苦蕎16kD過敏原基因啟動(dòng)子的克隆及功能分析
發(fā)布時(shí)間：2018-07-25 20:13
【摘要】：蕎麥富含蛋白質(zhì)和多種黃酮類物質(zhì),食用和藥用價(jià)值較高,苦蕎引發(fā)的過敏反應(yīng)日益受到重視�？嗍w16kD過敏原(TBW16)是引起苦蕎過敏癥狀的原因之一。本研究克隆并分析了TBW16啟動(dòng)子序列,將啟動(dòng)子融合β-葡萄糖醛酸酶(GUS)基因用基因槍法轟入苦蕎外植體瞬時(shí)表達(dá)研究其組織特異性。從5′端進(jìn)行啟動(dòng)子截短,將各段啟動(dòng)子缺失片融合熒光素酶轉(zhuǎn)入苦蕎葉片原生質(zhì)體瞬時(shí)表達(dá)以測(cè)定其活性,并根據(jù)各片段上相應(yīng)的激素元件對(duì)啟動(dòng)子進(jìn)行脅迫處理,研究其對(duì)激素調(diào)控的響應(yīng)規(guī)律。該研究對(duì)揭示TBW16啟動(dòng)子的組織特異性和基因在苦蕎中的代謝調(diào)控機(jī)制及其生物學(xué)功能具有重要意義。另外,本研究對(duì)TBW16基因的組織特異性進(jìn)行了分析,并構(gòu)建了TBW16基因的干擾和過表達(dá)載體,分別轉(zhuǎn)化苦蕎和擬南芥,從分子生物學(xué)途徑開展TBW16基因沉默和過表達(dá),對(duì)進(jìn)一步研究TBW16的功能和消除過敏原、改良苦蕎有重要意義。結(jié)論如下:(1)實(shí)時(shí)定量分析TBW16基因在苦蕎莖、根、葉和種子中的表達(dá)量,結(jié)果表明該基因在苦蕎種子中大量表達(dá),在苦蕎根、葉、莖中表達(dá)量較低。初步判定該基因的啟動(dòng)子為種子特異性啟動(dòng)子。(2)利用基因組步移法獲得1876 bp TBW16啟動(dòng)子序列,經(jīng)生物信息學(xué)分析發(fā)現(xiàn)其具有以下啟動(dòng)子基本元件:轉(zhuǎn)錄起始位點(diǎn)(-38bp)、TATA-box(-72bp)、CAAT-box(-50);三種激素響應(yīng)元件:2個(gè)茉莉酸甲酯響應(yīng)元件(TGACG-motif,-1584bp;CGTCA-motif,-1407bp)、2個(gè)赤霉素響應(yīng)元件(GARE-motif,-198bp、-1815bp)和1個(gè)脫落酸響應(yīng)元件(ABRE,-156bp);兩個(gè)種子特異性元件(RYrepeat,-92bp、-134bp)。(3)TBW16全長(zhǎng)啟動(dòng)子構(gòu)建重組載體TBW16P-pCAPMBIA1301,用基因槍法將重組質(zhì)粒轉(zhuǎn)入苦蕎外植體根、莖、葉和種子中并啟動(dòng)β-葡萄糖醛酸酶(β-glucuronidase,GUS)基因瞬時(shí)表達(dá),被轟擊的外植體經(jīng)GUS染色后觀察表明,種子中GUS的表達(dá)量最高,葉片中有微量GUS表達(dá),苦蕎的根和莖中均無肉眼可見的GUS表達(dá)。(4)將全長(zhǎng)啟動(dòng)子進(jìn)行5′端缺失形成4段啟動(dòng)子分析片段,并分別構(gòu)建截短啟動(dòng)子的重組雙熒光報(bào)告載體GP-TBW16P1、GP-TBW16P2、GP-TBW16P3和GP-TBW16P4。分別將4種重組載體轉(zhuǎn)入苦蕎葉片原生質(zhì)體中進(jìn)行瞬時(shí)表達(dá),結(jié)果顯示在TBW 16啟動(dòng)子上存在調(diào)節(jié)基因轉(zhuǎn)錄的區(qū)域,約位于-1564 bp至-1876bp。報(bào)告基因在GA、MeJA和ABA激素處理時(shí)表達(dá)量增加,證明TBW16啟動(dòng)子上存在響應(yīng)三種激素脅迫的作用元件。(5)向pTCK303載體中插入TBW16反向和正向干擾片,構(gòu)建苦蕎TBW16干擾載體(TBW16i-pTCK303)。將TBW16過表達(dá)片段插入到pTCK303載體中構(gòu)建了苦蕎過表達(dá)載體(TBW16m-pTCK303),為16kDa過敏原的進(jìn)一步研究苦蕎和低敏種質(zhì)的創(chuàng)制奠定了基礎(chǔ)。
[Abstract]:Buckwheat is rich in protein and flavonoids, and its edible and medicinal value is high. The allergic reaction caused by Tartary buckwheat has been paid more and more attention. Tartary buckwheat 16kD allergen (TBW16) is one of the causes of buckwheat allergy symptoms. In this study, the TBW16 promoter sequence was cloned and analyzed. The promoter fusion 尾 -glucuronidase (GUS) gene was bombarded into Tartary buckwheat explants by gene bombardment to study its tissue specificity. The promoter was truncated from the 5 'end of the promoter and fused with luciferase from each segment of the promoter to determine its activity in the protoplast of Tartary buckwheat leaves. The promoter was treated with stress according to the corresponding hormone elements in each fragment. To study its response to hormone regulation. This study is of great significance in revealing the tissue specificity of TBW16 promoter and the metabolic regulation mechanism and biological function of the gene in Tartary buckwheat. In addition, the tissue specificity of TBW16 gene was analyzed, and the interference and overexpression vectors of TBW16 gene were constructed, which were transformed into Tartary buckwheat and Arabidopsis thaliana, respectively. TBW16 gene silencing and overexpression were carried out by molecular biological pathway. It is of great significance to further study the function of TBW16, eliminate allergens and improve Tartary buckwheat. The