海濱錦葵耐鹽生理特性及脯氨酸代謝相關(guān)基因的研究
發(fā)布時(shí)間：2018-11-26 10:07
【摘要】：土壤鹽漬化已成為一個(gè)制約農(nóng)作物產(chǎn)量和質(zhì)量的全球性問題。為了充分開發(fā)鹽土資源和發(fā)展鹽土農(nóng)業(yè),耐鹽經(jīng)濟(jì)植物的篩選和培育越來越受到人們的重視。耐鹽植物在長(zhǎng)期適應(yīng)鹽漬環(huán)境的過程中進(jìn)化出獨(dú)特的耐鹽機(jī)制,也為人類提供了取之不盡的耐鹽基因資源庫。因此,研究耐鹽植物對(duì)鹽脅迫的生理響應(yīng)機(jī)制,從中挖掘關(guān)鍵耐鹽基因,并利用基因工程等技術(shù)培育耐鹽新品種,對(duì)鹽土農(nóng)業(yè)和農(nóng)業(yè)的可持續(xù)發(fā)展都具有重要的理論和現(xiàn)實(shí)意義。本論文以耐鹽植物海濱錦葵為實(shí)驗(yàn)材料,在系統(tǒng)地研究海濱錦葵對(duì)鹽脅迫的生理響應(yīng)的基礎(chǔ)上,針對(duì)鹽脅迫導(dǎo)致脯氨酸顯著積累的生理特點(diǎn),我們克隆了脯氨酸代謝相關(guān)基因,并對(duì)其表達(dá)模式和功能進(jìn)行了初步分析。主要研究結(jié)果如下:(1)用不同濃度的Na Cl對(duì)海濱錦葵幼苗進(jìn)行20d的脅迫處理,對(duì)其生長(zhǎng)參數(shù)及相關(guān)生理生化指標(biāo)進(jìn)行測(cè)定。結(jié)果表明:100和200 mmol/L Na Cl的鹽脅迫對(duì)海濱錦葵幼苗的生長(zhǎng)影響不大,雖然導(dǎo)致海濱錦葵幼苗葉片和根內(nèi)的Na+含量顯著增加,但更多的Na+被區(qū)隔化在根內(nèi),而葉片內(nèi)保持較高的K+/Na+比值和脯氨酸含量,說明海濱錦葵可以利用無機(jī)離子和有機(jī)溶質(zhì)的協(xié)同滲透調(diào)節(jié)作用以對(duì)抗鹽脅迫。同時(shí),抗氧化酶活性的增加能夠及時(shí)清除活性氧的積累,避免了氧化傷害的發(fā)生,表現(xiàn)在葉綠素?zé)晒鈪?shù)、MDA含量和細(xì)胞膜透性與對(duì)照沒有顯著差異。在300 mmol/L和400 mmol/L Na Cl的高鹽處理下,海濱錦葵幼苗的生長(zhǎng)受到嚴(yán)重抑制,各項(xiàng)生理指標(biāo)顯示海濱錦葵幼苗受到嚴(yán)重的滲透脅迫和離子毒害,光合機(jī)構(gòu)、細(xì)胞膜系統(tǒng)受到破壞。由此可見,海濱錦葵幼苗具有一定的耐鹽能力,低于200 mmol/L Na Cl脅迫處理基本不影響其生長(zhǎng),具有良好的滲透調(diào)節(jié)、離子平衡和區(qū)隔化及抗氧化能力。(2)針對(duì)鹽脅條件下海濱錦葵脯氨酸顯著積累的生理特征,采用RT-PCR及RACE技術(shù)克隆了脯氨酸代謝的相關(guān)基因,包括Kv P5CS、Kv OAT、Kv PDH和Kv Pro T基因。生物信息學(xué)分析表明,這些基因及其編碼的氨基酸序列與其他植物中已知的同源序列均具有較高的同源性。對(duì)這些基因在鹽脅迫條件下的表達(dá)模式進(jìn)行研究表明,海濱錦葵幼苗葉片中脯氨酸的積累主要是因?yàn)辂}脅迫促進(jìn)了脯氨酸的生物合成。其中調(diào)控谷氨酸合成途徑關(guān)鍵酶基因Kv P5CS相對(duì)于調(diào)控鳥氨酸合成途徑關(guān)鍵酶基因Kv OAT發(fā)揮了更重要的作用。Kv Pro T的表達(dá)與Kv P5CS類似,被鹽脅迫誘導(dǎo)顯著上調(diào),推測(cè)它可能在脯氨酸從其他部位向葉片中的運(yùn)輸和積累過程中發(fā)揮了重要作用,但這需要進(jìn)一步的試驗(yàn)來證實(shí)。(3)成功構(gòu)建了海濱錦葵脯氨酸合成酶關(guān)鍵基因Kv P5CS的過表達(dá)載體p BI121-Kv P5CS,并通過農(nóng)桿菌介導(dǎo)和葉盤轉(zhuǎn)化法獲得了轉(zhuǎn)基因煙草。利用200m M Na Cl對(duì)兩個(gè)株系的T1代轉(zhuǎn)基因煙草和野生型煙草進(jìn)行14天的鹽脅迫處理。結(jié)果表明,轉(zhuǎn)Kv P5CS基因煙草比野生型煙草具有較強(qiáng)的耐鹽能力,其葉綠素、脯氨酸含量及抗氧化酶活性明顯高于野生型,說明過表達(dá)Kv P5CS基因而引起脯氨酸的積累可能在其抵抗鹽脅迫過程發(fā)揮了重要作用。
[Abstract]:The salinization of soil has become a global problem that restricts the yield and quality of crops. In order to fully develop the salt-soil resources and to develop the salt-soil agriculture, the screening and cultivation of salt-tolerant economic plants are becoming more and more important. The salt tolerant plant has evolved a unique salt tolerance mechanism in the process of long-term adaptation to the saline environment, and also provides an inexhaustible resistant gene resource base for human. Therefore, it is of great theoretical and practical significance to study the physiological response mechanism of salt-tolerant plants to salt stress, to explore the key salt-resistant genes from them, and to use the techniques of genetic engineering to develop new varieties of salt-tolerant plants. Based on the study of the physiological response of the sea anemone to salt stress and the physiological characteristics of the significant accumulation of proline in the salt stress, we cloned the relevant genes of proline metabolism. The expression pattern and function were analyzed in this paper. The main results of the study were as follows: (1) The growth parameters and relevant