【摘要】：黃瓜(Cucumis sativus L.)是我國(guó)設(shè)施和露地主要種植的蔬菜作物之一,土壤次生鹽漬化已成為影響設(shè)施黃瓜生產(chǎn)的主要障礙之一,NO_3~-是土壤次生鹽漬化的的主要陰離子。植物通過(guò)硝酸鹽同化途徑以NO_3~-和NH4+的形式吸收N。硝酸鹽的同化是一個(gè)受到嚴(yán)格調(diào)控的過(guò)程,其中NR和NiR對(duì)初級(jí)氮的同化起主要調(diào)控作用。NR是整個(gè)硝態(tài)氮同化過(guò)程的關(guān)鍵酶、限速酶,在植物氮素代謝過(guò)程中起關(guān)鍵作用。NR也是植物體內(nèi)的NO合成酶,NO在一定程度上可提高植物的抗逆能力,緩解逆境脅迫。因此探討黃瓜硝酸還原酶基因(CsNR)對(duì)NO_3~-脅迫下植株幼苗抗氧化體系及氮代謝的影響,對(duì)指導(dǎo)黃瓜可持續(xù)生產(chǎn)具有重要的理論和現(xiàn)實(shí)意義。本試驗(yàn)通過(guò)構(gòu)建CsNR正義和反義表達(dá)載體,并進(jìn)行黃瓜和煙草的轉(zhuǎn)化,獲得轉(zhuǎn)Cs NR(+)和CsNR(-)基因的煙草植株,研究了NR在NO_3~-脅迫下植株的抗氧化系統(tǒng)、氮代謝相關(guān)酶表達(dá)特性及其內(nèi)源NO的分子調(diào)控機(jī)理的影響作用。主要研究結(jié)果如下:1.根據(jù)GeneBank中已發(fā)表的黃瓜CsNR序列(登錄號(hào)為NM_001280767)設(shè)計(jì)引物進(jìn)行RT-PCR,將PCR產(chǎn)物回收獲得長(zhǎng)度為2748bp的CDS全長(zhǎng)與360 bp的反義片段,分別構(gòu)建了CsNR正義和反義表達(dá)載體。同時(shí),黃瓜CsNR基因的氨基酸序列與甜瓜、煙草、擬南芥、油菜NR基因編碼的氨基酸序列高度同源,其中與甜瓜NR蛋白之間同源性最高,達(dá)到98.25%,與煙草NR蛋白的同源性達(dá)到78.67%,黃瓜CsNR反義片段與煙草NR基因編碼的氨基酸序列的同源性達(dá)到90.83%。2.本研究同時(shí)通過(guò)子房注射法和農(nóng)桿菌介導(dǎo)途徑進(jìn)行CsNR基因的遺傳轉(zhuǎn)化。PCR鑒定結(jié)果表明,利用子房注射與農(nóng)桿菌介導(dǎo)法在黃瓜上的遺傳轉(zhuǎn)化未成功,利用農(nóng)桿菌侵染法成功轉(zhuǎn)入煙草中,T0代轉(zhuǎn)基因煙草通過(guò)PCR檢測(cè)表明其目的基因已成功轉(zhuǎn)入再生植株體內(nèi),其轉(zhuǎn)化成功率高達(dá)92%。3.研究了硝酸鹽脅迫對(duì)轉(zhuǎn)基因煙草幼苗抗氧化系統(tǒng)的影響,發(fā)現(xiàn)NO_3~-脅迫降低了POD和CAT的活性,使得H2O2累積,從而對(duì)植株造成了傷害。與野生型相比較,Cs NR(+)煙草的CAT活性保持較高水平,能夠參與清除H2O2,保護(hù)植物不被傷害。Cs NR(-)基因植株的SOD、GR活性上升幅度較大,減少植物在逆境脅迫下產(chǎn)生的生理傷害。表明CsNR轉(zhuǎn)基因煙草通過(guò)提高煙草抗氧化酶活性來(lái)緩解NO_3~-脅迫。4.研究了硝酸鹽脅迫對(duì)轉(zhuǎn)基因煙草氮代謝的影響,發(fā)現(xiàn)NO_3~-脅迫下,CsNR(+)煙草可提高NR、NiR、GOGAT、GS、GDH活性以及其基因表達(dá)水平,CsNR(-)煙草可降低NR、NiR、GOGAT、GS、GDH活性以及其基因表達(dá)水平,基因表達(dá)量變化基本與酶活變化基本一致。5.研究了硝酸鹽脅迫對(duì)轉(zhuǎn)基因煙草NO影響,發(fā)現(xiàn)NO_3~-脅迫下,煙草幼苗中產(chǎn)生的NO通過(guò)調(diào)控NR表達(dá)激活NR活性的方式調(diào)節(jié)植物體內(nèi)NO含量,增強(qiáng)抗脅迫能力。
[Abstract]:Cucumber (Cucumis sativus L.) It is one of the main vegetable crops planted in facilities and open fields in China. Soil secondary salinization has become one of the main obstacles to cucumber production in facilities. NO_3~- is the main anion of soil secondary salinization. Plants absorb N. through nitrate assimilation in the form of NO_3~- and NH4. Nitrate assimilation is a strictly regulated process, in which NR and NiR play a major role in the assimilation of primary nitrogen. NR is the key enzyme in the whole process of nitrate assimilation, rate-limiting enzyme, which plays a key role in the process of plant nitrogen metabolism. NR is also NO synthase in plants. NO can improve the stress resistance of plants to a certain extent and alleviate stress. Therefore, it is of great theoretical and practical significance to investigate the effect of cucumber nitrate reductase gene (CsNR) on antioxidant system and nitrogen metabolism of plant seedlings under NO_3~- stress, which is of great theoretical and practical significance to guide the sustainable production of cucumber. In this experiment, CsNR sense and antisense expression vectors were constructed and transformed into cucumber and tobacco to obtain tobacco plants transgenic with