芍药类黄酮合成相关基因载体构建及对模式植物转化研究

发布时间：2019-01-18 21:09

【摘要】：花卉植物的花色直接影响了其观赏价值,是花卉育种的焦点。芍药(Paeonia lactiflora)属于芍药科芍药属,是具有可观的商品价值的观赏植物,但其颜色主要集中在粉色、红色、紫色、白色系,黄色系等品种非常稀少,这在一定程度上限制了其发展。对芍药花色的改良创新一直备受关注,主要报道集中在杂交育种,利用基因工程手段进行花色定向选育的研究受到限制,可能由于目前对芍药花色形成的分子机理了解甚少,同时相关基因的调节机制无深刻的认识。与花色密切相关的类黄酮合成途径中的许多关键基因已在许多植物中进行了克隆研究,因此,本研究基于课题组前期对芍药嵌合体品种花瓣的转录组测序结果,在明确CHI、ANS、DFR、FLS、PAL基因均为调控芍药类黄酮合成途径中调控花色的关键基因之后,克隆获得目的基因,并构建这五个基因的表达载体,以农杆菌为介导转化模式植物,以期对其功能进行初步探索,为开发芍药新品种和新异花色的分子育种提供可靠的理论依据和实践意义。主要研究结果如下:(1)克隆了芍药类黄酮途径相关基因。提取芍药'粉玉楼'花瓣总RNA,反转录合成cDNA第一链,根据课题组前期对相关基因的RACE结果,克隆获得五个目的基因CHI、ANS、DFR、FLS、PAL,均包含完整编码区,推测氨基酸序列相似性与NCBI数据库分别达 99%、98%、99%、100%、100%。(2)构建了芍药类黄酮途径相关基因超表达载体。在扩增获得带有合适的酶切位点的编码区后,用SmaⅠ和Sac Ⅰ双酶切目的基因CHI、ANS、DFR、PAL和表达载体质粒pCAMBIA1301,用BamH Ⅰ和KpnⅠ双酶切目的基因FLS和表达载体质粒pCAMBIA1301,回收、连接目的片段与载体,分别成功构建植物超表达载体pB1301-CHI、pB1301-ANS、pB1301-DFR、pB1301-FLS、pB1301-PAL共5个。测序验证后,采用冻融法将重组质粒以及空载体转化农杆菌EHA105,获得了用于植物转化的材料。(3)获得纯合转CHI、ANS、DFR、FLS、PAL基因拟南芥。利用农杆菌介导Floral-dip法将目的基因CHI、ANS、DFR、FLS、PAL和空载体导入拟南芥,经过T1代抗性筛选、GUS组织化学染色、T2和T3代分离比筛选,最终获得转CHI基因纯合株系6个,转ANS基因纯合株系2个,转DFR基因纯合株系6个,转FLS基因纯合株系3个,转PAL基因纯合株系5个,以及纯合的转空载株系2个。GUS染色、PCR鉴定及qRT-PCR分析显示,纯合转基因拟南芥均能在基因组中检测到目的基因,相对于对照组在转录水平表达水平明显提高。(4)纯合转基因拟南芥表型初步分析。对转基因株系的初步观察发现,转CHI、FLS及PAL基因拟南芥与对照组相比,均无显著差异。转DFR和ANS基因的株系,虽然花器官与对照组相比无明显差异,但在抽薹期,转ANS基因株系部分莲座叶叶背斑块状发红,主脉基部略微发红。转DFR基因株系莲座叶均叶色较深,部分叶片叶背发红,DFR表达量最高的株系2叶背为深紫红色。对转基因各株系总黄酮与花色苷含量的测定有待进一步研究。(5)获得转DFR、FLS基因烟草植株。利用农杆菌介导叶盘法将目的基因DFR、FLS和空载体导入烟草,通过抗性筛选,获得转DFR基因植株43株,转FLS基因植株66株,转空载对照5株。通过GUS组织化学染色、PCR鉴定,随机挑选的10株转DFR基因烟草中阳性植株为8株,12株转FLS基因烟草中阳性植株为7株,可认为目的基因已整合进入烟草基因组。通过qRT-PCR对阳性烟草中目的基因的转录水平进行分析,结果显示,与对照组相比,转基因烟草中目的基因表达量明显上升,表明目的基因均能在转基因植株中正常表达,且有一定促进作用。由于时间及温度原因,目前烟草还没有抽薹开花,其生长状况及生物学特性上还未发现明显差异,对其花色和花色苷的含量的变化有待进一步实验。
[Abstract]:The flower color of the flower plant directly influences the ornamental value of the flower, and is the focus of the flower breeding. Paeonia lactifla is a kind of ornamental plant with considerable commodity value, but its color is mainly concentrated in the pink, red, purple, white and yellow lines, which is limited to its development to a certain extent. The improvement and innovation of the Chinese medicine flower color has been paid attention to, the main report focuses on the cross breeding, the research on the selection and breeding of the flower color by using the genetic engineering means is limited, At the same time, the regulation mechanism of the related gene has no profound knowledge. Many of the key genes in the flavonoid synthesis pathway, which are closely related to the suit, have been cloned and studied in many plants. after the PAL gene is a key gene for regulating and controlling the color and color in the synthesis route of the peony flavonoid, the target gene is cloned and the expression vector of the five genes is constructed, and the agrobacterium is used as the mediated transformation mode plant, so as to carry out preliminary exploration on the function thereof, and provides a reliable theoretical basis and practical significance for the development of the new variety of the peony and the molecular breeding of the new heterotype. The main results of this study were as follows: (1) The related genes of paeonol pathway were cloned. extraction of Paeonia lactiflora "powder jade building 'The first strand of the cDNA was synthesized by reverse transcription of the total RNA and reverse transcription. Five target genes, CHI, ANS, DFR, FLS, and PAL, were obtained according to the RACE results of the related genes in the earlier stage of the research group. The results showed that the similarity of the amino acid sequence and the NCBI database were 99%, 98% and 99%, respectively. 