本文關(guān)鍵詞：海濱錦葵鹽脅迫轉(zhuǎn)錄組分析和抗鹽基因研究　出處：《中國科學(xué)院煙臺海岸帶研究所》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 海濱錦葵 鹽脅迫 轉(zhuǎn)錄組 KvLEA KvTIP3 KvHSP70

【摘要】：隨著全球土地鹽漬化程度的不斷加深和鹽漬化土地面積的不斷擴(kuò)張,土壤鹽脅迫發(fā)展成為制約糧食產(chǎn)量和質(zhì)量的主要限制因素。鹽脅迫能夠抑制植物的光合作用,造成植物體生長發(fā)育的遲緩和停滯。同時還能對植物體造成滲透脅迫和離子毒害,導(dǎo)致植物體水分平衡的失調(diào)和各種代謝活動的紊亂。面對鹽脅迫對植物體多方面的影響,植物特別是耐鹽植物進(jìn)化出了多種不同層次的響應(yīng)機(jī)制和策略。揭示這些機(jī)制對于人類充分利用植物資源、保護(hù)與生態(tài)恢復(fù)人類空間環(huán)境、增加與改善食品營養(yǎng)以及充分利用鹽漬土資源實(shí)現(xiàn)現(xiàn)代農(nóng)業(yè)可持續(xù)發(fā)展都具有重大理論與實(shí)踐意義。本論文以耐鹽植物海濱錦葵為研究材料,圍繞海濱錦葵在鹽脅迫下的應(yīng)答響應(yīng)分子機(jī)理展開研究工作。針對海濱錦葵超強(qiáng)的耐鹽特性,研究海濱錦葵的抗鹽機(jī)制和關(guān)鍵的抗鹽基因,并對抗鹽基因的功能進(jìn)行了深入研究。(1)對海濱錦葵幼苗進(jìn)行鹽脅迫處理、取材、提取RNA、高通量轉(zhuǎn)錄組測序。通過對3個樣品文庫的測序獲得了大量海濱錦葵的轉(zhuǎn)錄本信息,每個樣品文庫均有多達(dá)6千萬個轉(zhuǎn)錄本被測序,總測序長度達(dá)170多億個堿基。通過不同文庫之間的比較分析發(fā)現(xiàn),鹽脅迫誘導(dǎo)了海濱錦葵數(shù)千甚至上萬個轉(zhuǎn)錄本的改變。借助已有物種數(shù)據(jù)庫的數(shù)據(jù)信息,對海濱錦葵轉(zhuǎn)錄組數(shù)據(jù)分析發(fā)現(xiàn),鹽脅迫下海濱錦葵生長發(fā)育和光合途徑相關(guān)基因被明顯抑制,而編碼離子通道、載體蛋白的基因發(fā)生明顯上調(diào)。通過KEGG數(shù)據(jù)庫比對分析發(fā)現(xiàn),淀粉、蔗糖分解代謝,氨基酸、脂肪代謝以及激素代謝和信號傳導(dǎo)相關(guān)基因發(fā)生明顯上調(diào)。最后qPCR技術(shù)對轉(zhuǎn)錄組結(jié)果的驗(yàn)證,證明結(jié)果是準(zhǔn)確可靠的。(2)通過不同測序文庫之間的比較分析,篩選出海濱錦葵對鹽脅迫響應(yīng)明顯的基因。進(jìn)一步利用分子生物學(xué)技術(shù)克隆獲得了兩個鹽脅迫響應(yīng)基因的全長,并分別命名為KvLEA和KvTIP3。對KvLEA基因編碼蛋白的分析發(fā)現(xiàn),該蛋白具有許多已報(bào)道LEA蛋白的特性,與擬南芥中的兩個LEA蛋白基因At2g36640和At3g53040具有很高的同源性。組織表達(dá)模式顯示,該基因在海濱錦葵根中具有較高的表達(dá)水平。另外KvLEA基因的表達(dá)能夠被鹽脅迫明顯誘導(dǎo),隨著鹽離子濃度的升高表達(dá)水平逐漸遞增。同時該基因還受ABA、高溫和干旱的誘導(dǎo)表達(dá)。因此,該基因與海濱錦葵的抗逆性密切相關(guān)。對KvTIP3基因的分析表明,其編碼了一個水通道蛋白,分子量的大小,NPA基序以及保守的跨膜結(jié)構(gòu)域均與典型水通道蛋白的特征相一致。組織表達(dá)模式顯示,其編碼了海濱錦葵的一個根部特異的水通道蛋白。同時非生物脅迫下的表達(dá)模式顯示,其不僅參與了海濱錦葵對鹽脅迫的響應(yīng),同時低溫和ABA處理也均能誘導(dǎo)其表達(dá),是海濱錦葵一個重要的抗逆基因。(3)KvHSP70是從海濱錦葵中分離獲得的另一個鹽脅迫響應(yīng)基因,生物信息學(xué)分析表明該基因編碼了一個胞質(zhì)定位的熱激蛋白,在氨基酸序列上與其它物種中的HSP70蛋白具有高達(dá)96%的相似性。洋蔥表皮細(xì)胞瞬時轉(zhuǎn)化實(shí)驗(yàn)證明,該蛋白定位于細(xì)胞的胞質(zhì)中。表達(dá)分析結(jié)果表明,它不僅對鹽脅迫有響應(yīng),對高溫和ABA也都具有響應(yīng)。利用基因工程技術(shù)對該基因的生物學(xué)功能研究發(fā)現(xiàn),其在煙草中的表達(dá)增強(qiáng)了轉(zhuǎn)基因煙草鹽脅迫下各項(xiàng)生理生化指標(biāo)。證明該基因具有提高植物抵抗高鹽等非生物脅迫的生物學(xué)功能。
[Abstract]:With the continuous expansion of the global land salinization degree deepening and salinization land area, soil salt stress development has become the main restrict factors of grain yield and quality. Salt stress inhibited the photosynthesis of plants, resulting in plant growth retardation and stagnation. But also the plant caused by osmotic stress and ion toxicity, resulting in water the plant body balance disorders and metabolic activity disorder. In the face of the effect of salt stress on plants in many aspects, especially in plant salt tolerant plants have evolved a variety of response mechanisms and Strategies of different levels. These reveal the mechanism for the human to make full use of plant resources, protection and restoration of human environment, increase and improve food nutrition and make full use of the resources of saline soil is of great theoretical and practical significance to realize the sustainable development of modern agriculture. In this paper, salt tolerance Plant kostelezkyavirginica as research material, on the response of Kosteletzkya virginica under salt stress response mechanism to start the research. Strong salt tolerance for virginica, salt resistance mechanism and the key of kosteletzkyavirginica salt resistant gene, and the salt resistance gene function were studied. (1) of Kosteletzkya virginica seedlings for treatment, salt stress were extracted from RNA, high-throughput transcriptome sequencing. The sequencing of 3 samples obtained a library of transcription of kosteletzkyavirginica this information, each sample library has as many as 60 million transcripts