本文選題：葡萄 + VvABCG20基因　； 參考：《西北農(nóng)林科技大學(xué)》2017年碩士論文

【摘要】：本研究以有核與不同殘核大小的無核葡萄為材料克隆VvABCG20基因啟動子,并分析其序列間的差異,對VvABCG20基因啟動子進行不同長度缺失分析并構(gòu)建GUS報告基因融合載體,利用瞬時轉(zhuǎn)化法轉(zhuǎn)化煙草葉片,分析各片段啟動子的活性。同時通過激素處理分析啟動子的激素響應(yīng)元件。使用花序浸潤法侵染野生型擬南芥,并對得到的轉(zhuǎn)基因植株的各組織器官進行GUS組織染色,分析其GUS表達活性,并分析葡萄VvABCG20基因啟動子是否具有組織特異性。主要研究結(jié)果如下:1、獲得了15個葡萄品種VvABCG20基因的啟動子,發(fā)現(xiàn)有8個品種(包括有核和無核)同時得到了VvABCG20基因的啟動子序列(a)及其缺失序列(b),其余品種為缺失序列(b),且這兩種序列沒有有核或無核的規(guī)律性,其中黑比諾a序列與無核白a序列完全一致,記為P0。通過多序列比對發(fā)現(xiàn)所有的b序列均在802 bp處相比a序列缺失一段41bp的片段,且兩種序列與葡萄基因組網(wǎng)站預(yù)測的序列相似度均在97%以上,可能是同源染色體序列不同所導(dǎo)致的,出現(xiàn)兩種序列的材料為雜合體。將15個品種獲得的共23條序列進行多比對,分析結(jié)果顯示,所有序列間只有4處呈現(xiàn)出片段上的差異其余部位均是個別堿基的插入、缺失和突變等,有核葡萄品種和無核葡萄品種的啟動子序列間并無明顯差異。對23條啟動子序列的單核苷酸多態(tài)性進行分析,結(jié)果發(fā)現(xiàn)共有66個變異位點,其中僅721 bp處的變異導(dǎo)致失去O2-site元件,其余位點并未檢測到重要元件,說明15個葡萄品種的VvABCG20基因的啟動子序列表現(xiàn)出很高的保守性。2、通過分析無核白VvABCG20基因啟動子序列(b)的順式作用元件,可知該啟動子序列不僅包含啟動子基本的核心元件,還包含一些與種子的特異性表達相關(guān)的作用元件,如E-BOX、RY重復(fù)序列、-300 element、Skn-1-motif、SEF1、SEF 4等。對黑比諾VvABCG20基因啟動子序列順式作用元件分析發(fā)現(xiàn):調(diào)控元件與無核白相比沒有種類上的差異,只在一些元件的數(shù)量上有差異,如SEF1、SEF4和E-BOX等。通過瞬時轉(zhuǎn)化法將無核白和黑比諾VvABCG20基因啟動子全長(a、b兩種序列)轉(zhuǎn)化煙草葉片,通過GUS組織化學(xué)分析,結(jié)果表明兩者的VvABCG20基因得啟動子(a、b兩種序列)都具有啟動子活性,且黑比諾b序列啟動子(P1)的活性顯著高于無核白b序列(T1)和P0序列(P0.01);P0與T1組織染色與GUS定量均顯示啟動子活性沒有顯著差異(P0.01),推測P1序列中存在增強啟動子活性的作用元件。3、綜合分析黑比諾與無核白VvABCG20基因啟動子順勢元件及序列上的差異,對VvABCG20基因啟動子(b序列)進行5’端缺失分析,得到黑比諾全長及缺失片段5條(P1、P2、P3、P4、P5),無核白全長及缺失片段5條(T1、T2、T3、T4、T5)。將全長序列(P0、P1、T1)和各缺失片段與GUS的融合體瞬時轉(zhuǎn)化法轉(zhuǎn)化煙草葉片并噴施IAA、MeJA、ETH處理葉片,通過GUS組織化學(xué)分析,推測如下:黑比諾VvABCG20啟動子全長序列(P0、P1)的-1350 bp處和無核白VvABCG20基因啟動子的-1348 bp處的ERE元件可響應(yīng)ETH的誘導(dǎo),且對啟動子的活性起負調(diào)控的作用;黑比諾和無核白缺失片段P2和T2的-1009 bp和-1008 bp處的RY-motif元件可能應(yīng)答ETH的誘導(dǎo),且其響應(yīng)可增強啟動子活性;黑比諾VvABCG20基因啟動子全長P1的-1350 bp處和無核白VvABCG20基因啟動子全長T1的-1348 bp處的BOX-Ⅰ元件可以響應(yīng)MeJA的誘導(dǎo)且其對MEJA的響應(yīng)對啟動子活性有上調(diào)作用;位于黑比諾和無核白各啟動子片段末端的TGA-element元件可對IAA的誘導(dǎo)產(chǎn)生響應(yīng)并對啟動子活性起上調(diào)作用;黑比諾啟動子-807 bp上游和無核白啟動子-806 bp上游有受IAA誘導(dǎo)可增強啟動子活性的元件,但具體哪些元件及所在位置還需要進行更進一步的研究;P0序列上的ELI-box3元件響應(yīng)激素對啟動子活性有上調(diào)作用,推測ELI-box3元件可能不響應(yīng)MeJA但響應(yīng)ERE和IAA的誘導(dǎo)。4、對葡萄VvABCG20基因啟動子P0轉(zhuǎn)基因擬南芥進行GUS組織染色染色,結(jié)果顯示35S和VvABCG20基因啟動子均可驅(qū)動GUS基因在擬南芥各器官表達,野生型植株各器官均沒有GUS活性。但VvABCG20驅(qū)動的GUS基因在各組織表達量有無顯著差異,且Vv ABCG20基因啟動子的特異性是否表現(xiàn)在黑比諾和無核白另一條序列還需下一步試驗。分析推測,可能是葡萄中啟動子被整合到擬南芥中,其轉(zhuǎn)錄因子的結(jié)合受到了異源植株的影響而使其種子特異性受到影響,或者我們獲得的啟動子長度不夠,因而沒有表現(xiàn)出種子特異性,而VvABCG20基因啟動子是否為種子特異性啟動子及其控制種子特異表達的核心區(qū)段還需進一步驗證。
[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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S663.1