100%, 100%. and (2) constructing the hyperexpression vector of the peony flavonoid pathway related gene. after the coding region with the appropriate enzyme cutting site is obtained, the target gene CHI, ANS, DFR, PAL and the expression vector plasmid pCAMBIA1301 are cut with SmaI and Sac I double-enzyme digestion target genes CHI, ANS, DFR, PAL and expression vector pCAMBIA1301, The plant superexpression vectors pB1301-CHI, pB1301-ANS, pB1301-DFR, pB1301-FLS and pB1301-PAL were successfully constructed. After the sequencing and verification, the recombinant plasmid and the empty vector were transformed into Agrobacterium EHA105 by a freeze-thaw method, and a material for plant transformation was obtained. (3) obtaining pure-in-rotation CHI, ANS, DFR, FLS and PAL gene Arabidopsis. The target genes CHI, ANS, DFR, FLS, PAL and empty vector were introduced into arabidopsis by Agrobacterium-mediated Flourral-dip method, and then were selected by T1 generation resistance screening, GUS histochemical staining, T2 and T3 generation. 6 of the pure strain of the transgenic DFR gene, 3 of the pure strain of the transgenic FLS gene, 5 of the pure strain of the transgenic PAL gene, and 2 of the pure-in-one transgenic no-load strains. GUS staining, PCR identification and qRT-PCR analysis showed that both the pure and transgenic Arabidopsis can detect the target gene in the genome, and the expression level of the transcription level in the control group is obviously improved. (4) Preliminary analysis of the phenotype of pure-in-transgenic Arabidopsis. The results of the preliminary observation on the transgenic lines showed no significant difference in the expression of CHI, FLS and PAL and the control group. The lines of the DFR and ANS genes, although there was no significant difference between the flower organ and the control group, in the pumping stage, the leaf of the leaf of the lotus leaf of the ANS gene line was red, and the base of the main vein was slightly reddish. The leaf color of the leaf of the RFR gene was deep, the leaves of the leaf were red, and the 2-leaf back of the strain with the highest degree of DFR expression was deep-purplish. The determination of the content of total flavone and flower color of all the transgenic lines is to be further studied. (5) obtaining the transformed DFR and FLS gene tobacco plants. The target gene DFR, FLS and the empty vector were introduced into the tobacco by Agrobacterium-mediated leaf disc method, and the transgenic plants of the transgenic DFR gene were obtained by resistance screening, and 66 strains of the transgenic FLS gene and 5 strains of no-load control were obtained. By means of GUS histochemical staining and PCR identification, 8 of the 10 transgenic DFR gene plants selected randomly, and 7 of the 12 transgenic FLS genes, can be considered to be integrated into the tobacco genome. Through the analysis of the transcription level of the target gene in the positive tobacco by qRT-PCR, the results showed that the expression of the target gene in the transgenic tobacco obviously increased compared with the control group, indicating that the target gene can be normally expressed in the transgenic plant and has a certain promoting effect. Because of the time and temperature, there is no significant difference in the growth and biological characteristics of the tobacco, and the change of the content of the color and the flower color of the tobacco is to be further tested.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S682.12

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