本文關(guān)鍵詞： 甘薯 基因差異表達(dá) 淀粉合成 果糖-1 6-二磷酸醛縮酶 逆境脅迫　出處：《中國農(nóng)業(yè)科學(xué)院》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：轉(zhuǎn)錄組測序技術(shù)是獲取功能基因簡單快捷的方法,并可根據(jù)轉(zhuǎn)錄組測序結(jié)果估算出基因的差異表達(dá)水平。本實驗室對淀粉含量中等的徐薯18和高淀粉含量的徐781進(jìn)行轉(zhuǎn)錄組測序,從中獲得14個與淀粉合成相關(guān)的差異表達(dá)片段。為進(jìn)一步了解這些基因的功能特性,本研究采用qRT-PCR方法研究這些基因在甘薯膨大各時期的表達(dá)模式,分析基因表達(dá)量與淀粉含量的內(nèi)在聯(lián)系,并對IbFBA2和IbFBA5兩個基因進(jìn)行克隆與初步功能驗證。主要結(jié)果如下:1.根據(jù)qRT-PCR結(jié)果,這14個基因共呈現(xiàn)出6種不同的表達(dá)模式,即表達(dá)量逐漸上升、表達(dá)量不斷下降、先上升-再下降、先下降-再上升、上升-下降-上升和下降-上升-下降。同一基因在不同品種之間、以及同一品種不同組織之間呈現(xiàn)不同的表達(dá)模式。其中NMT3在兩個品種的兩個部位間表現(xiàn)出4種不同的表達(dá)模式,其他基因均呈現(xiàn)出2到3種不同的表達(dá)模式。2.徐薯18葉片中的淀粉含量變化趨勢與其葉片中IbFBA2的表達(dá)模式相同,皆為先下降后上升的模式;徐薯18薯塊中淀粉含量的變化趨勢與其薯塊中GLDP1、IbAGPb1A、SBPas、T1-30106和T2-33205的表達(dá)模式相同,皆為逐漸增加。徐781葉片中淀粉含量變化與其葉片中IbFBA2、NMT3、SBPase、XYL1、T1-23698和T2-33205的表達(dá)模式相同,皆為先下降后上升;徐781薯塊中淀粉含量的變化趨勢與其薯塊中IbAGPb1A、IbFBA5、MAP65-1、NMT2、T1-23698和T2-33205的表達(dá)模式相同,皆為先上升后下降。綜合兩個品種來看,IbFBA2在兩個品種葉片中表達(dá)模式均與淀粉變化情況吻合;IbAGPb1A和T2-33205在兩個品種薯塊中表達(dá)模式均與淀粉變化情況吻合。另外,XYL1在不同時期的表達(dá)水平與蔗糖積累的變化趨勢一致。3.從徐781中克隆到IbFBA2和IbFBA5。IbFBA2的cDNA全長1617 bp,開放閱讀框為1197bp,編碼398個氨基酸殘基,含5個外顯子和4個內(nèi)含子,預(yù)測其亞細(xì)胞定位到質(zhì)體中;IbFBA5的cDNA全長1341 bp,開放閱讀框1074 bp,編碼357個氨基酸殘基,含3個外顯子和2個內(nèi)含子,預(yù)測其亞細(xì)胞定位到胞質(zhì)中。4.分析IbFBA2和IbFBA5在栽后70天徐781的7個部位的表達(dá)情況發(fā)現(xiàn),兩個基因均具有組織表達(dá)特異性,IbFBA2主要在地上部表達(dá)量高,塊根中表達(dá)量低;IbFBA5在葉片、儲藏根、牛蒡根中表達(dá)量高,在花中表達(dá)量最低。5.IbFBA2和IbFBA5均能響應(yīng)ABA處理以及鹽、模擬干旱脅迫,但兩基因響應(yīng)脅迫的表達(dá)模式不同,IbFBA2的表達(dá)量皆呈先下降再上升的模式;而IbFBA5的表達(dá)量則顯著高于0 h的表達(dá)量。6.構(gòu)建過表達(dá)載體pGWB12::IbFBA2和pGWB12::IbFBA5,并瞬時轉(zhuǎn)化煙草葉片,由碘染結(jié)果推測IbFBA可能不是淀粉合成的主效基因;轉(zhuǎn)基因擬南芥的互補(bǔ)實驗表明,IbFBA5響應(yīng)ABA處理,并使轉(zhuǎn)基因株系的根長恢復(fù)到野生型的表型。
[Abstract]:Transcriptome sequencing is a simple and rapid method for obtaining functional genes. The differentially expressed level of the gene could be estimated according to the results of transcriptome sequencing. Xushu 18 with medium starch content and Xu 781 with high starch content were sequenced in our laboratory. Fourteen differentially expressed fragments related to starch synthesis were obtained to further understand the functional characteristics of these genes. In this study, qRT-PCR method was used to study the expression patterns of these genes in different stages of sweet potato expansion, and to analyze the relationship between the amount of gene expression and the content of starch. The main results are as follows: 1. According to the results of qRT-PCR. These 14 genes showed six different expression patterns, that is, the expression level gradually increased, the expression level decreased, first ascended-then decreased, first-then increased-then increased. Rise-down-up-and-down-down-down-down-down. the same gene is between different varieties. The expression patterns of NMT3 were different among different tissues of the same variety, and there were 4 different expression patterns between the two parts of the two cultivars. All the other genes showed 2 to 3 different expression patterns. 