【參考文獻】

相關(guān)期刊論文 前10條

1 李濯雪;陳信波;;植物組織特異性啟動子及相關(guān)順式作用元件研究進展[J];生物學(xué)雜志;2015年06期

2 張劍俠;牛茹萱;;無核葡萄胚挽救技術(shù)的研究現(xiàn)狀與展望[J];園藝學(xué)報;2013年09期

3 宋揚;周軍會;張永強;;植物組織特異性啟動子研究[J];生物技術(shù)通報;2007年06期

4 王躍進;江淑平;劉小寧;張劍俠;;假單性結(jié)實無核葡萄胚敗育機理研究[J];西北植物學(xué)報;2007年10期

5 林炳英;李梅;林德欽;金志強;;番茄果實特異性啟動子2A11的基因克隆及功能研究[J];中國農(nóng)學(xué)通報;2006年10期

6 楊英軍,王躍進,周鵬,王西平,張劍俠;葡萄無核基因的SCAR標(biāo)記及Southern blot分析[J];西北農(nóng)林科技大學(xué)學(xué)報(自然科學(xué)版);2002年06期

7 林潔,來茂德;HMLH1啟動子甲基化和hMLH1表達及微衛(wèi)星不穩(wěn)定性[J];浙江大學(xué)學(xué)報(醫(yī)學(xué)版);2002年04期

8 王躍進,楊英軍,周鵬,張劍俠,王西平;用DNA探針檢測我國栽培的無核葡萄及輔助育種初探[J];園藝學(xué)報;2002年02期

9 王躍進,Lamikanra Olusola;檢測葡萄無核基因DNA探針的合成與應(yīng)用[J];西北農(nóng)林科技大學(xué)學(xué)報(自然科學(xué)版);2002年03期

10 王躍進,ＬａｍｉｋａｎｒａＯ,盧江,ＲａｍｍｉｎｇＤ;葡萄無核基因的RAPD遺傳標(biāo)記[J];西北農(nóng)業(yè)大學(xué)學(xué)報;1996年05期

相關(guān)博士學(xué)位論文 前1條

1 徐偉榮;中國華東葡萄抗白粉病芪合成酶基因及啟動子克隆與功能分析[D];西北農(nóng)林科技大學(xué);2010年

相關(guān)碩士學(xué)位論文 前2條

1 唐玉瑾;葡萄ABCG基因亞家族半分子成員克隆及表達分析[D];西北農(nóng)林科技大學(xué);2016年

2 魏蓉;葡萄βVPE基因啟動子的克隆與功能分析[D];西北農(nóng)林科技大學(xué);2014年

，

本文編號：2066970