conclusions are as follows: (1) quantitative analysis of the expression of TBW16 gene in roots, stems, leaves and seeds of Tartary buckwheat in real time. The results showed that the expression of TBW16 gene in Tartary buckwheat seeds was much higher than that in roots, leaves and stems of Tartary buckwheat. The promoter of the gene was identified as seed specific promoter. (2) 1876 BP TBW16 promoter sequence was obtained by genomic step method. Bioinformatics analysis showed that it had the following basic promoter elements: transcriptional initiation site (-38bp) and TATA-box (-72bp) CAAT-box (-50), three hormone response elements: two methyl jasmonate response elements (TGACG-motif-1584bpCGTCA-motif-1407bp), two gibberellin response elements (GARE-motif-198bp-1815bp) and one abscisic acid response element (ABREE-156bp). The recombinant vector TBW16P-pCAPMBIA1301was constructed by two seed specific elements (RY repeat -92bpSn-134bp). (3) TBW16 promoter. The recombinant plasmid was transferred into the roots of Tartary buckwheat explants by gene gun method. The transient expression of 尾 -glucuronidase (Gus) gene was initiated in stem, leaf and seed. The results of GUS staining showed that the expression of GUS was the highest in the explants, and there was a trace of GUS expression in the leaves. There was no naked GUS expression in the roots and stems of Tartary buckwheat. (4) the full-length promoter was deleted at the 5'end to form four fragments of promoter analysis, and the recombinant double-fluorescence report vectors GP-TBW16P1, GP-TBW16P2GP-TBW16P16P3 and GP-TBW16P4 were constructed respectively. Four recombinant vectors were transferred into the protoplasts of Tartary buckwheat for transient expression. The results showed that there was a region regulating gene transcription on the promoter of TBW 16, which was located between -1564 BP and -1876 BP. The expression of the reporter gene was increased during the treatment of GAN Meja and ABA hormones, which indicated that there were three hormone stress response elements in the TBW16 promoter. (5) insert TBW16 reverse and forward interfering pieces into the pTCK303 vector to construct TBW16 interference vector (TBW16i-pTCK303) of Tartary Buckwheat (Tartary Buckwheat). The overexpression fragment of TBW16 was inserted into the pTCK303 vector to construct the over expression vector (TBW16m-pTCK303) of Tartary buckwheat, which laid a foundation for the further study of 16kDa allergen and the creation of hypersensitive germplasm.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q943.2;S517
本文編號(hào)：2144951
[Abstract]:Buckwheat is rich in protein and flavonoids, and its edible and medicinal value is high. The allergic reaction caused by Tartary buckwheat has been paid more and more attention. Tartary buckwheat 16kD allergen (TBW16) is one of the causes of buckwheat allergy symptoms. In this study, the TBW16 promoter sequence was cloned and analyzed. The promoter fusion 尾 -glucuronidase (GUS) gene was bombarded into Tartary buckwheat explants by gene bombardment to study its tissue specificity. The promoter was truncated from the 5 'end of the promoter and fused with luciferase from each segment of the promoter to determine its activity in the protoplast of Tartary buckwheat leaves. The promoter was treated with stress according to the corresponding