2. The change trend of starch content in Xushu 18 leaves was the same as the IbFBA2 expression pattern in its leaves. The change trend of starch content in Xushu 18 potato was the same as the expression pattern of IbAGPb1 / IbAGPb1 / SB Pasa T1-30106 and T2-33205 in tuber. The changes of starch content in X781 leaves and IbFBA2NMT3NMT3NMT3 SBPasePaseXYL1 in X781 leaves were all increased gradually. The expression patterns of T1-23698 and T2-33205 were the same. The change trend of starch content in Xu781 tuber and IbAGPb1An IbFBA5 MAP65-1 NMT2 in tuber. The expression patterns of T1-23698 and T2-33205 were the same. The expression patterns of IbFBA2 in the leaves of the two varieties were consistent with the changes of starch. The expression patterns of IbAGPb1A and T2-33205 in the two varieties were consistent with the changes of starch. The expression level of XYL1 at different stages was consistent with the change trend of sucrose accumulation. The total length of cDNA 1617 was cloned from Xu 781 and cloned into IbFBA2 and IbFBA5.IbFBA2. Bp. The open reading frame was 1197bp, encoding 398 amino acid residues, including 5 exons and 4 introns, which were predicted to be located in the plastids. The cDNA of IbFBA5 is 1341 BP, open reading frame 1074 BP, encoding 357 amino acid residues, including 3 exons and 2 introns. The expression of IbFBA2 and IbFBA5 in 7 parts of Xu781 were analyzed 70 days after transplanting. The results showed that the two genes were tissue-specific. The expression of IbFBA2 was high in shoot and low in root. IbFBA5 expression was high in leaves, storage roots and burdock roots, and the lowest in flowers. IbFBA2 and IbFBA5 could respond to ABA treatment and salt, simulating drought stress. However, the expression patterns of IbFBA2 were different in response to stress, and the expression level of IbFBA2 decreased first and then increased. The expression of IbFBA5 was significantly higher than that of 0 h. 6. The overexpression vectors pGWB12::IbFBA2 and pGWB12::IbFBA5 were constructed. The results of iodine staining suggested that IbFBA might not be the main gene of starch synthesis. The complementary experiment of transgenic Arabidopsis thaliana showed that IbFBA5 responded to ABA treatment and restored the root length of transgenic lines to the wild-type phenotype.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S531;Q943.2

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