本文關(guān)鍵詞： 陸地棉 基因克隆 遺傳轉(zhuǎn)化 脂肪酸 抗寒性　出處：《石河子大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：本研究以Gh SAD2為目的基因,采用電子拼接結(jié)合RT-PCR的方法克隆得到陸地棉GhSAD2基因的c DNA全長并對其進行相關(guān)的生物信息學(xué)分析;通過qRT-PCR研究了Gh SAD2基因在棉花不同組織中的表達特點,并探究目的基因在棉子發(fā)育過程中和低溫脅迫下的表達模式;本研究構(gòu)建了Gh SAD2基因的過表達載體,并通過農(nóng)桿菌介導(dǎo)遺傳轉(zhuǎn)化到煙草和新陸早33號,初步分析目的基因的功能;此外,以轉(zhuǎn)GhSAD2干涉載體的棉花T3代和受體材料2074B為研究材料,分析其幼苗抗寒能力及脂肪酸成分變化,主要研究結(jié)果如下:1.克隆了陸地棉GhSAD2基因,并對該基因進行了生物信息學(xué)分析。Gh SAD2在NCBI注冊的登錄號為KX197920,其cDNA全長1188 bp,編碼396個氨基酸,含有2個組氨酸簇是一個典型的脂肪酸去飽和酶家族蛋白。GhSAD2基因編碼的分子量大小為45.3 kD,理論等電點為5.88,平均親水系數(shù)(GRAVY)為-0.464,為親水性蛋白。系統(tǒng)進化分析表明,該基因與可可樹的同源基因進化關(guān)系非常接近。2.構(gòu)建了目的基因的過表達載體,并轉(zhuǎn)化了煙草和棉花。將植物過表達載體pBI121-GhSAD2通過根瘤農(nóng)桿菌介導(dǎo)的葉盤法和愈傷組織轉(zhuǎn)化法將其分別轉(zhuǎn)入‘山西煙草’和‘新陸早33號’,共得到9株GhSAD2基因過表達的轉(zhuǎn)基因煙草植株,其中煙草的轉(zhuǎn)化率為75%;得到了棉花陽性愈傷組織,并進行了GUS檢測。3.通過熒光定量PCR技術(shù),研究了目的基因的組織表達特征。結(jié)果表明,GhSAD2基因在棉花葉片中的表達量高于莖和根;Gh SAD2基因在棉花種子發(fā)育過程中的表達量呈現(xiàn)出先上升后逐步下降的變化趨勢,在花后25 d的種子中表達量最高,在成熟種子中的表達相對比較微弱;Gh SAD2基因能夠響應(yīng)低溫脅迫,在4℃和15℃脅迫6 h時其表達量最大,脅迫12h后,表達量基本保持穩(wěn)定狀態(tài),是對照組的1.3-2.3倍。4℃低溫脅迫48h后,GhSAD2過表達的轉(zhuǎn)基因煙草的電導(dǎo)率顯著低于野生型煙草,由此可以推測Gh SAD2基因可能在棉花冷脅迫調(diào)控中起一定的作用。4.測定了轉(zhuǎn)GhSAD2干涉載體的棉花T3代株系的農(nóng)藝性狀及棉子中脂肪酸的含量。結(jié)果表明:轉(zhuǎn)基因棉花與受體2074B相比,衣分增長了7%-8%,衣指含量增長了0.8-1.6g,籽指含量減少了1.3-2.4g。其轉(zhuǎn)基因棉子中的總脂肪酸含量、棕櫚酸含量及亞油酸含量顯著降低,總蛋白含量、硬脂酸含量及油酸含量顯著升高。5.通過熒光定量PCR技術(shù),研究了目的基因在轉(zhuǎn)基因植株種子發(fā)育過程中的表達特性,結(jié)果顯示,除花后15d其它時期均低于受體2074B,表達模式和受體植株相同,呈現(xiàn)出先上升后下降的變化趨勢,在花后30d的種子中表達量最高。6.測定了轉(zhuǎn)基因植株葉片的電導(dǎo)率、丙二醛含量及脯氨酸含量。結(jié)果表明,RNAi的轉(zhuǎn)基因植株相比CK,其抗寒能力減弱,耐熱能力增強,推測棉花幼苗的抗寒能力可能會與GhSAD2基因在棉花幼苗中的表達量相關(guān)。
[Abstract]:In this study, Gh SAD2 was used as the target gene. The full length of c DNA of GhSAD2 gene of Upland cotton was cloned by electronic splicing combined with RT-PCR and its bioinformatics analysis was carried out. The expression characteristics of Gh SAD2 gene in different tissues of cotton were studied by qRT-PCR, and the expression pattern of Gh SAD2 gene during cotton seed development and under low temperature stress was explored. In this study, the overexpression vector of Gh SAD2 gene was constructed and transformed into tobacco and Xinluzao 33 by Agrobacterium tumefaciens to analyze the function of the target gene. In addition, the T 3 generation and receptor material 2074B of cotton transformed with GhSAD2 interference vector were used as the research materials to analyze the changes of cold resistance and fatty acid composition of cotton seedlings. The main results are as follows: 1. The GhSAD2 gene of Upland cotton was cloned and analyzed by bioinformatics. Gh SAD2 was registered as KX197920 in NCBI. Its cDNA length is 1188bpand encodes 396 amino acids. Two histidine clusters are a typical fatty acid desaturase family protein. GhSAD2 gene encodes a molecular weight of 45.3 KD and a theoretical isoelectric point of 5.88. The average hydrophilic coefficient (GRAVY) is -0.464, which is a hydrophilic protein. Phylogenetic analysis shows that GRAVY is a hydrophilic protein. The evolutionary relationship between this gene and the coca homologous gene is very close to .2. the overexpression vector of the target gene was constructed. The plant overexpression vector pBI121-GhSAD2 was transformed into 'Shanxi tobacco' and 'Xinluzao 3' by Agrobacterium tumefaciens mediated leaf disk and callus transformation, respectively. Three. Nine transgenic tobacco plants with overexpression of GhSAD2 gene were obtained, and the conversion rate of tobacco was 75%. The positive callus of cotton was obtained and detected by GUS. The expression characteristics of the target gene were studied by fluorescence quantitative PCR. The expression of GhSAD2 gene in cotton leaves was higher than that in stems and roots. The expression of Gh SAD2 gene increased firstly and then decreased gradually during the development of cotton seeds, and the highest expression was found in the seeds 25 days after anthesis. The expression in mature seeds was relatively weak. The expression of Gh SAD2 gene was the highest at 4 鈩，

本文編號：1462116