小黑楊Poptr;CYCD1;1與擬南芥CYCD1;1、CYCD2;1、CTCD3;1基因在煙草中的遣傳轉化
發(fā)布時間：2019-06-22 11:59
【摘要】：細胞分裂是生物的基本特征之一,在植物生長發(fā)育的過程中,發(fā)揮著極其重要的作用,細胞周期和生長調控均離不開一類重要的細胞分裂調控因子一細胞周期蛋白(Cyclin)。 D類細胞周期蛋白(CYCD)基因作為調控因子,對調節(jié)細胞周期具有重要作用。為了探究小黑楊和擬南芥CYCD基因的功能,我們利用轉基因技術將小黑楊Poptr;CYCD1;1和擬南芥AtCYCD1;1、AtCYCD2;1、AtCYCD3;1基因轉入煙草,通過觀察轉基因植株的表型變化,驗證了小黑楊與擬南芥CYCD周期蛋白的生物學功能。主要結論如下：1.構建pER8-GFP植物表達載體并進行煙草遺傳轉化。利用不同濃度和不同時間的雌激素處理轉基因植株,并結合定量PCR和NightSHADE植物活體成像系統(tǒng)對轉基因植株表達水平進行檢測。結果表明GFP基因在煙草胚軸與根尖細胞出現(xiàn)了熒光信號,說明在煙草中XVE化學誘導激活系統(tǒng)也可以高效、嚴格的依賴雌激素的誘導來控制目的基因表達。2.構建植物表達載體pER8-Poptr;CYCD1;1與pER8-Poptr;CYCD1;1-RNAi并轉化煙草。雌激素處理后,過表達Poptr;CYCD1;1的轉基因煙草與野生型比,出現(xiàn)了花瓣變大、花柱變長和子房變大的顯著表型變化。此外,組成型表達Poptr;CYCD1;1的株系受到RNAi干擾后,部分轉基因株系能恢復為原來的野生型表型。以上結果表明Poptr;CYCD1;1的過量表達導致轉基因株系的生殖器官形態(tài)發(fā)生變化。3.將AtCYCD1;1、AtCYCD2;1、AtCYCD3;1構建于植物表達載體pROKII中并轉化野生型煙草。qRT-PCR檢測顯示, AtCYCD1;1、AtCYCD2;1、AtCYCD3;1在mRNA水平均有表達。此外,過量表達AtCYCD1;1、AtCYCD2;1、AtCYCD3;1的轉基因株系在生殖器官中存在顯著的表型變化,與野生型相比主要表現(xiàn)為轉基因植株花冠寬度變大,花瓣和萼片長度變長,果實變大。上述結果表明AtCYCD1;1、AtCYCD2;1、AtCYCD3;1基因影響花的發(fā)育。4.pROKⅡ--AtCYCD3;1轉基因植株與野生型相比,不僅能夠引起N tICK, NtCDK和NtCDKC等相關基因的轉錄水平變化,轉基因植株還表現(xiàn)出根尖細胞變小、莖干彎曲、葉片卷曲、種子變小等表型。莖段石蠟切片表明,轉基因植株彎曲部位的形成層細胞及木質部細胞增多。同時,莖段的徒手切片經(jīng)間苯三酚染色實驗表明轉基因煙草與野生型對照相比,木質化細胞增多。上述結果暗示AtCYCD3;1基因的過表達可能影響細胞的分裂并加速了轉基因煙草植株細胞壁的木質化進程。
[Abstract]:Cell division is one of the basic characteristics of organisms. It plays an extremely important role in the process of plant growth and development. Cell cycle and growth regulation can not be separated from a class of important cell division regulator-cell cycle (Cyclin). Class D cell cycle protein (CYCD) gene, as a regulatory factor, plays an important role in regulating cell cycle. In order to investigate the function of CYCD gene in Populus tomentosa and Arabidopsis thaliana, the Poptr;CYCD1;1 and AtCYCD1;1,AtCYCD2;1,AtCYCD3;1 genes of Populus tomentosa and Arabidopsis thaliana were transferred into tobacco by transgenic technique. The biological functions of CYCD cyclones of Populus tomentosa and Arabidopsis thaliana were verified by observing the phenotypic changes of transgenic plants. The main conclusions are as follows: 1. PER8-GFP plant expression vector was constructed and tobacco genetic transformation was carried out. Transgenic plants were treated with estrogen at different concentrations and different time, and the expression level of transgenic plants was detected by quantitative PCR and NightSHADE in vivo imaging system. The results showed that GFP gene showed fluorescence signal in tobacco Hypocotyl and root tip cells, which indicated that XVE chemical induction activation system could also be efficient and strictly dependent on estrogen induction to control the expression of the target gene. 2. Plant expression vectors pER8-Poptr;CYCD1;1 and pER8-Poptr;CYCD1;1-RNAi were constructed and transformed into tobacco. After estrogen treatment, the ratio of transgenic tobacco overexpressing Poptr;CYCD1;1 to wild type showed significant phenotypic changes in petal enlargement, style length and ovary enlargement. In addition, after the lines expressing Poptr;CYCD1;1 were interfered by RNAi, some transgenic lines could return to the original wild type phenotype. These results suggest that the overexpression of Poptr;CYCD1;1 leads to morphological changes in reproductive organs of transgenic lines. 3. AtCYCD1;1,AtCYCD2;1,AtCYCD3;1 was constructed into plant expression vector pROKII and transformed into wild type tobacco. QRT-PCR showed that AtCYCD1;1,AtCYCD2;1,AtCYCD3;1 was expressed at mRNA level. In addition, there were significant phenotypic changes in reproductive organs of transgenic lines overexpressing AtCYCD1;1,AtCYCD2;1,AtCYCD3;1. Compared with wild type, the Corolla width of transgenic plants became larger, the length of petals and sepals became longer, and the fruit became larger. These results suggest that AtCYCD1;1,AtCYCD2;1,AtCYCD3;1 gene affects flower development. 4. Compared with wild type, pROK II-AtCYCD3;1 transgenic plants can not only cause changes in transcription level of N tICK, NtCDK and NtCDKC and other related genes, but also show phenotypes such as root tip cells, stem bending, leaf curl, seed reduction and so on. Paraffin sections of stem segment showed that the cambium cells and xylem cells in the curved parts of transgenic plants increased. At the same time, resorcinol staining showed that the number of ligated cells in transgenic tobacco was higher than that in wild type control. These results suggest that the overexpression of AtCYCD3;1 gene may affect cell division and accelerate the lignification of cell wall in transgenic tobacco plants.
