烟草DXS及FPS双基因克

发布时间：2018-04-09 22:00

本文选题：DXS基因　切入点：FPS基因　出处：《郑州大学》2017年硕士论文

【摘要】：萜类化合物在植物代谢产物中种类繁多,除了在植物生长、发育、杀虫、避食、防御等方面有着重要的生理和生态作用外,大多成分还具有应用价值。近年来人们的研究重点逐渐转移到对其生物合成途径中关键酶的研究。关键酶一般位于合成途径的分支处,催化各种中间体以及后续产物的合成。有效调控某些关键酶的合成与表达,可以相应提高有关萜类化合物的产量。本实验主要对萜类合成途径中关键酶基因DXS和FPS进行克隆及生物信息学分析,构建p BI121-DXS、p BI121-FPS和p BI121-DXS-FPS过表达载体,通过烟草遗传转化进行基因表达和茄尼醇含量分析,探讨茄尼醇代谢的调控机制。主要获得以下研究结果:1.首次从普通烟草K326中克隆得到1-脱氧-木酮糖-5-磷酸合成酶(DXS)的完整编码区序列,长2142 bp,并较全面地对DXS基因与蛋白序列进行了生物信息学分析,结果表明其与枸杞、马铃薯、番茄、辣椒等的一致性在85%以上;该酶属于亲水性蛋白,没有信号肽及跨膜结构域,预测其具有叶绿体转运肽并定位于叶绿体,含有保守结构域TPP_synthase_N、TPP_PYR_DXS_TK_like、Transketolase_C及多功能结构域PLNO2582特征序列;系统发育树分析显示K326 DXS与多种植物的DXS2家族成员聚集在一个大分支上,在此分支内部又与茄科植物DXS2高度聚集在一小支中,推测该基因序列具有DXS2类型基因的功能,参与烟草类萜化合物的代谢过程。2.首次克隆了烟草K326法尼基焦磷酸合酶(FPS)完整编码区序列,长1029bp,生物信息学分析表明该核酸和蛋白质序列与番茄、辣椒、南非睡茄等的同源性在85%以上;该蛋白的分子质量约39.5 k D,属于稳定蛋白及亲水性蛋白,不含信号肽和跨膜结构域,具有底物Mg2+结合位点、活性位点、链长度决定区、催化位点等7个结构域。系统发育树分析显示烟草K326 FPS与同茄科的辣椒、番茄、马铃薯和催眠睡茄亲缘关系最近,聚集在一个大的分支,支持率为100%,并且与其中的烟草属植物高度聚集在一小支,这与植物系统发育的亲缘关系相一致。推测该基因应该具备异戊烯焦磷酸合酶的功能。3.进行了DXS、FPS基因的3′RACE序列克隆分析,DXS基因3′RACE序列长291 bp;FPS基因3′RACE扩增出两条分别长235 bp和261 bp的不同序列,BLAST比对分析显示两条序列与烟草属普通烟草其他品种的同源性100%,说明FPS基因序列在烟草种内比较稳定,不同种间有较小差异,同时也表明烟草K326中FPS基因存在多个基因家族成员。4.DXS基因、FPS基因表达模式分析表明,两基因在烟草根、茎、叶和花中都有表达,并且表达量均表现出叶茎根花的规律,与植物类萜合成代谢主要在叶片中相符。5.分别成功构建了单基因过表达载体PBI121-DXS、PBI121-FPS以及双基因过表达载体PBI121-DXS-FPS,成功进行了烟草遗传转化,获得过表达DXS转基因烟苗19株,过表达FPS转基因烟苗23株,双基因过表达转基因烟苗8株。6.不同转基因烟草,旺长期其DXS或FPS表达量均有显著性增强,双基因共表达的转基因植株DXS、FPS基因的表达均小于单基因转化植株量基因表达量增加的幅度。现蕾期单基因转化植株DXS、FPS基因表达量均有显著提高,双基因共表达转化植株与单基因转化植株基因表达增加幅度基本一致。单基因转化和双基因共转化植株都表明,超表达FPS基因的植株更易获得。旺长期,DXS或FPS单基因转化烟草均可显著促进茄尼醇的合成,而双基因过表达植株茄尼醇含量与非转基因植株相比无显著差异。现蕾期,烟草茄尼醇含量显著高于旺长期;与对照相比,单基因转化烟草与双基因转化烟草茄尼醇含量均显著增加,双基因转化植株的增加效果更显著。可见茄尼醇代谢受生育期以及基因间互作等多种因素影响,过表达合成途径中关键酶基因可以有效调控茄尼醇的合成代谢。同时实验表明茄尼醇含量与基因表达量不是简单的正相关关系,存在不一致现象,茄尼醇代谢复杂的调控机制还有待进一步研究。
[Abstract]:Terpenoids in plant metabolites in various species, except in plant growth, development, insecticidal, avoid eating, have important physiological and ecological effects of defense and other aspects, most components also has application value. In recent years, people's research focus gradually shifted to the research on the key enzymes of the biosynthetic pathway in branch. Key enzymes generally located in the synthesis of various catalytic intermediates and synthesis of subsequent products. Synthesis and expression of effective regulation of some key enzymes, can increase the yield of terpene compounds. The experiment of terpenoid synthesis key enzyme genes in the pathway of DXS and FPS cloning and bioinformatics analysis of P construction BI121-DXS, P BI121-FPS and P BI121-DXS-FPS expression vector, genetic transformation of tobacco through analysis of gene expression and the content of solanesol, discuss the regulation mechanism of solanesol metabolism. The main access to Research results: 1. for the first time from the ordinary tobacco K326 cloned 1- deoxy xylulose -5- phosphate synthase (DXS) sequence of the complete encoding region, 2142 BP long, and comprehensively on DXS gene and protein sequences were analyzed by bioinformatics, and the results show that the Chinese wolfberry, Ma Ling potato, tomato, consistent of chili in more than 85%; the enzyme protein is hydrophilic, no signal peptide and transmembrane domain prediction with chloroplast transit peptide and located in chloroplast containing the conserved domains of TPP_synthase_N, TPP_PYR_DXS_TK_like, Transketolase_C and multi functional domains of PLNO2582 sequences; phylogenetic tree analysis showed that K326 DXS with a variety of plants DXS2 family members gathered in a large branch, this branch of internal and Solanaceae plant height of DXS2 gathered in a small, speculated that the gene sequence with DXS2 type gene function in tobacco .2. metabolism