本文關(guān)鍵詞：基于轉(zhuǎn)錄組測(cè)序的辣木內(nèi)參基因篩選和三個(gè)代謝途徑相關(guān)基因的表達(dá)分析　出處：《華南農(nóng)業(yè)大學(xué)》2016年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 辣木 轉(zhuǎn)錄組 內(nèi)參基因 黃酮 萜類(lèi) 脂肪酸

【摘要】：辣木(Moringa oleifera Lam.,)是辣木科(Moringaceae)多年生速生喬木。辣木全株都含有豐富的營(yíng)養(yǎng)和藥用成分,具有重要經(jīng)濟(jì)價(jià)值,尤其在保健品、油料和飼料的開(kāi)發(fā)利用上前景廣闊。本研究通過(guò)測(cè)定辣木葉片的轉(zhuǎn)錄組,構(gòu)建了高質(zhì)量的辣木轉(zhuǎn)錄組數(shù)據(jù)庫(kù),篩選了一批內(nèi)參基因,獲得了調(diào)控辣木黃酮類(lèi)、萜類(lèi)和脂肪酸3種化合物合成途徑中的相關(guān)基因及其在不同組織和發(fā)育周期的表達(dá)情況。主要研究結(jié)果如下:1、對(duì)辣木葉片進(jìn)行轉(zhuǎn)錄姐測(cè)序得到38,672,496條clean reads,45,052條基因,豐富了辣木轉(zhuǎn)錄組數(shù)據(jù)庫(kù);逑2、從辣木轉(zhuǎn)錄組數(shù)據(jù)庫(kù)中篩選到一批常用的候選內(nèi)參基因,包括GAPDH、PEPC、ACP1、ACP2、UBCE、TUA1、TUA2、RPL1、RPL2、MDH1、MDH2、ACT、UEP、EF1、EF2、TUB、CYP1、CYP2;3、利用RT-qPCR(Real-time quantitative PCR)技術(shù)和3個(gè)常用內(nèi)參基因穩(wěn)定性分析軟件(geNorm,NormFinder,Bestkeeper)分析和評(píng)價(jià)各內(nèi)參基因在不同發(fā)育階段的果實(shí)、不同的組織、4種不同的脅迫(低溫、高溫、鹽和干旱),共6個(gè)實(shí)驗(yàn)條件下的穩(wěn)定性,結(jié)果表明基因RPL1和ACP2在所有樣品中具有最好的穩(wěn)定性,而在不同的實(shí)驗(yàn)條件下,最穩(wěn)定的內(nèi)參基因會(huì)發(fā)生變化。另外,鹽和干旱兩種脅迫下篩選的內(nèi)參基因被用于對(duì)應(yīng)條件下3個(gè)過(guò)氧化物歧化酶同型基因(Cu/Zn-SOD,Fe-SOD和Mn-SOD)的RT-qPCR表達(dá)定量,三個(gè)基因的表達(dá)量隨著處理時(shí)間的延長(zhǎng)而增加,這個(gè)結(jié)果和前人的研究相符,驗(yàn)證了所篩選內(nèi)參基因的可行性;4、篩選了和辣木黃酮類(lèi)化合物合成相關(guān)的關(guān)鍵基因,包括PAL、4CL、CHS、F3H、DFR、LAR。通過(guò)RT-qPCR技術(shù)定量這些基因在辣木不同組織(根、莖、成熟葉、嫩葉和幼果)中的表達(dá)量,表明黃酮類(lèi)化合物合成相關(guān)的基因在辣木的根和葉中表達(dá)量更高,黃酮類(lèi)化合物的合成可能在辣木的根和葉中更活躍;5、篩選了一批和辣木萜類(lèi)化合物合成相關(guān)的關(guān)鍵基因,包括HMGR、DXS、DXR、GPPS、CPR。這些基因在辣木根、莖、成熟葉、嫩葉和幼果中的定量結(jié)果表明,MEP途徑萜類(lèi)化合物的合成可能在辣木的成熟葉片中最活躍;6、篩選了和辣木脂肪酸合成相關(guān)的關(guān)鍵基因,包括ACCase、FAD2、KASI、SAD、FATA、FATB、LACS。根據(jù)這些基因在辣木不同發(fā)育時(shí)期的幼果的表達(dá)情況,說(shuō)明在辣木幼果的不同發(fā)育時(shí)期,幼果中脂肪酸的合成可能主要用于果實(shí)的發(fā)育,而和脂肪酸不飽和度相關(guān)的基因表達(dá)在幼果發(fā)育時(shí)期變化程度不大,可能在果實(shí)成熟階段發(fā)揮作用。
[Abstract]:Moringa (Moringa oleifera Lam.) Moringaceae (Moringaceae) is a fast-growing perennial tree. Moringa all plants contain rich nutrition and medicinal components, has important economic value, especially in health care, the vast prospect of development and utilization of oil and feed. This study by measuring the transcriptome set constructed Moringa leaves, Moringa the transcriptome database of high quality, select a set of reference genes, the regulation of Moringa flavonoids, terpenoids and fatty acid related genes of 3 compounds in the biosynthesis pathway and its expression in different tissues and development cycle. The main results are as follows: 1, the Moringa leaves were sequenced to obtain 38672496 clean transcription sister reads, 45052 genes, enrich the transcriptome database for 2 Moringa oleifera;, screened from Moringa transcriptome database to a number of common candidate reference genes, including GAPDH, PEPC, ACP1, ACP2, UBCE, TUA1, TUA2, RP L1, RPL2, MDH1, MDH2, ACT, UEP, EF1, EF2, TUB, CYP1, CYP2; 3, by RT-qPCR (Real-time quantitative PCR) software technology and analysis of 3 commonly used reference genes (geNorm, NormFinder, Bestkeeper stability) analysis and evaluation of various reference genes in different developmental stages of fruit, different organizations 4, different stress (low temperature, high temperature, salt and drought), the stability of a total of 6 experimental conditions, results showed that RPL1 gene and ACP2 has the best stability in all samples, and under different experimental conditions, in the most stable reference gene will change. In addition, salt and drought in two a stress screening reference gene was used for corresponding conditions of 3 superoxide dismutase gene with type (Cu/Zn-SOD, Fe-SOD and Mn-SOD) RT-qPCR quantitative expression, the expression of three genes increased with the prolonging of treatment time, the research results and the previous results, verified The feasibility of screening of reference genes; 4, screening key genes and Moringa flavonoids synthesis related including PAL, 4CL, CHS, F3H, DFR, LAR. by quantitative RT-qPCR of these genes in different tissues of Moringa (root, stem, mature leaves, young leaves and young fruit) expression in the indicated genes the synthesis of flavonoids is more highly expressed in the roots and leaves, the synthesis of flavonoids may be more active in the roots and leaves; 5, screening key genes, and a group of M.oleifera terpenoids synthesis related including HMGR, DXS, DXR, GPPS, CPR. of these genes in spicy the root, stem, mature leaves, young leaves and young fruit and the quantitative results show that the synthesis of MEP pathway of terpenoids may be the most active in the mature leaves of Moringa oleifera; 6, screening and M.oleifera fatty acid synthesis related genes, including ACCase, FAD2, KASI, SAD, FATA, FATB, according to LACS. The expression of these genes in different developmental stages of Moringa fruit, in different developmental stages of Moringa fruit, mainly used for fatty acid synthesis may young fruit and fruit development, the expression of related genes and the degree of unsaturation of fatty acids in the period of fruit development degree of change is small, may play a role in the ripening stage.

【學(xué)位授予單位】：華南農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：S792.99

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