發(fā)布時(shí)間：2018-03-13 18:27

  本文選題：蓖麻　切入點(diǎn)：鹽脅迫　出處：《內(nèi)蒙古民族大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：土壤鹽堿化一直是亟待解決的世界性農(nóng)業(yè)難題之一,嚴(yán)重影響了全球農(nóng)業(yè)經(jīng)濟(jì)的發(fā)展及生態(tài)環(huán)境的保護(hù)。利用基因工程選育耐鹽作物品種,已成為利用和改良鹽堿地的一條經(jīng)濟(jì)有效途徑。近年來,隨著蓖麻產(chǎn)業(yè)的發(fā)展,人們對(duì)蓖麻的生產(chǎn)和加工的重視程度越來越高,而針對(duì)蓖麻耐鹽相關(guān)基因的發(fā)掘和利用的研究較少。為了挖掘耐鹽相關(guān)基因,本研究以當(dāng)?shù)刂魍频墓歉勺越幌低ū?號(hào)為材料,利用RACE-PCR的方法成功克隆了蓖麻液泡膜Na~+/H~+逆向轉(zhuǎn)運(yùn)蛋白(vacuolar Na~+/H~+antiporter,NHX)基因RcNHX1。RcNHX1基因包括一個(gè)完整的開放閱讀框,推測編碼544個(gè)氨基酸。亞細(xì)胞定位試驗(yàn)表明RcNHX1融合蛋白主要積累于細(xì)胞膜與液泡膜。以蓖麻子葉節(jié)為外植體,構(gòu)建植物過表達(dá)載體、干擾載體,利用農(nóng)桿菌進(jìn)行遺傳轉(zhuǎn)化。PCR和Southern blot檢測表明共獲得2個(gè)穩(wěn)定遺傳的過表達(dá)株系。對(duì)過表達(dá)RcNHX1轉(zhuǎn)基因蓖麻在苗期抗鹽表型鑒定發(fā)現(xiàn):150 mmol/L NaCl處理14 d后,轉(zhuǎn)基因植株的株高和干重均顯著高于野生型。鹽脅迫后,轉(zhuǎn)基因植株脯氨酸的含量顯著高于野生型,而丙二醛的含量及相對(duì)電解質(zhì)滲透率顯著降低。此外,相比于野生型,鹽脅迫后轉(zhuǎn)基因植株葉片和根中Na~+與K~+離子含量的升高為RcNHX1提高轉(zhuǎn)基因植株的抗鹽性提供了可能的分子機(jī)制。
[Abstract]:Soil salinization has always been one of the world agricultural problems to be solved, which has seriously affected the development of global agricultural economy and the protection of ecological environment. In recent years, with the development of castor industry, people pay more and more attention to the production and processing of castor. In order to excavate salt-tolerant genes, the local backbone inbred line Tonggri 5 was used as the material. RACE-PCR method was used to clone the RcNHX1.RcNHX1 gene of the vacuolar Na ~ / / H ~ antiporterus NH _ Xgene of castor, including a complete open reading frame. Subcellular localization test showed that RcNHX1 fusion protein was mainly accumulated in cell membrane and vacuolar membrane. Plant overexpression vector was constructed with castor cotyledon as explant. The results of Agrobacterium tumefaciens genetic transformation. PCR and Southern blot analysis showed that two stable genetic overexpression lines were obtained. After 14 days of treatment, the overexpressed RcNHX1 transgenic castor plants were identified as salt-tolerant phenotypes at seedling stage and were treated with 1: 150 mmol/L NaCl for 14 days. The plant height and dry weight of transgenic plants were significantly higher than that of wild type. After salt stress, the content of proline was significantly higher than that of wild type, while the content of malondialdehyde and the relative electrolyte permeability of transgenic plant were significantly lower than that of wild type. The increase of Na ~ and K ~ + contents in leaves and roots of transgenic plants after salt stress may provide a possible molecular mechanism for RcNHX1 to improve the salt tolerance of transgenic plants.
【學(xué)位授予單位】：內(nèi)蒙古民族大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S565.6;Q943.2

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