基于转录组测序的辣木内参基因筛选和三个代谢途径相关基因的表达分析

发布时间：2018-01-14 01:23

本文关键词：基于转录组测序的辣木内参基因筛选和三个代谢途径相关基因的表达分析　出处：《华南农业大学》2016年硕士论文　论文类型：学位论文

【摘要】：辣木(Moringa oleifera Lam.,)是辣木科(Moringaceae)多年生速生乔木。辣木全株都含有丰富的营养和药用成分,具有重要经济价值,尤其在保健品、油料和饲料的开发利用上前景广阔。本研究通过测定辣木叶片的转录组,构建了高质量的辣木转录组数据库,筛选了一批内参基因,获得了调控辣木黄酮类、萜类和脂肪酸3种化合物合成途径中的相关基因及其在不同组织和发育周期的表达情况。主要研究结果如下:1、对辣木叶片进行转录姐测序得到38,672,496条clean reads,45,052条基因,丰富了辣木转录组数据库;逑2、从辣木转录组数据库中筛选到一批常用的候选内参基因,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)技术和3个常用内参基因稳定性分析软件(geNorm,NormFinder,Bestkeeper)分析和评价各内参基因在不同发育阶段的果实、不同的组织、4种不同的胁迫(低温、高温、盐和干旱),共6个实验条件下的稳定性,结果表明基因RPL1和ACP2在所有样品中具有最好的稳定性,而在不同的实验条件下,最稳定的内参基因会发生变化。另外,盐和干旱两种胁迫下筛选的内参基因被用于对应条件下3个过氧化物歧化酶同型基因(Cu/Zn-SOD,Fe-SOD和Mn-SOD)的RT-qPCR表达定量,三个基因的表达量随着处理时间的延长而增加,这个结果和前人的研究相符,验证了所筛选内参基因的可行性;4、筛选了和辣木黄酮类化合物合成相关的关键基因,包括PAL、4CL、CHS、F3H、DFR、LAR。通过RT-qPCR技术定量这些基因在辣木不同组织(根、茎、成熟叶、嫩叶和幼果)中的表达量,表明黄酮类化合物合成相关的基因在辣木的根和叶中表达量更高,黄酮类化合物的合成可能在辣木的根和叶中更活跃;5、筛选了一批和辣木萜类化合物合成相关的关键基因,包括HMGR、DXS、DXR、GPPS、CPR。这些基因在辣木根、茎、成熟叶、嫩叶和幼果中的定量结果表明,MEP途径萜类化合物的合成可能在辣木的成熟叶片中最活跃;6、筛选了和辣木脂肪酸合成相关的关键基因,包括ACCase、FAD2、KASI、SAD、FATA、FATB、LACS。根据这些基因在辣木不同发育时期的幼果的表达情况,说明在辣木幼果的不同发育时期,幼果中脂肪酸的合成可能主要用于果实的发育,而和脂肪酸不饱和度相关的基因表达在幼果发育时期变化程度不大,可能在果实成熟阶段发挥作用。
[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.

【学位授予单位】：华南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S792.99

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