physiological and biochemical indexes were determined by using Na Cl at different concentrations to treat the seedlings of Abelmoschus esculenta. The results showed that the salt stress of 100 and 200 mmol/ L Na Cl had no significant effect on the growth of the seedlings of the sea anemones, although the Na + content in the leaves and roots of the mallow seedlings was significantly increased, but more Na + was separated into the roots, while the higher K +/ Na + ratio and proline content in the leaves were maintained. The co-osmotic effect of inorganic ion and organic solute can be used to counter salt stress. At the same time, the increase of antioxidant enzyme activity can remove the accumulation of active oxygen in time, avoid the occurrence of oxidative damage, and show no significant difference in chlorophyll fluorescence parameter, MDA content and cell membrane permeability and control. Under the high salt treatment of 300 mmol/ L and 400 mmol/ L Na Cl, the growth of the seedlings of the Abelmoschus esculenta was severely inhibited, and various physiological indexes showed that the seedlings of the sea anemone were subjected to severe osmotic stress and ionic toxicity, and the photosynthetic mechanism and the cell membrane system were destroyed. It can be seen that the seedling of the sea anemone has a certain salt tolerance, less than 200 mmol/ L Na Cl stress treatment basically does not affect the growth of the seedling, and has good penetration regulation, ion balance and separation and oxidation resistance. (2) The relative genes of proline metabolism were cloned by RT-PCR and RACE, including Kv P5CS, Kv OAT, Kv PDH and Kv Pro T gene. Bioinformatics analysis shows that these genes and their encoded amino acid sequences have higher homology with the homologous sequences known in other plants. The study of the expression pattern of these genes under the condition of salt stress indicated that the accumulation of proline in the leaves of the moschus manihot was mainly because of salt stress and promoted the biosynthesis of proline. The key enzyme gene Kv P5CS, which regulates the synthesis of glutamic acid, plays a more important role in the control of the key enzyme gene Kv OAT of the synthetic pathway of the ornithine. The expression of Kv Pro T was similar to that of Kv P5CS, and was significantly upregulated by salt stress, suggesting that it could play an important role in the transport and accumulation of proline from other sites to the leaves, but this requires further testing to confirm. (3) The expression vector pBI121-Kv P5CS of Kv P5CS, a key gene of the Kv P5CS, was successfully constructed, and the transgenic tobacco was obtained by Agrobacterium-mediated and leaf-plate transformation. The T1-generation transgenic tobacco and wild-type tobacco of two strains were treated with 200m M Na Cl for 14 days. The results showed that the transgenic tobacco with Kv P5CS had a strong salt tolerance to wild type tobacco, and the content of chlorophyll, proline and antioxidant enzymes was higher than that of wild type, and it was shown that the accumulation of proline caused by the expression of Kv P5CS gene could play an important role in its resistance to salt stress.
