本文選題：紅花籽粒 + 轉(zhuǎn)錄組高通量測(cè)序��； 參考：《吉林農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：紅花Carthamus tinctorius L.是菊科(Asteraceae)紅花屬一年生或二年生草本植物,在我國(guó)已有2100多年的栽培歷史,也是我國(guó)唯一的栽培品種。紅花是一種集藥材、油料、飼料于一身的新興特種經(jīng)濟(jì)作物。目前,對(duì)紅花種子的油脂及花冠的色素已經(jīng)進(jìn)行了廣泛而深入的研究,而對(duì)紅花飼用價(jià)值的基礎(chǔ)研究極少見(jiàn)報(bào)道。紅花籽粒飼用的營(yíng)養(yǎng)價(jià)值主要體現(xiàn)在以天冬氨酸為前體的必需氨基酸含量上。然而在紅花去殼去油后籽粕中賴氨酸等必需氨基酸含量極低,制約了其在動(dòng)物飼料工業(yè)上的應(yīng)用。因此,提高紅花籽粒中必需氨基酸含量,培育紅花新品種顯得尤為重要。但是,紅花現(xiàn)有的遺傳信息十分有限,不能滿足我們從代謝工程的角度開(kāi)展改善紅花籽粒營(yíng)養(yǎng)品質(zhì)的研究。因此,挖掘與紅花天冬氨酸代謝途徑有關(guān)的基因資源對(duì)于提高紅花籽粒必需氨基酸含量具有十分重要的研究意義和應(yīng)用價(jià)值。鑒于上述分析,本研究開(kāi)展了紅花籽粒發(fā)育后期轉(zhuǎn)錄組文庫(kù)高通量測(cè)序工作,在轉(zhuǎn)錄水平全面了解紅花籽粒發(fā)育成熟過(guò)程中基因表達(dá)的變化情況,發(fā)掘紅花籽粒天冬氨酸代謝途徑重要候選功能基因,并分析這些基因在紅花籽粒發(fā)育后期的表達(dá)及其與總游離氨基酸含量的關(guān)系,同時(shí)分離得到天冬氨酸代謝途徑第一個(gè)關(guān)鍵限速酶CtAK1基因并進(jìn)行功能初步分析。本研究一方面豐富了紅花的遺傳信息資源,為闡明CtAK1的生物學(xué)功能及解析其在紅花籽粒天冬氨酸代謝途徑中的作用機(jī)制奠定基礎(chǔ),另一方面也為紅花籽粒品質(zhì)改良分子育種工作提供了重要的遺傳背景基礎(chǔ)和科學(xué)依據(jù)。主要研究結(jié)論如下:1、應(yīng)用IlluminaHiseq2000測(cè)序平臺(tái)對(duì)紅花籽粒發(fā)育后期3份材料進(jìn)行轉(zhuǎn)錄組文庫(kù)高通量測(cè)序,共獲得超過(guò)6GB數(shù)據(jù)量,共138193個(gè)Unigenes,平均長(zhǎng)度700bp,為后續(xù)分析提供基礎(chǔ)數(shù)據(jù)�；蚬δ茏⑨尫治霭l(fā)現(xiàn)所有的Unigenes均得到注釋,說(shuō)明轉(zhuǎn)錄組數(shù)據(jù)拼接質(zhì)量較高。Unigenes的CDS區(qū)預(yù)測(cè)共獲得54291個(gè)CDS,占39.29%,其中長(zhǎng)度在1000bp以上的序列有14509個(gè),占26.74%。皮爾森系數(shù)法分析樣品間基因表達(dá)水平相關(guān)性發(fā)現(xiàn)各樣品間的相關(guān)系數(shù)在0.661-0.782之間,說(shuō)明在紅花籽粒不同發(fā)育時(shí)期基因的表達(dá)是不同的,即樣品間存在一定程度的相似性,又有各自發(fā)育時(shí)期的特點(diǎn)。2、通過(guò)基因差異表達(dá)分析,共鑒定出1528個(gè)差異基因參與到131條代謝途徑,其中參與到天冬氨酸代謝途徑的Unigene有179個(gè),參與到蛋氨酸代謝途徑的Unigene有264個(gè),參與到支鏈氨基酸代謝途徑的Unigene有68個(gè),參與到芳香族氨基酸代謝途徑的Unigene有149個(gè)。在179個(gè)天冬氨酸代謝途徑候選基因中去除注釋信息重復(fù)的序列,共篩選到11條Unigene,它們分別是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技術(shù)篩選出紅花籽粒不同發(fā)育時(shí)期穩(wěn)定表達(dá)的CtEF1A和Ct60S組合為內(nèi)參基因,選取上述9個(gè)候選基因進(jìn)行表達(dá)分析,同時(shí)檢測(cè)總游離氨基酸的含量,通過(guò)二者相關(guān)性分析發(fā)現(xiàn)4個(gè)候選基因(c55378_g1,c67326_g1,c12631_g1,c48258_g3)的表達(dá)與總游離氨基酸的含量關(guān)系十分密切。同時(shí)與轉(zhuǎn)錄組測(cè)序數(shù)據(jù)進(jìn)行相關(guān)性分析,發(fā)現(xiàn)候選基因的表達(dá)模式與轉(zhuǎn)錄組測(cè)序分析結(jié)果一致,證實(shí)轉(zhuǎn)錄組高通量測(cè)序分析的準(zhǔn)確性,同時(shí)也獲得紅花天冬氨酸代謝途徑重要候選基因,為進(jìn)一步研究奠定基礎(chǔ)。4、本研究選取紅花籽粒天冬氨酸代謝途徑第一個(gè)關(guān)鍵限速酶CtAK1進(jìn)行基因克隆和功能驗(yàn)證。首先通過(guò)RACE和定點(diǎn)突變技術(shù)獲得CtAK1及其突變體mCtAK1;利用DNA重組技術(shù)構(gòu)建植物超表達(dá)載體pCAMBIA3301-CTP-(m)CtAK1,通過(guò)凍融法獲得陽(yáng)性農(nóng)桿菌工程菌;采用Flora dip法轉(zhuǎn)化擬南芥,分別收獲5株CtAK1-OE和3株mCt AK1-OE T2代擬南芥種子。RT-qPCR分析表明:在T2代轉(zhuǎn)基因擬南芥中,CtAK1-OE和mCtAK1-OE在轉(zhuǎn)錄水平的表達(dá)量分別提高2.94倍和4.4倍�？傆坞x氨基酸含量分析表明:與野生型擬南芥相比,mCtAK1-OE擬南芥種子總游離氨基酸含量提高2.38倍,CtAK-OE擬南芥種子總游離氨基酸提高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.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S567.219

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