading Chinese cabbage. This study is based on two different strains of different strains of Chinese cabbage system biology Laboratory of Nanjing Agricultural University. The Chinese cabbage is a test material, NJZX1-3 and NJZX1-0., in which the front of NJZX1-3 in the growth process is purple, the back is green, and the back also produces some purple on the back, and NJZX1-0 is a green mutant of NJZX1-3. No matter under which conditions, the color change is not obvious; after the treatment of the two materials with DTT (two disulfidol), hair is made. There will be a different response in morphology and physiology. Then it focuses on the analysis of the DTT induced endoplasmic reticulum stress (ER stress) and the relationship between autophagy and anthocyanins. Then, the same gene BrcbZIP60, which is related to the stress response related to the endoplasmic reticulum in Arabidopsis, is obtained by BLAST, and through bioinformatics and molecular birth. The results of these studies lay a foundation for further study of the relationship between anthocyanin and autophagy. The results are as follows: the morphological observation and physiological indexes after 1.DTT treatment were based on two kinds of non heading Chinese cabbage, NJZX1-0 and NJZX1-3, with DTT spraying leaves at 5 leaf age, and in 0h, 2 h, 12 h, 2. Sampling at 4 h. Then the content of leaf anthocyanins, antioxidant enzyme SOD, POD and CAT activity, and the expression of BrcCHS gene related to anthocyanin synthesis and related nuclear gene expression caused by ROS were recorded. The photo was recorded at 2 h. The results showed that the color of the leaf on the back of the NJZX1-3 blade was deepened and the antioxidant enzyme activity was observed. On the other hand, the two kinds of materials have different trends, although all of them have an upward trend, but the activity of POD, SOD, CAT and other enzymes of NJZX1-0 is obviously higher than that of NJZX1-3; the expression of BrcCHS gene in NJZX1-3 is also significantly increased, but there is no significant change in NJZX1-0; moreover, the anti oxidative gene BrcGPXH in the nucleus is rising in two different strains. The change in NJZX1-3 was significantly lower than that of NJZX1-0, which was the same as that of previous enzyme activity, and the expression of BrcGST in the other nuclear antioxidant gene in the two strains was different from that before, that is, in NJZX1-3, the increase in the expression of the gene was significant with that of NJZX1-0. In summary, the anthocyanin in the purple non heading Chinese cabbage treated by DTT was more than 2.5 times higher than that in the non heading Chinese cabbage and the maximum yield reached 2.7mg.L-1; the variant NJZX1-0 of NJZX1-3 did not change basically. In addition, anthocyanins played an anti oxygen effect.2.DTT induced endoplasmic reticulum stress biologic mark. Determination of gene expression and autophagy related gene expression using Arabidopsis related genes to obtain homologous gene BrcIRE1, BrcbZIP60, BrcERO1 and BrcPDI of non heading Chinese cabbage endoplasmic reticulum stress marker gene by BLAST. The expression of the above gene at different time points was measured by qRT-PCR; the non heading White was obtained and measured by the same method. The expression of autophagy related genes, BrcATG8C-1, BrcATG8C-2, BrcATG8C-3, BrcATG8D and BrcATI1, showed that the endoplasmic reticulum stress marker gene and autophagy related genes were expressed in the non heading Chinese cabbage treated by DTT, and the expression pattern of four endoplasmic reticulum stress marker genes was similar, but in the NJZX1-3 strain, The expression of autophagy related genes is more obvious, but the expression in NJZX1-0 is not significant. This indicates that the synthesis of anthocyanins and autophagy in the process of endoplasmic reticulum stress induced autophagy is positively related to autophagy. The cloning and preliminary analysis of the BrcbZIP60 gene of.3. non heading Chinese cabbage is to further clarify anthocyanins. The relationship between synthesis and autophagy induced by endoplasmic reticulum stress, the BrcbZIP60 gene of non heading Chinese cabbage was obtained by BLAST, and its preliminary analysis by bioinformatics and molecular biological methods showed that the ORF sequence of BrcbZI 60 was successfully obtained by cloning technology, which includes 450 BP bases and 149 amino acids. It is used to construct the carrier of the GFP label; the BrcbZIP60 gene is similar to that of the bZIP60 gene by software analysis. Then the plasmids obtained before are transferred into the protoplasm of Arabidopsis thaliana by using the transient technique. After the treatment of ycomycin, the gene has a large number of expression.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S634.3

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