【學(xué)位授予單位】：中國(guó)科學(xué)院煙臺(tái)海岸帶研究所
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q945.78
本文編號(hào)：2358248
[Abstract]:The salinization of soil has become a global problem that restricts the yield and quality of crops. In order to fully develop the salt-soil resources and to develop the salt-soil agriculture, the screening and cultivation of salt-tolerant economic plants are becoming more and more important. The salt tolerant plant has evolved a unique salt tolerance mechanism in the process of long-term adaptation to the saline environment, and also provides an inexhaustible resistant gene resource base for human. Therefore, it is of great theoretical and practical significance to study the physiological response mechanism of salt-tolerant plants to salt stress, to explore the key salt-resistant genes from them, and to use the techniques of genetic engineering to develop new varieties of salt-tolerant plants. Based on the study of the physiological response of the sea anemone to salt stress and the physiological characteristics of the significant accumulation of proline in the salt stress, we cloned the relevant genes of proline metabolism. The expression pattern and function were analyzed in this paper. The main results of the study were as follows: (1) The growth parameters and relevant physiological and biochemical indexes were determined by using Na Cl at different concentrations to treat the seedlings of Abelmoschus esculenta. The results showed that the salt stress of 100 and 200 mmol/ L Na Cl had no significant effect on the growth of the seedlings of the sea anemones, although the Na + content in the leaves and roots of the mallow seedlings was significantly increased, but more Na + was separated into the roots, while the higher K +/ Na + ratio and proline content in the leaves were maintained. The co-osmotic effect of inorganic ion and organic solute can be used to counter salt stress. At the same time, the increase of antioxidant enzyme activity can remove the accumulation of active oxygen in time, avoid the occurrence of oxidative damage, and show no significant difference in chlorophyll fluorescence parameter, MDA content and cell membrane permeability and control. Under the high salt treatment of 300 mmol/ L and 400 mmol/ L Na Cl, the growth of the seedlings of the Abelmoschus esculenta was severely inhibited, and various physiological indexes showed that the seedlings of the sea anemone were subjected to severe osmotic stress and ionic toxicity, and the photosynthetic mechanism and the cell membrane system were destroyed. It can be seen that the seedling of the sea anemone has a certain salt tolerance, less than 200 mmol/ L Na Cl stress treatment basically does not affect the growth of the seedling, and has good penetration regulation, ion balance and separation and oxidation resistance. (2) The relative genes of proline metabolism were cloned by RT-PCR and RACE, including Kv P5CS, Kv OAT, Kv PDH and Kv Pro T gene. Bioinformatics analysis shows that these genes and their encoded amino acid sequences have higher homology with the homologous sequences known in other plants. The study of the expression pattern of these genes under the condition of salt stress indicated that the accumulation of proline in the leaves of the moschus manihot was mainly because of salt stress and promoted the biosynthesis of proline. The key enzyme gene Kv P5CS, which regulates the synthesis of glutamic acid, plays a more important role in the control of the key enzyme gene Kv OAT of the synthetic pathway of the ornithine. The expression of Kv Pro T was similar to that of Kv P5CS, and was significantly upregulated by salt stress, suggesting that it could play an important role in the transport and accumulation of proline from other sites to the leaves, but this requires further testing to confirm. (3) The expression vector pBI121-Kv P5CS of Kv P5CS, a key gene of the Kv P5CS, was successfully constructed, and the transgenic tobacco was obtained by Agrobacterium-mediated and leaf-plate transformation. The T1-generation transgenic tobacco and wild-type tobacco of two strains were treated with 200m M Na Cl for 14 days. The results showed that the transgenic tobacco with Kv P5CS had a strong salt tolerance to wild type tobacco, and the content of chlorophyll, proline and antioxidant enzymes was higher than that of wild type, and it was shown that the accumulation of proline caused by the expression of Kv P5CS gene could play an important role in its resistance to salt stress.