of diterpenes was cloned in tobacco K326 farnesyl diphosphate synthase (FPS) sequence, complete encoding region 1029bp, bioinformatics analysis showed that the nucleotide and protein sequences with tomato, pepper, more than 85% homology in South Africa and other Solanaceae; the molecular weight of the protein is about 39.5 K D, which belongs to stable protein and hydrophilic protein without signal peptide and transmembrane domain with substrate Mg2+ binding site, active site, chain length is determined, 7 domains of catalytic sites. The phylogenetic tree analysis showed that tobacco K326 FPS and with the Solanaceae capsicum, tomato, potato and eggplant related recent sleep hypnosis, gathered in a large branch, the support rate of 100%, and the genus highly concentrated in a small branch of the tobacco, which is consistent with the relationship between development of plant system. It is speculated that the gene should have isopentenyl pyrophosphate synthase function .3. DXS, 3 'RACE sequence analysis cloning of FPS gene, DXS gene 3' long sequences of RACE 291 BP; FPS gene 3 'RACE amplified two different sequences were 235 BP and 261 BP, BLAST sequence analysis showed that two sequences and Nicotiana tobacco varieties of other 100% homology. FPS gene sequence is relatively stable in tobacco species, there are small differences in different species, and also indicates the existence of multiple gene family members.4.DXS gene K326 in tobacco FPS gene, FPS gene expression pattern analysis showed that two genes in tobacco roots, stems, leaves and flowers are expressed, and the expression level showed stem root flowers and plants of terpenoid metabolism mainly in leaves in compliance with.5. were successfully constructed. Single gene expression vector PBI121-DXS, PBI121-FPS and the double gene expression vector PBI121-DXS-FPS, the success of the tobacco genetic transformation, won 19 strains of DXS transgenic tobacco seedlings expression, overexpression of FPS transgenic tobacco seedlings of 23 strains, double gene transgenic tobacco seedlings of 8 different strains of.6. transgenic tobacco, growing its DXS or FPS expression were significantly enhanced in transgenic plants, DXS gene co expression, gene expression of FPS were less than single gene transgenic plants quantity gene expression increase. The amplitude of single gene transgenic plants DXS budding stage, FPS gene expression was significantly increased, co expression of transgenic plants and single gene transgenic plants gene expression increased basically. Plants were transferred to the single gene transformation and double gene showed that over expression of FPS gene in plants is obtained more easily Wang. Long, DXS or FPS single gene in transgenic tobacco could significantly promote the synthesis of solanesol, and double gene overexpression plants the content of solanesol and non transgenic plants showed no significant difference. The budding stage, the content of Solanesol in tobacco. Compared with the vigorous; compared with the control, single and double transgenic tobacco transgenic tobacco solanesol content increased significantly, the double gene transgenic plants increased more significant effect. The visible solanesol metabolism by stages and interaction among genes and other factors, the over expression of metabolism as the key enzyme in the pathway the gene can effectively control the solanesol. The experiments show that the content of solanesol and gene expression is not a simple positive correlation, are inconsistent, the regulation mechanism of solanesol metabolism remains to be further studied.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2

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