葡萄VvABCG20基因启动子的克隆及活性分析

发布时间：2018-06-25 17:52

本文选题：葡萄 + VvABCG20基因　；参考：《西北农林科技大学》2017年硕士论文

【摘要】：本研究以有核与不同残核大小的无核葡萄为材料克隆VvABCG20基因启动子,并分析其序列间的差异,对VvABCG20基因启动子进行不同长度缺失分析并构建GUS报告基因融合载体,利用瞬时转化法转化烟草叶片,分析各片段启动子的活性。同时通过激素处理分析启动子的激素响应元件。使用花序浸润法侵染野生型拟南芥,并对得到的转基因植株的各组织器官进行GUS组织染色,分析其GUS表达活性,并分析葡萄VvABCG20基因启动子是否具有组织特异性。主要研究结果如下:1、获得了15个葡萄品种VvABCG20基因的启动子,发现有8个品种(包括有核和无核)同时得到了VvABCG20基因的启动子序列(a)及其缺失序列(b),其余品种为缺失序列(b),且这两种序列没有有核或无核的规律性,其中黑比诺a序列与无核白a序列完全一致,记为P0。通过多序列比对发现所有的b序列均在802 bp处相比a序列缺失一段41bp的片段,且两种序列与葡萄基因组网站预测的序列相似度均在97%以上,可能是同源染色体序列不同所导致的,出现两种序列的材料为杂合体。将15个品种获得的共23条序列进行多比对,分析结果显示,所有序列间只有4处呈现出片段上的差异其余部位均是个别碱基的插入、缺失和突变等,有核葡萄品种和无核葡萄品种的启动子序列间并无明显差异。对23条启动子序列的单核苷酸多态性进行分析,结果发现共有66个变异位点,其中仅721 bp处的变异导致失去O2-site元件,其余位点并未检测到重要元件,说明15个葡萄品种的VvABCG20基因的启动子序列表现出很高的保守性。2、通过分析无核白VvABCG20基因启动子序列(b)的顺式作用元件,可知该启动子序列不仅包含启动子基本的核心元件,还包含一些与种子的特异性表达相关的作用元件,如E-BOX、RY重复序列、-300 element、Skn-1-motif、SEF1、SEF 4等。对黑比诺VvABCG20基因启动子序列顺式作用元件分析发现:调控元件与无核白相比没有种类上的差异,只在一些元件的数量上有差异,如SEF1、SEF4和E-BOX等。通过瞬时转化法将无核白和黑比诺VvABCG20基因启动子全长(a、b两种序列)转化烟草叶片,通过GUS组织化学分析,结果表明两者的VvABCG20基因得启动子(a、b两种序列)都具有启动子活性,且黑比诺b序列启动子(P1)的活性显著高于无核白b序列(T1)和P0序列(P0.01);P0与T1组织染色与GUS定量均显示启动子活性没有显著差异(P0.01),推测P1序列中存在增强启动子活性的作用元件。3、综合分析黑比诺与无核白VvABCG20基因启动子顺势元件及序列上的差异,对VvABCG20基因启动子(b序列)进行5’端缺失分析,得到黑比诺全长及缺失片段5条(P1、P2、P3、P4、P5),无核白全长及缺失片段5条(T1、T2、T3、T4、T5)。将全长序列(P0、P1、T1)和各缺失片段与GUS的融合体瞬时转化法转化烟草叶片并喷施IAA、MeJA、ETH处理叶片,通过GUS组织化学分析,推测如下:黑比诺VvABCG20启动子全长序列(P0、P1)的-1350 bp处和无核白VvABCG20基因启动子的-1348 bp处的ERE元件可响应ETH的诱导,且对启动子的活性起负调控的作用;黑比诺和无核白缺失片段P2和T2的-1009 bp和-1008 bp处的RY-motif元件可能应答ETH的诱导,且其响应可增强启动子活性;黑比诺VvABCG20基因启动子全长P1的-1350 bp处和无核白VvABCG20基因启动子全长T1的-1348 bp处的BOX-Ⅰ元件可以响应MeJA的诱导且其对MEJA的响应对启动子活性有上调作用;位于黑比诺和无核白各启动子片段末端的TGA-element元件可对IAA的诱导产生响应并对启动子活性起上调作用;黑比诺启动子-807 bp上游和无核白启动子-806 bp上游有受IAA诱导可增强启动子活性的元件,但具体哪些元件及所在位置还需要进行更进一步的研究;P0序列上的ELI-box3元件响应激素对启动子活性有上调作用,推测ELI-box3元件可能不响应MeJA但响应ERE和IAA的诱导。4、对葡萄VvABCG20基因启动子P0转基因拟南芥进行GUS组织染色染色,结果显示35S和VvABCG20基因启动子均可驱动GUS基因在拟南芥各器官表达,野生型植株各器官均没有GUS活性。但VvABCG20驱动的GUS基因在各组织表达量有无显著差异,且Vv ABCG20基因启动子的特异性是否表现在黑比诺和无核白另一条序列还需下一步试验。分析推测,可能是葡萄中启动子被整合到拟南芥中,其转录因子的结合受到了异源植株的影响而使其种子特异性受到影响,或者我们获得的启动子长度不够,因而没有表现出种子特异性,而VvABCG20基因启动子是否为种子特异性启动子及其控制种子特异表达的核心区段还需进一步验证。
[Abstract]:In this study, the VvABCG20 gene promoter was cloned with nucleated and different nucleus size seedless grapes, and the differences in the sequence were analyzed. The VvABCG20 gene promoter was analyzed with different length deletion and the GUS reporter gene fusion carrier was constructed. The transient transformation method was used to transform the tobacco leaf blade, and the activity of the promoter was analyzed. The hormone response element was used to analyze the hormone response element of the promoter by the hormone treatment. The wild type Arabidopsis was infected with the method of inflorescence, and the tissues and organs of the transgenic plants were stained by GUS tissue. The expression of GUS was analyzed and the VvABCG20 gene promoter of grape was analyzed. The main results are as follows: 1, 1 The promoter of the VvABCG20 gene of 5 grape varieties found that 8 varieties (including nuclear and nuclear free) were simultaneously obtained the promoter sequence of the VvABCG20 gene (a) and its deletion sequence (b), and the others were missing sequence (b), and the two sequences had no nuclear or nuclear regulation, and the black Pinot a sequence was exactly the same as the non nucleated a sequence. P0. through multiple sequence alignment found that all b sequences were missing a segment of 41bp compared with the a sequence at 802 BP, and the sequence similarity between the two sequences and the grape genome site was more than 97%, which might be caused by different homologous chromosomes, and two sequences were found to be heterozygous. The total of 15 varieties was 23. The results showed that there were only 4 fragments between all the sequences, and the other parts were the insertion, deletion and mutation of the other bases, and there was no significant difference between the promoter sequences of the seedless and seedless grape varieties. The single nucleotide polymorphisms of the 23 promoter sequences were analyzed. There are 66 mutation sites in the fruit, of which