【學(xué)位授予單位】：中國(guó)科學(xué)院煙臺(tái)海岸帶研究所
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q945.78
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 閆道良;余婷;徐菊芳;黃堅(jiān)欽;;鹽脅迫對(duì)海濱錦葵生長(zhǎng)及Na~+、K~+離子積累的影響[J];生態(tài)環(huán)境學(xué)報(bào);2013年01期
2 賈永霞;孫錦;王麗萍;束勝;郭世榮;;低氧脅迫下黃瓜植株熱耗散途徑[J];應(yīng)用生態(tài)學(xué)報(bào);2011年03期
3 馬清;包愛科;伍國(guó)強(qiáng);王鎖民;;質(zhì)膜Na~+/H~+逆向轉(zhuǎn)運(yùn)蛋白與植物耐鹽性[J];植物學(xué)報(bào);2011年02期
4 呂湘芳;李利;徐新文;韓張雄;;準(zhǔn)噶爾盆地4種鹽生植物耐鹽機(jī)制分析[J];干旱區(qū)研究;2010年01期
5 桂仁意;劉亞迪;郭小勤;季海寶;賈月;余明增;方偉;;不同劑量~(137)Cs-γ輻射對(duì)毛竹幼苗葉片葉綠素?zé)晒鈪?shù)的影響[J];植物學(xué)報(bào);2010年01期
6 楊帆;丁菲;杜天真;;鹽脅迫下構(gòu)樹幼苗各器官中K~+、Ca~(2+)、Na~+和Cl~-含量分布及吸收特征[J];應(yīng)用生態(tài)學(xué)報(bào);2009年04期
7 黨瑞紅;周俊山;范海;;海濱錦葵的抗鹽特性[J];植物生理學(xué)通訊;2008年04期
8 郭予琦;田曾元;閆道良;張潔;欽佩;;鹽生植物海濱錦葵幼苗鹽脅迫下基因差異表達(dá)分析[J];遺傳;2008年07期
9 韓志平;郭世榮;馮吉慶;高新昊;;鹽脅迫對(duì)西瓜幼苗生長(zhǎng)、葉片光合色素和脯氨酸含量的影響[J];南京農(nóng)業(yè)大學(xué)學(xué)報(bào);2008年02期
10 聶小安;蔣劍春;高一葦;常俠;;海濱錦葵油生物柴油的制備及性能分析[J];南京林業(yè)大學(xué)學(xué)報(bào)(自然科學(xué)版);2008年01期
相關(guān)碩士學(xué)位論文 前3條
1 高亞迪;農(nóng)桿菌介導(dǎo)的P5CS基因轉(zhuǎn)化東農(nóng)303馬鈴薯的研究[D];東北林業(yè)大學(xué);2010年
2 趙曉惠;鹽脅迫下旱稻鄭2愈傷組織的生理生化響應(yīng)[D];蘭州大學(xué);2007年
3 馬東建;幾種鹽生植物對(duì)鹽脅迫反應(yīng)的研究[D];新疆大學(xué);2004年
，本文編號(hào)：2358248
本文鏈接：http://sikaile.net/kejilunwen/jiyingongcheng/2358248.html
最近更新
教材專著
