苦荞16kD过敏原基因启动子的克隆及功能分析

发布时间：2018-07-25 20:13

【摘要】：荞麦富含蛋白质和多种黄酮类物质,食用和药用价值较高,苦荞引发的过敏反应日益受到重视。苦荞16kD过敏原(TBW16)是引起苦荞过敏症状的原因之一。本研究克隆并分析了TBW16启动子序列,将启动子融合β-葡萄糖醛酸酶(GUS)基因用基因枪法轰入苦荞外植体瞬时表达研究其组织特异性。从5′端进行启动子截短,将各段启动子缺失片融合荧光素酶转入苦荞叶片原生质体瞬时表达以测定其活性,并根据各片段上相应的激素元件对启动子进行胁迫处理,研究其对激素调控的响应规律。该研究对揭示TBW16启动子的组织特异性和基因在苦荞中的代谢调控机制及其生物学功能具有重要意义。另外,本研究对TBW16基因的组织特异性进行了分析,并构建了TBW16基因的干扰和过表达载体,分别转化苦荞和拟南芥,从分子生物学途径开展TBW16基因沉默和过表达,对进一步研究TBW16的功能和消除过敏原、改良苦荞有重要意义。结论如下:(1)实时定量分析TBW16基因在苦荞茎、根、叶和种子中的表达量,结果表明该基因在苦荞种子中大量表达,在苦荞根、叶、茎中表达量较低。初步判定该基因的启动子为种子特异性启动子。(2)利用基因组步移法获得1876 bp TBW16启动子序列,经生物信息学分析发现其具有以下启动子基本元件:转录起始位点(-38bp)、TATA-box(-72bp)、CAAT-box(-50);三种激素响应元件:2个茉莉酸甲酯响应元件(TGACG-motif,-1584bp;CGTCA-motif,-1407bp)、2个赤霉素响应元件(GARE-motif,-198bp、-1815bp)和1个脱落酸响应元件(ABRE,-156bp);两个种子特异性元件(RYrepeat,-92bp、-134bp)。(3)TBW16全长启动子构建重组载体TBW16P-pCAPMBIA1301,用基因枪法将重组质粒转入苦荞外植体根、茎、叶和种子中并启动β-葡萄糖醛酸酶(β-glucuronidase,GUS)基因瞬时表达,被轰击的外植体经GUS染色后观察表明,种子中GUS的表达量最高,叶片中有微量GUS表达,苦荞的根和茎中均无肉眼可见的GUS表达。(4)将全长启动子进行5′端缺失形成4段启动子分析片段,并分别构建截短启动子的重组双荧光报告载体GP-TBW16P1、GP-TBW16P2、GP-TBW16P3和GP-TBW16P4。分别将4种重组载体转入苦荞叶片原生质体中进行瞬时表达,结果显示在TBW 16启动子上存在调节基因转录的区域,约位于-1564 bp至-1876bp。报告基因在GA、MeJA和ABA激素处理时表达量增加,证明TBW16启动子上存在响应三种激素胁迫的作用元件。(5)向pTCK303载体中插入TBW16反向和正向干扰片,构建苦荞TBW16干扰载体(TBW16i-pTCK303)。将TBW16过表达片段插入到pTCK303载体中构建了苦荞过表达载体(TBW16m-pTCK303),为16kDa过敏原的进一步研究苦荞和低敏种质的创制奠定了基础。
[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.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2;S517

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