水稻胞间连丝相关的突变体分离与鉴定
发布时间：2019-01-05 15:20
【摘要】：本文使用两种方法进行突变体的筛选,分别为荧光显微镜法和激光扫描共聚焦显微镜法,从中国农科院提供的T-DNA插入的突变体800个株系。使用共质性荧光染料CFDA,筛选出水稻胞间连丝相关的突变体,并考察了突变体的光合生理指标。通过TAIL-PCR技术,扩增T-DNA的侧翼序列,以分析T-DNA插入位点信息。最后,使用实时荧光定量PCR法,检测T-DNA插入位点上下游基因的表达量变化,从而找出是由于哪个基因表达量的改变,导致胞间连丝的改变。主要研究结果如下:(1)初筛获得89份,复筛获得25份突变体材料。(2)同一植株在不同的生长时期,其胞间连丝的通透性、数量不同。(3)利用TAIL-PCR技术扩增突变体侧翼序列,经过序列比对发现:有2个植株的T-DNA同时插入到日本晴3号染色体上,且均插入同一基因内。(4)通过TAIL-PCR获得的两个突变体的农艺性状均低于野生型,如株高、穗长、千粒重等。可能是由于T-DNA的插入导致农艺性状的改变。(5)通过实时荧光定量PCR技术发现,T-DNA插入位点附近的基因表达量均低于野生型,与相关文献报道不符,其原因还需进行后续实验来深入探究。
[Abstract]:In this paper, two methods were used to screen the mutants, fluorescence microscope and laser scanning confocal microscope respectively. 800 mutants were inserted from T-DNA supplied by the Chinese Academy of Agricultural Sciences. The mutants associated with intercellular connective filaments in rice were screened by CFDA, and the photosynthetic physiological indexes of the mutants were investigated. The flanking sequence of T-DNA was amplified by TAIL-PCR technique to analyze the insertion site information of T-DNA. Finally, real-time fluorescence quantitative PCR method was used to detect the expression changes of upstream and downstream genes at T-DNA insertion site, so as to find out which gene expression changed, which led to the change of intercellular ligaments. The main results are as follows: (1) 89 mutants were obtained by primary screening and 25 mutants were obtained by double screening. (2) the permeability of intercellular connective filaments of the same plant at different growth stages. (3) TAIL-PCR technique was used to amplify the flanking sequence of the mutant. After sequence alignment, it was found that the T-DNA of two plants was inserted into chromosome 3 of Nippon at the same time. All of them were inserted into the same gene. (4) the agronomic characters of the two mutants obtained by TAIL-PCR were lower than those of wild type, such as plant height, ear length, 1000-grain weight and so on. It may be that the insertion of T-DNA results in the change of agronomic characters. (5) the expression of genes near the insertion site of T-DNA is lower than that of wild type by real-time fluorescence quantitative PCR, which is not consistent with the related literature. Its reason still needs to carry on the follow-up experiment to delve deeply.
【学位授予单位】：河北科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S511
[Abstract]:In this paper, two methods were used to screen the mutants, fluorescence microscope and laser scanning confocal microscope respectively. 800 mutants were inserted from T-DNA supplied by the Chinese Academy of Agricultural Sciences. The mutants associated with intercellular connective filaments in rice were screened by CFDA, and the photosynthetic physiological indexes of the mutants were investigated. The flanking sequence of T-DNA was amplified by TAIL-PCR technique to analyze the insertion site information of T-DNA. Finally, real-time fluorescence quantitative PCR method was used to detect the expression changes of upstream and downstream genes at T-DNA insertion site, so as to find out which gene expression changed, which led to the change of intercellular ligaments. The main results are as follows: (1) 89 mutants were obtained by primary screening and 25 mutants were obtained by double screening. (2) the permeability of intercellular connective filaments of the same plant at different growth stages. (3) TAIL-PCR technique was used to amplify the flanking sequence of the mutant. After sequence alignment, it was found that the T-DNA of two plants was inserted into chromosome 3 of Nippon at the same time. All of them were inserted into the same gene. (4) the agronomic characters of the two mutants obtained by TAIL-PCR were lower than those of wild type, such as plant height, ear length, 1000-grain weight and so on. It may be that the insertion of T-DNA results in the change of agronomic characters. (5) the expression of genes near the insertion site of T-DNA is lower than that of wild type by real-time fluorescence quantitative PCR, which is not consistent with the related literature. Its reason still needs to carry on the follow-up experiment to delve deeply.
【学位授予单位】：河北科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S511
【参考文献】
相关期刊论文 前10条
1 符德保;李燕;肖景华;张启发;吴昌银;;中国水稻基因组学研究历史及现状[J];生命科学;2016年10期
2 何炜;朱永生;连玲;周平;蔡秋华;王颖Y，
本文编号：2401944
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