紫苏HPPR基因启动子的克隆与功能分析
发布时间：2018-09-07 19:18
【摘要】：迷迭香酸是天然多功能酚酸类化合物的一种,是紫苏中一种具有多种活性的次生代谢产物。对紫苏营养成分和活性物质的了解与深入研究发现,紫苏中迷迭香酸的含量竟高达1.29%。本实验以唇形科属植物紫苏(Perilla frutecens)为实验对象,首次从紫苏中克隆得到了迷迭香酸合成途径酪氨酸支路中的关键酶基因羟苯基丙酮酸还原酶(Hydroxyphenylpyruvate reductase gene,HPPR)的启动子序列,由于 HPPR作用的底物4-羟基苯丙酮酸是迷迭香酸的前体物,同时也是尿黑酸的前体物,所以HPPR被认为是迷迭香酸合成途径中第一个特异性关键酶。因此分析HPPR基因启动子具有的顺式作用元件,为进一步验证HPPR启动子在逆境应答中的功能奠定基础,同时也为紫苏的遗传育种奠定了理论基础。(1)根据数据库中HPPR基因cDNA序列(Genebank登录号:HM587131.1),设计引物通过PCR扩增得到HPPR基因DNA,测序得长度为2525bp。利用基因组步移(genomic walking)的方法分离得到启动子并测序,长度为2216bp。利用P1antCare数据库在线预测显示,该启动子除含有TATA-Box和CAAT-Box等最基本的元件外,还含有BoxI,G-box,GTl-motif,MNF1,SP1等光调控元件,赤霉素(P-box),水杨酸(TCA-element),茉莉酸甲酯(CGTCA-motif),脱落酸(ABRE)及生长素(TGA-element)等激素响应元件,以及真菌诱导子响应元件(BoxW1),MYBHv1结合位点(CCAAT-box),热应力(HSE),低温响应(LTR),干旱可诱导性结合位点(MBS)等抗逆境胁迫响应元件,多种顺式作用元件的存在充分体现了启动子对基因表达调控在转录水平上具有高效性和复杂性。(2)设计引物对HPPR启动子5'端连续缺失,得到带有酶切位点的目的片段,并将其连接到酶切掉CaMV 35S启动子的双元表达载体PBI121上,得到重组质粒P1、P2、P3。利用农杆菌介导法转化本氏烟草,通过卡那霉素的抗性筛选,获得阳性菌落。农杆菌侵染烟草后进行三天共培养,并通过GUS染色法对烟草组织化学染色,从而判定启动子核心序列的位置,为深入研究该启动子组织特异性表达及胁迫诱导机理分析奠定基础。
[Abstract]:Rosemary acid is one of the natural multifunctional phenolic acids and a secondary metabolite with many activities in perilla. The contents of rosemary acid in perilla were found to be as high as 1.29%. In this study, the promoter sequence of the key enzyme gene, hydroxyphenyl pyruvate reductase (Hydroxyphenylpyruvate reductase gene,HPPR), which is a tyrosine branch of rosemary acid synthesis pathway, was cloned from perilla (Perilla frutecens) for the first time. Because 4-hydroxyphenylpyruvate (4-hydroxyphenylpyruvate), the substrate of HPPR, is the precursor of rosemary acid and the precursor of uric acid, HPPR is considered to be the first specific key enzyme in the biosynthesis of rosemary acid. Therefore, the analysis of the cis-acting elements of HPPR promoter lays a foundation for further verification of the function of HPPR promoter in stress response. It also laid a theoretical foundation for the inheritance and breeding of perilla. (1) according to the cDNA sequence of HPPR gene (Genebank accession number: HM587131.1), primers were designed to amplify HPPR gene DNA, by PCR amplification and the length of HPPR gene DNA, was 2525bp. The promoter was isolated by genomic step (genomic walking) and sequenced. The length of promoter was 2216bp. The on-line prediction of P1antCare database showed that the promoter contained not only the most basic elements, such as TATA-Box and CAAT-Box, but also the photoregulatory elements such as BoxI,G-box,GTl-motif,MNF1,SP1, P-box, TCA-element, CGTCA-motif, (ABRE) and TGA-element, and other hormone response elements, such as gibberellin (P-box), salicylic acid (TCA-element), methyl jasmonate (CGTCA-motif), abscisic acid (ABRE) and auxin (TGA-element). The fungal elicitor response elements (BoxW1) and MYBHv1 binding site (CCAAT-box), thermal stress (HSE), low temperature response to (LTR), drought inducible binding site (MBS), etc. The existence of various cis-acting elements fully demonstrates the high efficiency and complexity of promoter regulation of gene expression at the transcriptional level. (2) Design primers for the continuous deletion of the 5'end of HPPR promoter and obtain the target fragment with restriction site. It was ligated to the double expression vector PBI121 of CaMV 35s promoter, and the recombinant plasmid P1P2P2P3 was obtained. Positive colonies were obtained by screening kanamycin resistance by Agrobacterium tumefaciens mediated transformation of tobacco. Agrobacterium tumefaciens were co-cultured for three days and were stained by GUS staining to determine the position of promoter core sequence. The results laid a foundation for further study on the specific expression of the promoter and the mechanism of stress induction.
