黄瓜CsNR基因的克

发布时间：2019-06-20 14:52

【摘要】：黄瓜(Cucumis sativus L.)是我国设施和露地主要种植的蔬菜作物之一,土壤次生盐渍化已成为影响设施黄瓜生产的主要障碍之一,NO_3~-是土壤次生盐渍化的的主要阴离子。植物通过硝酸盐同化途径以NO_3~-和NH4+的形式吸收N。硝酸盐的同化是一个受到严格调控的过程,其中NR和NiR对初级氮的同化起主要调控作用。NR是整个硝态氮同化过程的关键酶、限速酶,在植物氮素代谢过程中起关键作用。NR也是植物体内的NO合成酶,NO在一定程度上可提高植物的抗逆能力,缓解逆境胁迫。因此探讨黄瓜硝酸还原酶基因(CsNR)对NO_3~-胁迫下植株幼苗抗氧化体系及氮代谢的影响,对指导黄瓜可持续生产具有重要的理论和现实意义。本试验通过构建CsNR正义和反义表达载体,并进行黄瓜和烟草的转化,获得转Cs NR(+)和CsNR(-)基因的烟草植株,研究了NR在NO_3~-胁迫下植株的抗氧化系统、氮代谢相关酶表达特性及其内源NO的分子调控机理的影响作用。主要研究结果如下:1.根据GeneBank中已发表的黄瓜CsNR序列(登录号为NM_001280767)设计引物进行RT-PCR,将PCR产物回收获得长度为2748bp的CDS全长与360 bp的反义片段,分别构建了CsNR正义和反义表达载体。同时,黄瓜CsNR基因的氨基酸序列与甜瓜、烟草、拟南芥、油菜NR基因编码的氨基酸序列高度同源,其中与甜瓜NR蛋白之间同源性最高,达到98.25%,与烟草NR蛋白的同源性达到78.67%,黄瓜CsNR反义片段与烟草NR基因编码的氨基酸序列的同源性达到90.83%。2.本研究同时通过子房注射法和农杆菌介导途径进行CsNR基因的遗传转化。PCR鉴定结果表明,利用子房注射与农杆菌介导法在黄瓜上的遗传转化未成功,利用农杆菌侵染法成功转入烟草中,T0代转基因烟草通过PCR检测表明其目的基因已成功转入再生植株体内,其转化成功率高达92%。3.研究了硝酸盐胁迫对转基因烟草幼苗抗氧化系统的影响,发现NO_3~-胁迫降低了POD和CAT的活性,使得H2O2累积,从而对植株造成了伤害。与野生型相比较,Cs NR(+)烟草的CAT活性保持较高水平,能够参与清除H2O2,保护植物不被伤害。Cs NR(-)基因植株的SOD、GR活性上升幅度较大,减少植物在逆境胁迫下产生的生理伤害。表明CsNR转基因烟草通过提高烟草抗氧化酶活性来缓解NO_3~-胁迫。4.研究了硝酸盐胁迫对转基因烟草氮代谢的影响,发现NO_3~-胁迫下,CsNR(+)烟草可提高NR、NiR、GOGAT、GS、GDH活性以及其基因表达水平,CsNR(-)烟草可降低NR、NiR、GOGAT、GS、GDH活性以及其基因表达水平,基因表达量变化基本与酶活变化基本一致。5.研究了硝酸盐胁迫对转基因烟草NO影响,发现NO_3~-胁迫下,烟草幼苗中产生的NO通过调控NR表达激活NR活性的方式调节植物体内NO含量,增强抗胁迫能力。
[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.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S642.2

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