only 721 BP variation leads to the loss of O2-site elements, and the rest of the loci do not detect important components, indicating that the promoter sequence of the VvABCG20 gene of the 15 grape varieties shows a high conservatism.2, by analyzing the cis acting element of the VvABCG20 based promoter sequence (b) of the non nuclear white VvABCG20 The promoter sequence not only contains the core components of the promoter, but also contains some elements related to the specific expression of the seed, such as E-BOX, RY repeat, -300 element, Skn-1-motif, SEF1, SEF 4. The cis element analysis of the promoter sequence of the black Pinot VvABCG20 gene found that the regulatory element is not different from that of the non nuclear white. There are differences in the number of components, such as SEF1, SEF4 and E-BOX. Through transient transformation, the whole length of the promoter of the VvABCG20 gene (a, B, two sequences) is transformed into tobacco leaves. The results of GUS histochemical analysis show that the promoter of the two VvABCG20 genes (a, B two sequences) all have promoter. Activity, and the activity of the promoter (P1) of the black Pinot b sequence was significantly higher than that of the non nuclear white b sequence (T1) and P0 sequence (P0.01). P0 and T1 tissue staining and GUS quantified that there was no significant difference in promoter activity (P0.01). 5 'terminal deletion analysis of the VvABCG20 gene promoter (b sequence) was carried out in the sequence of homeopathic elements and sequences, and 5 strips of black Pinot whole length and missing fragments (P1, P2, P3, P4, P5), and 5 strips of total length of non nuclear white and 5 missing fragments (T1, T2, T3, T4, T5) were obtained. The leaves were treated with IAA, MeJA and ETH. Through the histochemical analysis of GUS, we speculated that the -1350 BP at P0, P1, and the -1348 BP of the non nuclear VvABCG20 gene promoter could respond to the induction and negatively regulate the activity of the promoter; black Pinot Noir and the absence of nuclear white deletion fragments. The RY-motif elements at the -1009 BP and -1008 BP of T2 may respond to the induction of ETH, and their response can enhance the promoter activity; the -1350 BP at the P1 VvABCG20 gene promoter of the black Pinot VvABCG20 gene promoter and the total length of the non nuclear white VvABCG20 gene promoter can respond to the induction and its response to the promoter. There is an up-regulated sex; the TGA-element element at the end of the promoters of the black Pinot and non nuclear white promoters can respond to the induction of IAA and up the promoter activity; the upstream of the black Pinot promoter and the upstream of the non nuclear white promoter -806 BP have the components that are induced by IAA to enhance the activity of the initiator, but which components and the components are specific. Further studies are needed in the position, and the response hormone on the P0 sequence has an up-regulated response to the promoter activity. It is speculated that ELI-box3 elements may not respond to MeJA but respond to ERE and IAA induced.4 and stain GUS tissue of the grape VvABCG20 gene promoter P0 transgenic Arabidopsis thaliana. The results show 35S and VvABCG20 base. The expression of GUS gene in various organs of Arabidopsis can be driven by promoter, but there is no GUS activity in all organs of wild type plants. But there is no significant difference in the expression of GUS gene driven by VvABCG20 in each tissue, and the specificity of the Vv ABCG20 gene promoter is still in the next step. It may be that the promoter of the grape is integrated into Arabidopsis thaliana. The binding of the transcription factors is affected by the heterologous plant, or the seed specificity of the promoter is affected, or the length of the promoter is not enough to show the seed specificity, and whether the promoter of the VvABCG20 gene is a seed specific promoter and its control species. The core segment of subspecific expression needs further verification.
【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】：S663.1

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