红花天冬氨酸代谢途径关键酶基因的挖掘及功能鉴定

发布时间：2018-05-21 09:27

本文选题：红花籽粒 + 转录组高通量测序　；参考：《吉林农业大学》2016年博士论文

【摘要】：红花Carthamus tinctorius L.是菊科(Asteraceae)红花属一年生或二年生草本植物,在我国已有2100多年的栽培历史,也是我国唯一的栽培品种。红花是一种集药材、油料、饲料于一身的新兴特种经济作物。目前,对红花种子的油脂及花冠的色素已经进行了广泛而深入的研究,而对红花饲用价值的基础研究极少见报道。红花籽粒饲用的营养价值主要体现在以天冬氨酸为前体的必需氨基酸含量上。然而在红花去壳去油后籽粕中赖氨酸等必需氨基酸含量极低,制约了其在动物饲料工业上的应用。因此,提高红花籽粒中必需氨基酸含量,培育红花新品种显得尤为重要。但是,红花现有的遗传信息十分有限,不能满足我们从代谢工程的角度开展改善红花籽粒营养品质的研究。因此,挖掘与红花天冬氨酸代谢途径有关的基因资源对于提高红花籽粒必需氨基酸含量具有十分重要的研究意义和应用价值。鉴于上述分析,本研究开展了红花籽粒发育后期转录组文库高通量测序工作,在转录水平全面了解红花籽粒发育成熟过程中基因表达的变化情况,发掘红花籽粒天冬氨酸代谢途径重要候选功能基因,并分析这些基因在红花籽粒发育后期的表达及其与总游离氨基酸含量的关系,同时分离得到天冬氨酸代谢途径第一个关键限速酶CtAK1基因并进行功能初步分析。本研究一方面丰富了红花的遗传信息资源,为阐明CtAK1的生物学功能及解析其在红花籽粒天冬氨酸代谢途径中的作用机制奠定基础,另一方面也为红花籽粒品质改良分子育种工作提供了重要的遗传背景基础和科学依据。主要研究结论如下:1、应用IlluminaHiseq2000测序平台对红花籽粒发育后期3份材料进行转录组文库高通量测序,共获得超过6GB数据量,共138193个Unigenes,平均长度700bp,为后续分析提供基础数据。基因功能注释分析发现所有的Unigenes均得到注释,说明转录组数据拼接质量较高。Unigenes的CDS区预测共获得54291个CDS,占39.29%,其中长度在1000bp以上的序列有14509个,占26.74%。皮尔森系数法分析样品间基因表达水平相关性发现各样品间的相关系数在0.661-0.782之间,说明在红花籽粒不同发育时期基因的表达是不同的,即样品间存在一定程度的相似性,又有各自发育时期的特点。2、通过基因差异表达分析,共鉴定出1528个差异基因参与到131条代谢途径,其中参与到天冬氨酸代谢途径的Unigene有179个,参与到蛋氨酸代谢途径的Unigene有264个,参与到支链氨基酸代谢途径的Unigene有68个,参与到芳香族氨基酸代谢途径的Unigene有149个。在179个天冬氨酸代谢途径候选基因中去除注释信息重复的序列,共筛选到11条Unigene,它们分别是c14707_g1,c52815_g4,c55378_g1,c54079_g2,c67402_g1,c67326_g1,c12631_g1,c53058_g1,c18700_g1,c48258_g3,c46041_g1。3、利用RT-qPCR技术筛选出红花籽粒不同发育时期稳定表达的CtEF1A和Ct60S组合为内参基因,选取上述9个候选基因进行表达分析,同时检测总游离氨基酸的含量,通过二者相关性分析发现4个候选基因(c55378_g1,c67326_g1,c12631_g1,c48258_g3)的表达与总游离氨基酸的含量关系十分密切。同时与转录组测序数据进行相关性分析,发现候选基因的表达模式与转录组测序分析结果一致,证实转录组高通量测序分析的准确性,同时也获得红花天冬氨酸代谢途径重要候选基因,为进一步研究奠定基础。4、本研究选取红花籽粒天冬氨酸代谢途径第一个关键限速酶CtAK1进行基因克隆和功能验证。首先通过RACE和定点突变技术获得CtAK1及其突变体mCtAK1;利用DNA重组技术构建植物超表达载体pCAMBIA3301-CTP-(m)CtAK1,通过冻融法获得阳性农杆菌工程菌;采用Flora dip法转化拟南芥,分别收获5株CtAK1-OE和3株mCt AK1-OE T2代拟南芥种子。RT-qPCR分析表明:在T2代转基因拟南芥中,CtAK1-OE和mCtAK1-OE在转录水平的表达量分别提高2.94倍和4.4倍。总游离氨基酸含量分析表明:与野生型拟南芥相比,mCtAK1-OE拟南芥种子总游离氨基酸含量提高2.38倍,CtAK-OE拟南芥种子总游离氨基酸提高1.87倍。
[Abstract]:Safflower Carthamus tinctorius L. is an annual or biennial herb of the genus Carthamus from the Compositae (Asteraceae). It has been cultivated for more than 2100 years in our country and is the only cultivar in China. The red flower is a new special economic crop, which combines medicinal materials, oil and feed. The basic research on the feeding value of safflower is rarely reported. The nutritional value of safflower seed feeding is mainly reflected in the essential amino acid content of aspartic acid as the precursor. However, the content of lysine and other essential amino acids in the seed meal after safflower deoiling is very low, which restricts its animal feed workers. Therefore, it is very important to improve the content of essential amino acids in the seed of safflower and to cultivate new safflower varieties. However, the existing genetic information of safflower is very limited, which can not satisfy our research on improving the nutritional quality of safflower seed from the angle of metabolic engineering. In view of the above analysis, the high throughput sequencing of the transcriptional Bank of the late growth of safflower seed was carried out in this study. The important candidate functional genes for the anaspartic acid metabolism pathway of flower seeds were analyzed. The expression of these genes in the late development of safflower seed and the relationship with the total free amino acid content were analyzed. At the same time, the first key speed limiting enzyme CtAK1 gene of aspartic acid metabolism pathway was isolated and its function was preliminarily analyzed. Genetic information resources have laid the foundation for clarifying the biological function of CtAK1 and analyzing its mechanism in the metabolic pathway of safflower aspartic acid. On the other hand, it also provides important genetic background and scientific basis for the improved