CsNR () and CsNR (-) genes. the antioxidant system of NR plants under NO_3~- stress, the expression characteristics of nitrogen metabolism related enzymes and the molecular regulation mechanism of endogenous NO were studied. The main results are as follows: 1. According to the published cucumber CsNR sequence (login number NM_001280767) in GeneBank, primers were designed and RT-PCR, was used to recover the PCR product to obtain the full length of CDS and the antisense fragment of 2748bp. The sense and antisense expression vectors of CsNR were constructed, respectively. At the same time, the amino acid sequence of cucumber CsNR gene is highly homologous to the amino acid sequence encoded by muskmelon, tobacco, Arabidopsis thaliana and rape NR gene, among which the homology with melon NR protein is the highest (98.25%), the homology with tobacco NR protein is 78.67%, and the homology between cucumber CsNR antisense fragment and amino acid sequence encoded by tobacco NR gene is 90.83%. 2. At the same time, the genetic transformation of CsNR gene was carried out by ovary injection and Agrobacterium tumefaciens mediated pathway. The results of PCR identification showed that the genetic transformation on cucumber by ovary injection and Agrobacterium tumefaciens was not successful, and the transgenic tobacco was successfully transferred into tobacco by Agrobacterium tumefaciens infection. T0 generation transgenic tobacco showed that the target gene had been successfully transferred into regenerated plants, and the success rate of transformation was as high as 92%. The effects of nitrate stress on antioxidant system of transgenic tobacco seedlings were studied. it was found that NO_3~- stress decreased the activities of POD and CAT and resulted in the accumulation of H2O2, thus causing damage to plants. Compared with wild type, Cs NR () tobacco maintained a high level of CAT activity, which could be involved in scavenging H2O2 and protecting plants from injury. The SOD,GR activity of Cs NR (-) gene plants increased greatly, which reduced the physiological damage caused by plants under stress. The results showed that CsNR transgenic tobacco alleviated NO_3~- stress by increasing the activity of antioxidant enzymes in tobacco. 4. The effects of nitrate stress on nitrogen metabolism of transgenic tobacco were studied. it was found that, CsNR () tobacco under NO_3~- stress could increase NR,NiR,GOGAT,GS,GDH activity and its gene expression level, CsNR (-) tobacco could decrease NR,NiR,GOGAT,GS,GDH activity and its gene expression level, and the change of gene expression was basically the same as that of enzyme activity. 5. The effects of nitrate stress on NO of transgenic tobacco were studied. it was found that under NO_3~- stress, NO produced in tobacco seedlings regulated NO content in plants by regulating NR expression and activating NR activity, and enhanced the ability of resistance to stress.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S642.2

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