紫蘇HPPR基因啟動子的克隆與功能分析
發(fā)布時間：2018-09-07 19:18
【摘要】：迷迭香酸是天然多功能酚酸類化合物的一種,是紫蘇中一種具有多種活性的次生代謝產(chǎn)物。對紫蘇營養(yǎng)成分和活性物質(zhì)的了解與深入研究發(fā)現(xiàn),紫蘇中迷迭香酸的含量竟高達1.29%。本實驗以唇形科屬植物紫蘇(Perilla frutecens)為實驗對象,首次從紫蘇中克隆得到了迷迭香酸合成途徑酪氨酸支路中的關(guān)鍵酶基因羥苯基丙酮酸還原酶(Hydroxyphenylpyruvate reductase gene,HPPR)的啟動子序列,由于 HPPR作用的底物4-羥基苯丙酮酸是迷迭香酸的前體物,同時也是尿黑酸的前體物,所以HPPR被認為是迷迭香酸合成途徑中第一個特異性關(guān)鍵酶。因此分析HPPR基因啟動子具有的順式作用元件,為進一步驗證HPPR啟動子在逆境應(yīng)答中的功能奠定基礎(chǔ),同時也為紫蘇的遺傳育種奠定了理論基礎(chǔ)。(1)根據(jù)數(shù)據(jù)庫中HPPR基因cDNA序列(Genebank登錄號:HM587131.1),設(shè)計引物通過PCR擴增得到HPPR基因DNA,測序得長度為2525bp。利用基因組步移(genomic walking)的方法分離得到啟動子并測序,長度為2216bp。利用P1antCare數(shù)據(jù)庫在線預(yù)測顯示,該啟動子除含有TATA-Box和CAAT-Box等最基本的元件外,還含有BoxI,G-box,GTl-motif,MNF1,SP1等光調(diào)控元件,赤霉素(P-box),水楊酸(TCA-element),茉莉酸甲酯(CGTCA-motif),脫落酸(ABRE)及生長素(TGA-element)等激素響應(yīng)元件,以及真菌誘導(dǎo)子響應(yīng)元件(BoxW1),MYBHv1結(jié)合位點(CCAAT-box),熱應(yīng)力(HSE),低溫響應(yīng)(LTR),干旱可誘導(dǎo)性結(jié)合位點(MBS)等抗逆境脅迫響應(yīng)元件,多種順式作用元件的存在充分體現(xiàn)了啟動子對基因表達調(diào)控在轉(zhuǎn)錄水平上具有高效性和復(fù)雜性。(2)設(shè)計引物對HPPR啟動子5'端連續(xù)缺失,得到帶有酶切位點的目的片段,并將其連接到酶切掉CaMV 35S啟動子的雙元表達載體PBI121上,得到重組質(zhì)粒P1、P2、P3。利用農(nóng)桿菌介導(dǎo)法轉(zhuǎn)化本氏煙草,通過卡那霉素的抗性篩選,獲得陽性菌落。農(nóng)桿菌侵染煙草后進行三天共培養(yǎng),并通過GUS染色法對煙草組織化學(xué)染色,從而判定啟動子核心序列的位置,為深入研究該啟動子組織特異性表達及脅迫誘導(dǎo)機理分析奠定基礎(chǔ)。
[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.
【學(xué)位授予單位】：天津科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：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.
【學(xué)位授予單位】：天津科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：Q943.2
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