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蔗糖是植物体中重要的碳源物质,参与植物的生长、发育乃至信号转导等多个生理过程。植物体在长距离运输蔗糖的过程中,蔗糖转运蛋白(sucrose transporters,SUT)基因起关键作用。为了解该基因在牡丹(Paeonia suffruticosa)植株体内的转运机制,以洛阳红牡丹(P.suffruticosa’Luoyanghong’)为材料,利用反转录PCR(reverse transcription-PCR,RT-PCR)和RACE(rapid-amplification of cDNA ends)方法从叶片中克隆了蔗糖转运蛋白基因,命名为PsSUT2。该基因全长2 347 bp,包含1 803 bp的开放阅读框,编码601个氨基酸,Gen Bank登录号为KC542396,相对分子量为64.6 k D,等电点为6.14,与葡萄(Vitis vinifera)中的蔗糖转运蛋白基因同源性最高,为75.27%。该蛋白属于易化子超家族(major facilitator superfamily,MFS)成员,包含12个跨膜螺旋。采用qRT-PCR和离子色谱法研究牡丹不同生育期与不同组织部位中PsSUT2基因的表达量及可溶性糖的含量变化,结果表明,在牡丹的年生长周期中,PsSUT2在牡丹的根、老茎、叶和鳞芽(花瓣)中均有表达,但主要在盛花期的花瓣中大量表达,相对表达量为11.18,是同时期根的4.26倍、小风铃期根的4.40倍、圆桃期花瓣的10.48倍、鳞芽分化期鳞芽的30.55倍,与同时期的其他部位乃至其他时期的所有部位相比差异显著,因此推测该基因可能参与了“库”端蔗糖的卸载。蔗糖、葡萄糖和果糖的含量在不同生育期、不同部位间存在较大差异,盛花期到来之前,鳞芽(花瓣)中蔗糖含量几乎为0,而葡萄糖和果糖水平较高,说明洛阳红牡丹花瓣属于己糖(葡萄糖和果糖)积累型器官,己糖的不断积累有利于花朵的开放。相关性分析表明,PsSUT2的表达量与与叶片中蔗糖的含量及根中葡萄糖含量成显著负相关,说明该基因的表达可能受叶片中蔗糖及根中葡萄糖的反馈抑制。本研究结果为进一步了解PsSUT2基因的功能、利用该基因进行牡丹分子育种和遗传改良提供理论依据。
Sucrose is an important carbon source in plants and participates in many physiological processes such as plant growth, development and even signal transduction. The plant sucrose transporters (SUT) genes play a key role in transporting sucrose over long distances. In order to understand the transport mechanism of this gene in Paeonia suffruticosa plants, P.suffruticosa’Luoyanghong ’was used as a material, and the reverse transcription-PCR (RT-PCR) and RACE -amplification of cDNA ends) was cloned from the leaf sucrose transporter gene, named PsSUT2. The full length cDNA was 2 347 bp in length and contained an open reading frame of 1 803 bp encoding a polypeptide of 601 amino acids. The GenBank accession number was KC542396, the relative molecular mass was 64.6 kD, the isoelectric point was 6.14, which was similar to that in Vitis vinifera Sucrose transporter gene has the highest homology of 75.27%. This protein is a member of the major facilitator superfamily (MFS) and contains 12 transmembrane helices. Using qRT-PCR and ion chromatography to study the changes of PsSUT2 gene expression and soluble sugar content in different growth stages and different tissues of peony, the results showed that in the peony’s annual growth cycle, Leaf and scale buds (petals), but they were mainly expressed in the petals of full-flowering stage. The relative expression level was 11.18, which was 4.26 times of that of the same period and 4.40 times of that of the small flowering stage, 10.48 folds, and 30.55 folds of scale buds during the differentiation of the buds. Compared with other parts of the same period and even all other parts of the same period, the difference was significant. Therefore, it is speculated that this gene may be involved in the unloading of sucrose. Sucrose, glucose and fructose content in different growth stages, there is a big difference between different parts before the full flowering stage, the bud (petals) in the sucrose content is almost 0, while the glucose and fructose levels, indicating the red petals of Luoyang Belonging to the accumulation of hexose (glucose and fructose) organs, the accumulation of hexose is conducive to the opening of flowers. Correlation analysis showed that the expression level of PsSUT2 was negatively correlated with the content of sucrose and the content of glucose in the root, indicating that the expression of PsSUT2 may be controlled by the feedback inhibition of sucrose and glucose in roots. The results of this study provide a theoretical basis for further understanding of the function of PsSUT2 gene and using the gene for molecular breeding and genetic improvement of peony.