【学位授予单位】：天津科技大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2
[Abstract]:Rosemary acid is one of the natural multifunctional phenolic acids and a secondary metabolite with many activities in perilla. The contents of rosemary acid in perilla were found to be as high as 1.29%. In this study, the promoter sequence of the key enzyme gene, hydroxyphenyl pyruvate reductase (Hydroxyphenylpyruvate reductase gene,HPPR), which is a tyrosine branch of rosemary acid synthesis pathway, was cloned from perilla (Perilla frutecens) for the first time. Because 4-hydroxyphenylpyruvate (4-hydroxyphenylpyruvate), the substrate of HPPR, is the precursor of rosemary acid and the precursor of uric acid, HPPR is considered to be the first specific key enzyme in the biosynthesis of rosemary acid. Therefore, the analysis of the cis-acting elements of HPPR promoter lays a foundation for further verification of the function of HPPR promoter in stress response. It also laid a theoretical foundation for the inheritance and breeding of perilla. (1) according to the cDNA sequence of HPPR gene (Genebank accession number: HM587131.1), primers were designed to amplify HPPR gene DNA, by PCR amplification and the length of HPPR gene DNA, was 2525bp. The promoter was isolated by genomic step (genomic walking) and sequenced. The length of promoter was 2216bp. The on-line prediction of P1antCare database showed that the promoter contained not only the most basic elements, such as TATA-Box and CAAT-Box, but also the photoregulatory elements such as BoxI,G-box,GTl-motif,MNF1,SP1, P-box, TCA-element, CGTCA-motif, (ABRE) and TGA-element, and other hormone response elements, such as gibberellin (P-box), salicylic acid (TCA-element), methyl jasmonate (CGTCA-motif), abscisic acid (ABRE) and auxin (TGA-element). The fungal elicitor response elements (BoxW1) and MYBHv1 binding site (CCAAT-box), thermal stress (HSE), low temperature response to (LTR), drought inducible binding site (MBS), etc. The existence of various cis-acting elements fully demonstrates the high efficiency and complexity of promoter regulation of gene expression at the transcriptional level. (2) Design primers for the continuous deletion of the 5'end of HPPR promoter and obtain the target fragment with restriction site. It was ligated to the double expression vector PBI121 of CaMV 35s promoter, and the recombinant plasmid P1P2P2P3 was obtained. Positive colonies were obtained by screening kanamycin resistance by Agrobacterium tumefaciens mediated transformation of tobacco. Agrobacterium tumefaciens were co-cultured for three days and were stained by GUS staining to determine the position of promoter core sequence. The results laid a foundation for further study on the specific expression of the promoter and the mechanism of stress induction.
【学位授予单位】：天津科技大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2
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