【摘要】：△9硬脂酰-ACP去飽和酶(Stearoyl-ACP desaturase,簡稱:SAD)催化脫氫形成第一個雙鍵,其參與細胞膜脂不飽和脂肪酸的合成,其含量的變化能夠改變飽和脂肪酸與不飽和脂肪酸之間的比例,從而影響植物的抗寒性。本試驗從植物分子水平上研究SAD基因在低溫脅迫下的表達模式,為深入研究菊花抗寒機制、篩選抗寒品種以及進一步進行抗性育種奠定基礎,也可用于抗寒品種的選育,并提高菊花的觀賞價值。以秋菊抗寒品種‘星光燦爛’為試材,通過改良的CTAB法從菊花葉片中克隆出菊花SAD基因的中間片段,并對其序列進行分析。利用實時熒光定量的方法對菊花SAD的表達量進行測定,分析此基因在不同溫度下的表達情況。主要研究結果如下:1.根據(jù)菊花SAD基因的同源序列設計簡并引物,從菊花‘星光燦爛’葉片中克隆出菊花SAD基因的中間片段,并設計特異引物擴增得到c DNA全長共有1203bp,編碼401個氨基酸,相對分子量為45.57 KD,在Gene Bank注冊號為KC529335。2.對菊花SAD基因進行同源序列比較,菊花SAD基因片段編碼的氨基酸與同科的新疆雪蓮(Saussurea involucrata Kar.et Kir.ex Maxim.,ABF66638)SAD基因的同源性為80.29%與向日葵(Helianthus annuus,AAB65145.1)SAD基因的同源性為79.81%。與其它物種SAD基因也具有較高的同源性,如麻瘋樹(77.62%),百脈根(75.91%),馬鈴薯(77.86%),水黃皮(77.62%),花生(77.13%),烏桕(76.89%),木薯(76.89%)。與其他植物的SAD基因有較高的同源性,因此命名為CmSAD。該蛋白是一個可溶性蛋白,沒有跨膜信號區(qū),N-端含有一段葉綠體轉運肽,蛋白活性位點分析發(fā)現(xiàn)在CmSAD編碼的蛋白C-端有1個SAD的保守區(qū)域序列,并對該蛋白3D結構進行預測。3.根系中CmSAD基因的表達量隨著溫度的降低而降低,16℃時表達量最高,5℃、-4℃和-8℃分別是16℃的0.87、0.11和0.03倍。葉片中CmSAD表達量隨著溫度的降低先升高再降低,5℃時最高,分別是16、-4、-8℃條件下的1.3、5.6、3.5倍。隨著溫度的下降,CmSAD基因的表達量呈下降趨勢。在16℃和5℃時,CmSAD基因在根系中的表達量高于葉片;-4℃和-8℃時,葉片中CmSAD的表達量高于根系。SAD基因使飽和脂肪酸脫氫形成第一個氫鍵,使不飽和脂肪酸的含量發(fā)生改變。CmSAD基因的表達量隨著溫度的降低而降低,因此,CmSAD基因的表達量與溫度有關,同時與抗寒性也有著密切的關系。
[Abstract]:9 stearyl-ACP desaturase (Stearoyl-ACP desaturase, for short) catalyzes dehydrogenation to form the first double bond, which is involved in the synthesis of unsaturated fatty acids of cell membrane. The change of the content of stearyl-ACP desaturase (Stearoyl-ACP desaturase,) can change the ratio of saturated fatty acids to unsaturated fatty acids. Thus affecting the cold resistance of plants. In this study, the expression pattern of SAD gene in chrysanthemum under low temperature stress was studied at the molecular level, which laid a foundation for further study on chrysanthemum cold resistance mechanism, selection of cold resistant varieties and further resistance breeding, and could also be used in breeding cold resistant varieties. And improve the ornamental value of chrysanthemum. The intermediate fragment of chrysanthemum SAD gene was cloned from chrysanthemum leaves by improved CTAB method and its sequence was analyzed with chrysanthemum cold resistant variety 'Xingguangguanghuang'. The expression of SAD in chrysanthemum was determined by real-time fluorescence quantitative method, and the expression of this gene at different temperature was analyzed. The main results are as follows: 1. According to the homologous sequence of chrysanthemum SAD gene, degenerate primers were designed and the intermediate fragment of SAD gene was cloned from chrysanthemum 'starlight brilliant' leaves. The total length of c DNA was 1203bp, encoding 401 amino acids. Relative molecular weight 45.57 KD, at Gene Bank registration number KC529335.2. The homology of SAD gene in chrysanthemum was 80.29% and 79.81% respectively in chrysanthemum SAD gene and Helianthus annuus,AAB65145.1 SAD gene. The amino acid sequence of chrysanthemum SAD gene was 80.29% compared with that of SAD gene of Saussurea Xinjiang Saussurea in the same family. There were also high homology with other species, such as Jatropha curcas (77.62%), Potato (77.86%), Peanut (77.13%), Sapium sebiferum (76.89%), cassava (76.89%), Chinese tallow (77.62%), Potato (77.86%), Peanut (77.13%), Sapium sebifera (76.89%) and cassava (76.89%). The SAD gene has high homology with other plants, so it is named CmSAD.. The protein is a soluble protein with no transmembrane signal region, and the N-terminal contains a chloroplast transport peptide. The protein activity site analysis shows that there is a conserved region of SAD at the C-terminal of the protein encoded by CmSAD, and the 3D structure of the protein is predicted. The expression of CmSAD gene in roots decreased with the decrease of temperature. The expression of CmSAD gene was the highest at 16 鈩，

本文編號：2287830