were sequenced, the total sequencing length of about 17000000000 nucleotides. Through the comparison between different library analysis discovery, salt stress induced kosteletzkyavirginica thousands or even tens of thousands of transcripts. The change of the existing species database data of Kosteletzkya virginica transcriptome data analysis found that the salt sea Jin Bin Sunflower Growth and significant inhibition of photosynthetic pathway related genes, and encoding ion channels, carrier protein gene was up-regulated by KEGG. Database analysis found that starch, sucrose metabolism, amino acid, fat metabolism and hormone metabolism and signal transduction related genes were up-regulated. To verify the final qPCR on the transcriptome results the results are accurate and reliable. (2) through the analysis and comparison between different sequencing library and screened response were gene of Kosteletzkya virginica under salt stress. Further cloning obtained two salt stress response gene sequences by molecular biology technology, and were named to analysis of the KvLEA gene encoding protein KvLEA and KvTIP3., the protein LEA protein has been reported to have many characteristics, has high homology with the Arabidopsis LEA protein two gene At2g36640 and At3g53040 group. Show the fabric expression pattern, the gene has a higher expression level in Kosteletzkya virginica root. KvLEA gene expression also can be significantly induced by salt stress, salt ion concentration increases with the expression level increased gradually. At the same time the gene is also affected by ABA expression induced by high temperature and drought. Therefore, the gene and the resistance of kosteletzkyavirginica closely related. Analysis of KvTIP3 gene showed that the encoding of a water channel protein, molecular weight, consistent with characteristics of NPA motifs and conserved transmembrane domains with typical aquaporins. Tissue expression pattern showed that the water channel protein encoding a specific root of kosteletzkyavirginica at the same time. Non display expression patterns of biological stress, which not only in kosteletzkyavirginica in response to salt stress, while low temperature and ABA treatment can induce the expression of kosteletzkyavirginica, is an important base of resistance . (3) KvHSP70 was isolated and another salt obtained from kosteletzkyavirginica in stress response genes, bioinformatics analysis showed that the gene encoding heat shock protein a cytoplasmic localization, and other species in the amino acid sequence of HSP70 protein with similarity as high as 96%. The instantaneous onion epidermal cells the transformation experiment proved that the protein located in the cytoplasm. The expression analysis results show that it is not only the salt stress response, both in response to high temperature and ABA. The study of biological function by gene engineering technology to the discovery of the gene, its expression in tobacco enhances the physiological and biochemical indexes of the transgenic tobacco salt stress. The gene can improve the biological function of high salt and other abiotic stress resistance in plants.

【學(xué)位授予單位】：中國科學(xué)院煙臺海岸帶研究所
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q945.78

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 李建國;濮勵杰;韓明芳;朱明;張潤森;向仰州;;中國土壤鹽漬化研究的現(xiàn)狀及未來(英文)[J];Journal of Geographical Sciences;2014年05期

2 洪立洲;王茂文;劉沖;丁海榮;朱小梅;邢錦城;趙寶泉;;氮、磷肥對蘇北沿海灘涂海濱錦葵生長及品質(zhì)的影響[J];河南農(nóng)業(yè)科學(xué);2011年05期

3 林鶯;李偉;范海;趙可夫;;海濱錦葵光合作用對鹽脅迫的響應(yīng)[J];山東師范大學(xué)學(xué)報(bào)(自然科學(xué)版);2006年02期

4 尹增芳;何禎祥;王麗霞;欽佩;;NaCl脅迫下海濱錦葵種子萌發(fā)和幼苗生長過程的生理特性變化[J];植物資源與環(huán)境學(xué)報(bào);2006年01期

，

本文編號：1357642