hormone elements in each fragment. To study its response to hormone regulation. This study is of great significance in revealing the tissue specificity of TBW16 promoter and the metabolic regulation mechanism and biological function of the gene in Tartary buckwheat. In addition, the tissue specificity of TBW16 gene was analyzed, and the interference and overexpression vectors of TBW16 gene were constructed, which were transformed into Tartary buckwheat and Arabidopsis thaliana, respectively. TBW16 gene silencing and overexpression were carried out by molecular biological pathway. It is of great significance to further study the function of TBW16, eliminate allergens and improve Tartary buckwheat. The conclusions are as follows: (1) quantitative analysis of the expression of TBW16 gene in roots, stems, leaves and seeds of Tartary buckwheat in real time. The results showed that the expression of TBW16 gene in Tartary buckwheat seeds was much higher than that in roots, leaves and stems of Tartary buckwheat. The promoter of the gene was identified as seed specific promoter. (2) 1876 BP TBW16 promoter sequence was obtained by genomic step method. Bioinformatics analysis showed that it had the following basic promoter elements: transcriptional initiation site (-38bp) and TATA-box (-72bp) CAAT-box (-50), three hormone response elements: two methyl jasmonate response elements (TGACG-motif-1584bpCGTCA-motif-1407bp), two gibberellin response elements (GARE-motif-198bp-1815bp) and one abscisic acid response element (ABREE-156bp). The recombinant vector TBW16P-pCAPMBIA1301was constructed by two seed specific elements (RY repeat -92bpSn-134bp). (3) TBW16 promoter. The recombinant plasmid was transferred into the roots of Tartary buckwheat explants by gene gun method. The transient expression of 尾 -glucuronidase (Gus) gene was initiated in stem, leaf and seed. The results of GUS staining showed that the expression of GUS was the highest in the explants, and there was a trace of GUS expression in the leaves. There was no naked GUS expression in the roots and stems of Tartary buckwheat. (4) the full-length promoter was deleted at the 5'end to form four fragments of promoter analysis, and the recombinant double-fluorescence report vectors GP-TBW16P1, GP-TBW16P2GP-TBW16P16P3 and GP-TBW16P4 were constructed respectively. Four recombinant vectors were transferred into the protoplasts of Tartary buckwheat for transient expression. The results showed that there was a region regulating gene transcription on the promoter of TBW 16, which was located between -1564 BP and -1876 BP. The expression of the reporter gene was increased during the treatment of GAN Meja and ABA hormones, which indicated that there were three hormone stress response elements in the TBW16 promoter. (5) insert TBW16 reverse and forward interfering pieces into the pTCK303 vector to construct TBW16 interference vector (TBW16i-pTCK303) of Tartary Buckwheat (Tartary Buckwheat). The overexpression fragment of TBW16 was inserted into the pTCK303 vector to construct the over expression vector (TBW16m-pTCK303) of Tartary buckwheat, which laid a foundation for the further study of 16kDa allergen and the creation of hypersensitive germplasm.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q943.2;S517
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