molecular breeding of safflower seed quality. The main conclusions are as follows: 1, the application of IlluminaHiseq2000 The sequence platform was used to carry out high throughput sequencing of 3 materials in the late stage of safflower seed development. A total of more than 6GB data, a total of 138193 Unigenes, with an average length of 700bp, was used to provide basic data for the follow-up analysis. The DS area forecast 54291 CDS, accounting for 39.29%, of which 14509 of the sequences above 1000bp, accounting for the correlation of gene expression level between samples by 26.74%. Pearson coefficient method, found that the correlation coefficient between various products was between 0.661-0.782, indicating that the expression of genes in different developmental stages of safflower grains was different, that is, the existence of samples between samples. A certain degree of similarity, and the characteristics of their respective developmental stages.2, through gene differential expression analysis, 1528 differentially expressed genes were identified to participate in 131 metabolic pathways, of which, there were 179 Unigene involved in aspartic acid metabolism pathway, 264 Unigene involved in methionine metabolic pathway, and participated in the pathway of the branched chain amino acid metabolism. There are 68 Unigene and 149 Unigene involved in the metabolic pathway of aromatic amino acids. In the 179 aspartic pathway candidate genes, the repeating sequence of annotated information is removed, and 11 Unigene are screened. They are c14707_g1, c52815_g4, c55378_g1, c54079_g2, c67402_g1, c67326_g1, c12631_g1, c53058_g1, c18700_g1, etc. 6041_g1.3, RT-qPCR technology was used to screen the CtEF1A and Ct60S combinations of the stable expression of safflower grains at different developmental stages. The 9 candidate genes were selected for expression analysis, and the content of total free amino acids was detected. 4 candidate genes (c55378_g1, c67326_g1, c12631_g1, c48258_g3) were detected by two correlation analysis. The expression was closely related to the content of total free amino acids. At the same time, the correlation analysis with the sequencing data of the transcriptional group showed that the expression pattern of the candidate gene was consistent with the result of the sequence analysis of the transcriptional group, confirmed the accuracy of the high throughput sequencing analysis in the transcriptional group, and also obtained the important candidate genes for the metabolic pathway of the safflower aspartic acid. Step research lays the foundation for.4. This study selects the first key speed limiting enzyme CtAK1 of the aspartic acid metabolism pathway of safflower seed to carry out gene cloning and functional verification. First, CtAK1 and its mutant mCtAK1 are obtained by RACE and site directed mutagenesis, and pCAMBIA3301-CTP- (m) CtAK1, a plant overexpression vector, is constructed by DNA recombination technology and obtained by freezing and thawing method. Positive Agrobacterium tumefaciens were obtained; the Arabidopsis thaliana was transformed by Flora dip method, and 5 CtAK1-OE and 3 strains of mCt AK1-OE T2 were harvested for.RT-qPCR analysis. The expression of CtAK1-OE and mCtAK1-OE at the transcriptional level was increased by 2.94 times and 4.4 times respectively in T2 generation transgenic Arabidopsis. The analysis of total free amino acids showed that the total free amino acid content was in the wild. Compared with Arabidopsis thaliana, the total free amino acid content of Arabidopsis thaliana seeds increased by 2.38 times, and the total free amino acids of CtAK-OE Arabidopsis thaliana increased 1.87 times than that of mCtAK1-OE.
【学位授予单位】：吉林农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S567.219

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