本文選題：紅麻　切入點(diǎn)：線粒體基因　出處：《廣西大學(xué)》2016年碩士論文

【摘要】：植物細(xì)胞質(zhì)雄性不育(cytoplasmic male sterility, CMS)普遍存在于植物界,植物CMS不育機(jī)理的闡明對于人工創(chuàng)制新的細(xì)胞質(zhì)雄性不育資源、調(diào)控CMS的育性具有重要意義。前人對植物CMS機(jī)理的研究,大多數(shù)是以胞質(zhì)差異較大的不育系和保持系為材料,比較二者線粒體基因組的差異,由此挖掘與植物CMS密切相關(guān)的基因。由于細(xì)胞質(zhì)雄性不育系和保持系胞質(zhì)來源不同細(xì)胞質(zhì)基因組遺傳信息差別很大,容易造成CMS以外的累贅遺傳信息的干擾。因此本研究以紅麻細(xì)胞質(zhì)近等基因系UG93A、UG93B及F1 (UG93A/福紅992)為研究材料,對紅麻線粒體基因nad3、nad9、nad4L、 nad6、nad9、sdh4、cob、 atpl、atp4等15個基因進(jìn)行了克隆和表達(dá)分析,以此挖掘與紅麻CMS密切相關(guān)的基因,獲得的結(jié)果如下：1.獲得不育系UG93A、保持系UG93B和F1代15個基因編碼區(qū),這些基因在UG93A、UG93B和F1中編碼區(qū)序列差異僅為單堿基突變。利用熒光定量PCR方法研究CMS相關(guān)基因在花藥發(fā)育雙核期的表達(dá)量,結(jié)果表明不育系中CMS相關(guān)基因的表達(dá)量總體上要低于保持系。2.通過比較分析紅麻線粒體基因RNA編輯與CMS的關(guān)系,結(jié)果表明15個基因在不育系和保持系中RNA編輯差別不大,推測這些基因的RNA編輯可能與紅麻UG93細(xì)胞質(zhì)雄性不育無關(guān)。3.紅麻CMS系和保持系atp4基因的克隆和序列對比發(fā)現(xiàn),細(xì)胞質(zhì)雄性不育系UG93A中atp4基因起始密碼子上游5’端存在4 bp堿基缺失,1 bp堿基插入和4個核苷酸序列差異,Northern Blot技術(shù)分析表明其轉(zhuǎn)錄本條帶并無差異,但在不育系、保持系和F1代雙核期花藥中表達(dá)量存在顯著差異,不育系UG93A atp4的表達(dá)量是保持系的0.31倍,F1代是保持系的1.32倍,且雙核期不育系UG93A復(fù)合體V的活性是保持系的0.90倍,推測atp4基因可能與紅麻細(xì)胞質(zhì)雄性不育相關(guān),atp4基因在紅麻花粉發(fā)育能量代謝過程中有著重要的作用。4.紅麻不育系、保持系和恢復(fù)系花藥不同發(fā)育時期線粒體復(fù)合體V活性和ATP含量測定結(jié)果顯示,在花藥發(fā)育的單核期和雙核期線粒體復(fù)合體V的活性均表現(xiàn)出不育系低于保持系和恢復(fù)系,呈遞增趨勢,而且單核期復(fù)合體V活性低于雙核期。紅麻不育系、保持系和恢復(fù)系在花藥發(fā)育的單核期ATP含量沒有顯著差異,而雙核期呈現(xiàn)出遞增趨勢,結(jié)果表明花藥發(fā)育的雙核期能量供給不足可能與雄性不育密切相關(guān)。
[Abstract]:Cytoplasmic male sterility (CMSs) exists widely in the botanical world. The elucidation of the mechanism of plant CMS sterility is of great significance in creating new cytoplasmic male sterility resources and regulating the fertility of CMS. Most of them were male sterile lines and maintainer lines with different cytoplasm, and the difference of mitochondrial genome between them was compared. Therefore, the genes closely related to plant CMS were excavated. The genetic information of cytoplasmic genomes of cytoplasmic male sterile lines and maintainer lines varied greatly from different cytoplasmic sources. It is easy to interfere with the unwanted genetic information other than CMS. Therefore, 15 genes of kenaf mitochondria, nad9nad9nad4L, nad6nad9sdh4cob and atplatp4, were cloned and expressed in kenaf cytoplasm, UG93A / Fuhong 992 and UG93A / FUG93B / F1 UG93A / Fuhong 992, respectively. The results were as follows: 1. The male sterile line UG93A, the maintainer line UG93B and F1 had 15 coding regions. The sequence difference of these genes in UG93AnUG93B and F1 was only a single base mutation. The expression of CMS related genes in anther developmental binucleate stage was studied by fluorescence quantitative PCR. The results showed that the expression of CMS related genes in male sterile lines was generally lower than that in maintainer lines. By comparing and analyzing the relationship between RNA editing and CMS of kenaf mitochondrial gene, the results showed that there was no significant difference between RNA editing of 15 genes in male sterile lines and maintainer lines. It is speculated that the RNA editing of these genes may not be related to the cytoplasmic male sterility of kenaf UG93. The cloning and sequence comparison of atp4 gene of kenaf CMS line and maintainer line were found. In cytoplasmic male sterile line UG93A, there were 4 BP deletion 1 BP insertion and 4 nucleotide sequence difference at the 5 'end of the initiation codon of atp4 gene. Northern Blot analysis showed that there was no difference in the transcriptional band, but there was no difference in the transcriptional band in the male sterile line. There was significant difference in the expression of UG93A atp4 between the maintainer line and the F1 generation. The expression of UG93A atp4 was 1.32 times of that of the maintainer line, and the activity of UG93A complex V was 0.90 times higher than that of the maintainer line. It is speculated that atp4 gene may play an important role in the energy metabolism of kenaf pollen development, which may be related to the cytoplasmic male sterility of kenaf. The activity of mitochondrial complex V and the content of ATP at different stages of anther development of maintainer and restorer line showed that the activity of mitochondrial complex V in anther development stage was lower than that in maintainer line and restorer line. The activity of complex V in mononuclear stage was lower than that in binuclear stage. There was no significant difference in ATP content between maintainer line and restorer line in anther development stage, but there was an increasing trend in binuclear stage in kenaf male sterile line, maintainer line and restorer line. The results showed that the lack of energy supply in the binuclear stage of anther development may be closely related to male sterility.
【學(xué)位授予單位】：廣西大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S563.5

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本文編號：1688942