题名: | 基于甲基化变异的杂草稻特异分子检测技术研发 |
作者: | |
学号: | 2021116084 |
保密级别: | 保密两年 |
语种: | chi |
学科代码: | 071008 |
学科: | 理学 - 生物学 - 发育生物学 |
学生类型: | 硕士 |
学位: | 理学硕士 |
学校: | 南京农业大学 |
院系: | |
专业: | |
研究方向: | 杂草稻甲基化检测 |
导师姓名: | |
导师单位: | |
完成日期: | 2023-08-31 |
答辩日期: | 2024-12-05 |
外文题名: | Development of Weed Rice-Specific Molecular Detection Technology Based on Methylation Variants |
关键词: | |
外文关键词: | Methylation group ; Methylation-specific PCR ; Grey scale analysis ; Rapid identification of weedy rice |
摘要: |
海南省是国家级南繁育种基地,全国水稻育种均会在此交集并将种子引种至各地。杂草稻(Oryza sativa f. spontanea)是最重要的稻田恶性杂草,其可以通过双向基因流与水稻品种间杂交产生,使得稻种中混入杂草稻,并伴随正常稻种输送至全国各地,加重我国杂草稻发生与危害,影响稻田生产。因此,南繁育种基地肩负内防杂草稻输出的责任,需要一种针对杂草稻的特异性分子检测技术。本研究通过对不同地理来源的杂草稻种群进行甲基化组分析与甲基化特异性PCR实验,开发出一种不同地理来源杂草稻种群中特异性鉴定海南省杂草稻的方法,希望为南繁基地内防杂草稻输出提供技术支持。 本研究通过杂草稻全基因的甲基化组分析,筛选出不同地理来源杂草稻种群甲基化差异较大的3个基因——Os12g0622850、OsICE1、OsIAGLU,获取到相应的甲基化差异序列,依此设计了19条引物、15个引物组合,5个退火温度(48-56Δ2 ℃)。先后利用9个甲基化组同地理来源杂草稻种群,再扩大到来自全国20省、市、自治区的61个杂草稻种群,最终扩大到来自全国24省、市、自治区的186个杂草稻种群进行初筛、优化、验证,最终确定引物组合OsG1-F1、OsG1-R2,在退火温度52 ℃的条件下能够特异性的将海南省杂草稻种群与供试的其它23个省、市、自治区杂草稻种群区分开。 研究过程中部分杂草稻种群在检测片段处存在单一条带,但亮度低于均匀恒定的海南种群,对区分杂草稻地理来源造成了干扰。因此,引入Image J软件进行条带灰度定量分析,设定海南杂草稻种群灰度值为100。实验结果显示,其它地理来源杂草稻种群灰度值始终小于一定值,据此可准确区分其它地理来源杂草稻种群与海南省杂草稻种群。其原理是不同地理来源杂草稻种群与Os12g0622850基因匹配度存在差异,因而扩增表现不同,体现于胶图中则表现为亮度不同,即Image J定量后的灰度值不同。引物组合OsG1-F1、OsG1-R2在186个杂草稻种群的检测实验结果显示,海南省杂草稻具有最亮的单一条带,灰度值最高,稳定在100左右;其它省份的杂草稻种群中,138个完全没有扩增条带,灰度值为零;30个可以扩增出亮度较低的条带,灰度值均不超过65;15个扩增出非特异性条带,142 bp处灰度值记为0。本研究针对杂草稻开发特异性分子检测技术,为杂草稻种群的快速地理来源鉴定提供技术方法,期望为海南省南繁基地杂草稻的内防外输管理和控制提供合适的技术支撑。 |
外摘要要: |
Hainan Province is a national-level southern breeding base, where national rice breeding is intersected and seeds are introduced to various places. Weedy rice (Oryza sativa f. spontanea) is the most important malignant weed in paddy fields, which can be produced through bidirectional gene flow and crosses between rice varieties, so that the rice seed is mixed with weedy rice and transported to all parts of the country along with the normal rice seed, which exacerbates the occurrence of weedy rice in our country and harms the rice field, affecting paddy field production. Therefore, the Southern Breeding Breeding Base shoulders the responsibility of preventing the export of weedy rice within the country, and a specific molecular detection technology for weedy rice is needed. In this study, we developed a method for the specific identification of weedy rice in Hainan Province through methylation group analysis and methylation-specific PCR experiments on weedy rice populations of different geographical origins, with the hope of providing technical support for the export of weedy rice to the Southern Breeding Base for internal control of weedy rice. In this study, we screened three genes, Os12g0622850, OsICE1, and OsIAGLU, with large methylation differences in weed rice populations of different geographical origins by methylation group analysis of the whole gene of weed rice, and obtained the corresponding methylation difference sequences, and accordingly designed 19 primers, 15 primer combinations, and five annealing temperatures (48-56Δ2 °C) were designed accordingly. Weed rice populations of the same geographical origin using nine methylation groups were successively expanded to 61 weed rice populations from 20 provinces, municipalities and autonomous regions of China, and finally to 186 weed rice populations from 24 provinces, municipalities and autonomous regions of China for initial screening, optimisation and validation, and the primer combinations OsG1-F1 and OsG1-R2 were finally identified, which were capable of specifically distinguishing the weed rice populations of Hainan Province from the other 23 provinces, municipalities and autonomous regions for testing at the annealing temperature of 52 °C. The primer combinations OsG1-F1 and OsG1-R2 were specifically able to distinguish the weedy rice populations in Hainan Province from those in the other 23 provinces, municipalities and autonomous regions under the annealing temperature of 52 ℃. During the study, some weedy rice populations had a single band at the detected segment, but the brightness was lower than that of Hainan populations, which interfered with the differentiation of geographic origin of weedy rice. Therefore, we introduced Image J software to quantitatively analyse the grey level of the bands, and set the grey level value of the Hainan population to 100, and the experimental results showed that the grey level value of the other geographic populations of weedy rice was always less than a certain value, so that we could accurately differentiate between the other geographic populations of weedy rice and those of Hainan province. The principle is that different geographic origin of weedy rice populations and Os12g0622850 gene match differences, so the amplification performance is different, which is reflected in the gelatin map as different brightness, that is, different grey values after Image J quantification. The results of the primer combinations OsG1-F1 and OsG1-R2 in 186 populations of weedy rice showed that the weedy rice in Hainan Province had the brightest single band with the highest grey value, which was stable at around 100; among the populations of weedy rice in other provinces, 138 populations did not amplify any band at all, with a grey value of zero; 30 populations could amplify a lower brightness band, with a grey value of no more than 65; and 15 populations amplified a non-specific band, with a grey value of 0 at 142 bp.In this study, we developed a specific molecular detection technique for weedy rice populations for rapid geographic origin identification. In this study, we developed a specific molecular detection technique for weedy rice, which provides a technical method for the rapid identification of the geographic origin of weedy rice populations, and is expected to provide a suitable technical support for the management and control of weedy rice at the southern propagation bases in Hainan Province. |
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中图分类号: | S45 |
开放日期: | 2027-02-17 |