苹果MdLBD13的基因克隆及其在花青苷合成和硝酸盐吸收中的作用

发布时间：2019-03-06 14:35

【摘要】：Lateral organ boundaries domain(LBD)家族是植物中特有的一类转录因子。LBD转录因子参与调控植物顶端分生组织和侧生器官原基的启动以及边界的建立、植物体内激素的积累以及花青苷和硝酸盐代谢途径。LBD蛋白家族作为一类新发现高等植物的转录因子,目前仅在拟南芥、水稻等模式植物中揭示了部分LBD基因的功能,而在苹果中的研究相对较少。花青苷作为苹果色泽的主要成分和重要营养物质,对果实品质起到重要作用。苹果全基因组测序的完成对苹果花青苷合成相关的LBD基因的鉴定具有重要的意义。本研究从苹果中分离出一个典型的LOB-Domain的基因,并进一步分析了其表达模式。通过转基因获得超表达MdLBD13的苹果愈伤和拟南芥分析其功能,研究工作揭示了苹果中LBD基因调控花青苷的合成以及硝酸盐代谢途径。主要研究结果和结论如下:1.从"嘎拉"苹果中分离出MdLBD13(MDP0000317227)。MdLBD13基因为753 bp,编码251个氨基酸。该蛋白具典型的LOB-Domain,在它的N端具有典型的CX2CX6CX3C zinc finger-like domain,是LBD基因家族的第二类成员。氨基酸序列比较分析与系统进化树分析表明,MdLBD13基因是拟南芥LBD37/38的同源基因。2.qRT-PCR实验结果表明,MdLBD13在苹果各个器官中都有表达,进一步分析其表达模式发现,MdLBD13在苹果苗中特异性响应硝酸盐,因此MdLBD13有可能参与到苹果硝酸盐代谢途径中。3.为了研究MdLBD13在苹果花青苷合成中的作用,在苹果愈伤组织过量表达MdLBD13基因。实验结果表明,转基因愈伤组织明显减少了花青苷的积累,并且MdLBD13能够抑制花青苷相关的MYB、b HLH以及花青苷合成途径的关键基因的表达。4.过量表达MdLBD13能够抑制苹果中硝酸盐吸收转运以及同化过程中基因的表达。检测苹果愈伤中的硝酸盐以及硝酸还原酶活性的结果验证了MdLBD13能够影响苹果硝酸盐代谢途径。5.为了进一步验证MdLBD13的功能,在拟南芥中异位表达MdLBD13,实验结果表明在拟南芥中,MdLBD13也可以抑制花青苷的合成和硝酸盐的吸收转运。6.在拟南芥中异位表达MdLBD13可以明显促进侧根的形成。
[Abstract]:Lateral organ boundaries domain (LBD) transcription factors regulate primordium initiation of apical meristem and lateral organ as well as establish boundary of plant apical meristem and lateral organ. The accumulation of hormones and the metabolism of anthocyanins and nitrate in plants. As a new transcription factor of higher plants, LBD protein family has only revealed the function of some LBD genes in Arabidopsis, rice and other model plants. There is relatively little research in apple. Anthocyanin, as the main component and important nutrient of apple color, plays an important role in fruit quality. The complete sequencing of apple genome is of great significance for the identification of LBD genes related to anthocyanin synthesis in apple. In this study, a typical LOB-Domain gene was isolated from apple and its expression pattern was further analyzed. The functions of apple calli overexpressing MdLBD13 and Arabidopsis thaliana were analyzed by transgenic method. The results showed that LBD gene regulated anthocyanin synthesis and nitrate metabolism in apple. The main findings and conclusions are as follows: 1. MdLBD13 (MDP0000317227) was isolated from Gala apple. The MdLBD13 gene encodes 251 amino acids for 753 bp,. The protein has a typical LOB-Domain, and a typical CX2CX6CX3C zinc finger-like domain, at its N-terminal is the second member of the LBD gene family. Amino acid sequence comparison and phylogenetic tree analysis showed that MdLBD13 gene was a homologous gene of Arabidopsis thaliana LBD37/38. The results of 2.qRT-PCR showed that MdLBD13 was expressed in all organs of apple, and further analysis of its expression pattern revealed that the gene was a homologue gene of Arabidopsis thaliana LBD37/38. MdLBD13 specifically responds to nitrate in apple seedlings, so MdLBD13 may be involved in the nitrate metabolism pathway of apple. 3. In order to study the role of MdLBD13 in the synthesis of apple anthocyanin, MdLBD13 gene was overexpressed in apple calli. The results showed that transgenic calli significantly reduced the accumulation of anthocyanin, and MdLBD13 could inhibit the expression of anthocyanin-related MYB,b HLH and the key genes of anthocyanin synthesis pathway. 4. Overexpression of MdLBD13 could inhibit nitrate uptake and transport and gene expression in apple during assimilation. The activity of nitrate and nitrate reductase in apple calli showed that MdLBD13 could affect the pathway of nitrate metabolism in apple. 5. In order to further verify the function of MdLBD13, the results of ectopic expression of MdLBD13, in Arabidopsis showed that MdLBD13 could also inhibit anthocyanin synthesis and nitrate uptake and transport in Arabidopsis thaliana. 6. Ectopic expression of MdLBD13 in Arabidopsis significantly promoted the formation of lateral roots.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S661.1

【相似文献】