【學位授予單位】：東北林業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：Q943.2
本文編號：2504558
[Abstract]:Cell division is one of the basic characteristics of organisms. It plays an extremely important role in the process of plant growth and development. Cell cycle and growth regulation can not be separated from a class of important cell division regulator-cell cycle (Cyclin). Class D cell cycle protein (CYCD) gene, as a regulatory factor, plays an important role in regulating cell cycle. In order to investigate the function of CYCD gene in Populus tomentosa and Arabidopsis thaliana, the Poptr;CYCD1;1 and AtCYCD1;1,AtCYCD2;1,AtCYCD3;1 genes of Populus tomentosa and Arabidopsis thaliana were transferred into tobacco by transgenic technique. The biological functions of CYCD cyclones of Populus tomentosa and Arabidopsis thaliana were verified by observing the phenotypic changes of transgenic plants. The main conclusions are as follows: 1. PER8-GFP plant expression vector was constructed and tobacco genetic transformation was carried out. Transgenic plants were treated with estrogen at different concentrations and different time, and the expression level of transgenic plants was detected by quantitative PCR and NightSHADE in vivo imaging system. The results showed that GFP gene showed fluorescence signal in tobacco Hypocotyl and root tip cells, which indicated that XVE chemical induction activation system could also be efficient and strictly dependent on estrogen induction to control the expression of the target gene. 2. Plant expression vectors pER8-Poptr;CYCD1;1 and pER8-Poptr;CYCD1;1-RNAi were constructed and transformed into tobacco. After estrogen treatment, the ratio of transgenic tobacco overexpressing Poptr;CYCD1;1 to wild type showed significant phenotypic changes in petal enlargement, style length and ovary enlargement. In addition, after the lines expressing Poptr;CYCD1;1 were interfered by RNAi, some transgenic lines could return to the original wild type phenotype. These results suggest that the overexpression of Poptr;CYCD1;1 leads to morphological changes in reproductive organs of transgenic lines. 3. AtCYCD1;1,AtCYCD2;1,AtCYCD3;1 was constructed into plant expression vector pROKII and transformed into wild type tobacco. QRT-PCR showed that AtCYCD1;1,AtCYCD2;1,AtCYCD3;1 was expressed at mRNA level. In addition, there were significant phenotypic changes in reproductive organs of transgenic lines overexpressing AtCYCD1;1,AtCYCD2;1,AtCYCD3;1. Compared with wild type, the Corolla width of transgenic plants became larger, the length of petals and sepals became longer, and the fruit became larger. These results suggest that AtCYCD1;1,AtCYCD2;1,AtCYCD3;1 gene affects flower development. 4. Compared with wild type, pROK II-AtCYCD3;1 transgenic plants can not only cause changes in transcription level of N tICK, NtCDK and NtCDKC and other related genes, but also show phenotypes such as root tip cells, stem bending, leaf curl, seed reduction and so on. Paraffin sections of stem segment showed that the cambium cells and xylem cells in the curved parts of transgenic plants increased. At the same time, resorcinol staining showed that the number of ligated cells in transgenic tobacco was higher than that in wild type control. These results suggest that the overexpression of AtCYCD3;1 gene may affect cell division and accelerate the lignification of cell wall in transgenic tobacco plants.
【學位授予單位】：東北林業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：Q943.2
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1 代麗娟;小黑楊Poptr;CYCD1;1與擬南芥CYCD1;1、CYCD2;1、CTCD3;1基因在煙草中的遣傳轉化[D];東北林業(yè)大學;2016年
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