小黑杨Poptr;CYCD1;1与拟南芥CYCD1;1、CYCD2;1、CTCD3;1基因在烟草中的遣传转化
发布时间：2019-06-22 11:59
【摘要】：细胞分裂是生物的基本特征之一,在植物生长发育的过程中,发挥着极其重要的作用,细胞周期和生长调控均离不开一类重要的细胞分裂调控因子一细胞周期蛋白(Cyclin)。 D类细胞周期蛋白(CYCD)基因作为调控因子,对调节细胞周期具有重要作用。为了探究小黑杨和拟南芥CYCD基因的功能,我们利用转基因技术将小黑杨Poptr;CYCD1;1和拟南芥AtCYCD1;1、AtCYCD2;1、AtCYCD3;1基因转入烟草,通过观察转基因植株的表型变化,验证了小黑杨与拟南芥CYCD周期蛋白的生物学功能。主要结论如下：1.构建pER8-GFP植物表达载体并进行烟草遗传转化。利用不同浓度和不同时间的雌激素处理转基因植株,并结合定量PCR和NightSHADE植物活体成像系统对转基因植株表达水平进行检测。结果表明GFP基因在烟草胚轴与根尖细胞出现了荧光信号,说明在烟草中XVE化学诱导激活系统也可以高效、严格的依赖雌激素的诱导来控制目的基因表达。2.构建植物表达载体pER8-Poptr;CYCD1;1与pER8-Poptr;CYCD1;1-RNAi并转化烟草。雌激素处理后,过表达Poptr;CYCD1;1的转基因烟草与野生型比,出现了花瓣变大、花柱变长和子房变大的显著表型变化。此外,组成型表达Poptr;CYCD1;1的株系受到RNAi干扰后,部分转基因株系能恢复为原来的野生型表型。以上结果表明Poptr;CYCD1;1的过量表达导致转基因株系的生殖器官形态发生变化。3.将AtCYCD1;1、AtCYCD2;1、AtCYCD3;1构建于植物表达载体pROKII中并转化野生型烟草。qRT-PCR检测显示, AtCYCD1;1、AtCYCD2;1、AtCYCD3;1在mRNA水平均有表达。此外,过量表达AtCYCD1;1、AtCYCD2;1、AtCYCD3;1的转基因株系在生殖器官中存在显著的表型变化,与野生型相比主要表现为转基因植株花冠宽度变大,花瓣和萼片长度变长,果实变大。上述结果表明AtCYCD1;1、AtCYCD2;1、AtCYCD3;1基因影响花的发育。4.pROKⅡ--AtCYCD3;1转基因植株与野生型相比,不仅能够引起N tICK, NtCDK和NtCDKC等相关基因的转录水平变化,转基因植株还表现出根尖细胞变小、茎干弯曲、叶片卷曲、种子变小等表型。茎段石蜡切片表明,转基因植株弯曲部位的形成层细胞及木质部细胞增多。同时,茎段的徒手切片经间苯三酚染色实验表明转基因烟草与野生型对照相比,木质化细胞增多。上述结果暗示AtCYCD3;1基因的过表达可能影响细胞的分裂并加速了转基因烟草植株细胞壁的木质化进程。
[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.
【学位授予单位】：东北林业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2
本文编号：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.
【学位授予单位】：东北林业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2
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1 代丽娟;小黑杨Poptr;CYCD1;1与拟南芥CYCD1;1、CYCD2;1、CTCD3;1基因在烟草中的遣传转化[D];东北林业大学;2016年
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