【摘要】：本文使用兩種方法進(jìn)行突變體的篩選,分別為熒光顯微鏡法和激光掃描共聚焦顯微鏡法,從中國農(nóng)科院提供的T-DNA插入的突變體800個(gè)株系。使用共質(zhì)性熒光染料CFDA,篩選出水稻胞間連絲相關(guān)的突變體,并考察了突變體的光合生理指標(biāo)。通過TAIL-PCR技術(shù),擴(kuò)增T-DNA的側(cè)翼序列,以分析T-DNA插入位點(diǎn)信息。最后,使用實(shí)時(shí)熒光定量PCR法,檢測(cè)T-DNA插入位點(diǎn)上下游基因的表達(dá)量變化,從而找出是由于哪個(gè)基因表達(dá)量的改變,導(dǎo)致胞間連絲的改變。主要研究結(jié)果如下:(1)初篩獲得89份,復(fù)篩獲得25份突變體材料。(2)同一植株在不同的生長時(shí)期,其胞間連絲的通透性、數(shù)量不同。(3)利用TAIL-PCR技術(shù)擴(kuò)增突變體側(cè)翼序列,經(jīng)過序列比對(duì)發(fā)現(xiàn):有2個(gè)植株的T-DNA同時(shí)插入到日本晴3號(hào)染色體上,且均插入同一基因內(nèi)。(4)通過TAIL-PCR獲得的兩個(gè)突變體的農(nóng)藝性狀均低于野生型,如株高、穗長、千粒重等�？赡苁怯捎赥-DNA的插入導(dǎo)致農(nóng)藝性狀的改變。(5)通過實(shí)時(shí)熒光定量PCR技術(shù)發(fā)現(xiàn),T-DNA插入位點(diǎn)附近的基因表達(dá)量均低于野生型,與相關(guān)文獻(xiàn)報(bào)道不符,其原因還需進(jìn)行后續(xù)實(shí)驗(yàn)來深入探究。
[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.
【學(xué)位授予單位】：河北科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S511

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 符德保;李燕;肖景華;張啟發(fā);吳昌銀;;中國水稻基因組學(xué)研究歷史及現(xiàn)狀[J];生命科學(xué);2016年10期

2 何煒;朱永生;連玲;周平;蔡秋華;王穎Y，

本文編號(hào)：2401944