【摘要】：我國(guó)海域遼闊,濱海鹽堿地只有小部分被利用耕種,大部分還為荒地和海涂地,海岸帶擁有大量的鹽堿土待開發(fā)利用,是重要的后備土地資源。濱海鹽堿地改良和生態(tài)綠化是我國(guó)沿海城市化進(jìn)程中一項(xiàng)異常艱巨的重要難題。根據(jù)近年來國(guó)內(nèi)外實(shí)踐經(jīng)驗(yàn),篩選耐鹽植物是進(jìn)行濱海鹽堿地生物改良和生態(tài)綠化最具光明前景的經(jīng)濟(jì)有效的方法。紫花苜蓿(Medicagosativa L.)是一種具有較強(qiáng)耐鹽性的豆科植物,栽培歷史悠久,在全球廣泛種植,具有一定的觀賞價(jià)值,是進(jìn)行鹽堿地改良和園林綠化的理想植物。本研究以高秋休眠級(jí)南方型紫花苜蓿‘Millennium’的耐鹽突變體為材料,進(jìn)行轉(zhuǎn)錄組測(cè)序分析,篩選鹽脅迫響應(yīng)差異基因,并對(duì)其進(jìn)行GO功能顯著性分析、KEGG Pathway顯著性富集分析和轉(zhuǎn)錄因子分子,已期探究耐鹽相關(guān)基因參與的主要生物學(xué)功能和代謝途徑,為進(jìn)一步研究紫花苜蓿耐鹽分子機(jī)制,培育耐鹽新品種奠定基礎(chǔ)。本研究結(jié)果如下:1、通過對(duì)南方型紫花苜蓿耐鹽突變體幼苗進(jìn)行250mmol/LNaCl處理0、24、48、72和96h時(shí)生理指標(biāo)的測(cè)定,我們發(fā)現(xiàn)在鹽脅迫72h內(nèi),葉片相對(duì)含水量持續(xù)降低,脯氨酸含量、相對(duì)電導(dǎo)率、MDA含量和SOD活性持續(xù)升高;在鹽脅迫大于72h后,葉綠素含量和SOD活性明顯下降,脯氨酸含量、相對(duì)電導(dǎo)率和MDA含量升高幅度增大。表明250mmol/LNa Cl處理72h是紫花苜蓿耐鹽突變體幼苗鹽脅迫響應(yīng)的最佳時(shí)間,為接下來耐鹽分子機(jī)制研究提供了依據(jù)。2、利用RNA-Seq技術(shù),經(jīng)過250mmol/LNa Cl脅迫72 h,南方型紫花苜蓿耐鹽突變體幼苗葉片樣品中共檢測(cè)到30 900個(gè)基因表達(dá)量發(fā)生改變,其中7 694個(gè)基因被定義為差異基因(4 187個(gè)基因上調(diào)表達(dá),3 507個(gè)基因下調(diào)表達(dá)),包括隸屬于50個(gè)轉(zhuǎn)錄家族的422個(gè)轉(zhuǎn)錄因子(268個(gè)轉(zhuǎn)錄因子上調(diào)表達(dá),154個(gè)轉(zhuǎn)錄因子下調(diào)表達(dá))。3、實(shí)時(shí)熒光定量PCR驗(yàn)證表明,隨機(jī)選擇的8個(gè)差異表達(dá)基因在鹽脅迫前后的表達(dá)特點(diǎn)與表達(dá)譜測(cè)序結(jié)果一致,表明了轉(zhuǎn)錄組測(cè)序數(shù)據(jù)的可靠性。4、GO功能顯著性分析顯示,差異表達(dá)基因的生物學(xué)功能主要表現(xiàn)在催化活性、結(jié)合、細(xì)胞組分、細(xì)胞、代謝過程和細(xì)胞過程。KEGG Pathway顯著性富集分析顯示,差異表達(dá)基因廣泛涉及代謝途徑和次生代謝產(chǎn)物生物合成。候選出Ms GST、Ms L-APX、Ms GPX、Ms RLK、Ms SIK、Ms Sn RK、Ms CMLs、Ms CMO、Ms MIP、Ms P5CS1、Ms P5CS3、Ms ABCB、Ms WBC、Ms LEA3和Ms PP2C等重要耐鹽相關(guān)基因。轉(zhuǎn)錄因子分析發(fā)現(xiàn),應(yīng)答基因數(shù)量最多的是MYB基因家族,其次是WRKY、NAC、b HLH、AP2-EREBP和GRAS等基因家族。候選出Ms Alfin-like、Ms ERF110、Ms ANT、Msb HLH36、Ms NAI1、Msb ZIP、Ms C3H、Ms GRAS、Ms NAD、Ms MYB、Ms MYB85、Ms NAC、Ms RWP-RK、Ms Trihelix和Ms WRKY等耐鹽相關(guān)轉(zhuǎn)錄因子。
[Abstract]:China has a vast sea area, and only a small part of the coastal saline land is used for cultivation, most of which are also wasteland and coastal land. The coastal zone has a large number of saline-alkali soil to be developed and used, which is an important reserve land resource. Coastal saline-alkali land improvement and ecological greening is an extremely difficult problem in the process of coastal urbanization in China. According to the practical experience at home and abroad in recent years, screening salt-tolerant plants is the most promising economic and effective method for biological improvement and ecological greening of coastal saline-alkali land. Alfalfa (Medicagosativa L.) It is a kind of leguminous plants with strong salt tolerance. It has a long cultivation history and is widely planted in the world. It has some ornamental value. It is an ideal plant for saline-alkali land improvement and garden greening. In this study, the salt-tolerant mutants of southern alfalfa 'Millennium' with high autumn dormancy level were used as materials. The transcriptional sequence analysis was carried out to screen the different genes of salt stress response, and the significance of GO function was analyzed. Significant enrichment analysis of KEGG Pathway and transcription factor molecules have been carried out to explore the main biological functions and metabolic pathways of salt-tolerance related genes in order to lay a foundation for further study on the molecular mechanism of salt tolerance in alfalfa and the cultivation of new salt-tolerant varieties. The results were as follows: 1. The relative water content of leaves and proline content in leaves of southern type alfalfa seedlings were continuously decreased within 72 h and 96 h after 250mmol/LNaCl treatment. The relative conductivity, MDA content and SOD activity continued to increase. When salt stress was more than 72 hours, chlorophyll content and SOD activity decreased obviously, proline content, relative conductivity and MDA content increased. The results showed that 250mmol/LNa Cl treatment for 72 h was the best time for salt stress response of alfalfa salt tolerant mutants, which provided a basis for the study of molecular mechanism of salt tolerance in alfalfa seedlings. 2. Using RNA-Seq technique, 72 h of 250mmol/LNa Cl stress was used. A total of 30 900 genes were detected in the leaves of southern alfalfa salt tolerant mutants, of which 7 694 genes were defined as differential genes (4 187 genes up-regulated and 3 507 genes down-regulated). Including 422 transcription factors belonging to 50 transcription families (268 transcription factors up-regulated and 154 transcription factors down-regulated). The expression characteristics of the eight differentially expressed genes selected at random before and after salt stress were consistent with the sequencing results of the expression profiles, which indicated the reliability of the transcriptional sequence data. The biological functions of differentially expressed genes were mainly manifested in catalytic activity, binding, cell composition, cell, metabolic process and. KEGG Pathway enrichment in cellular processes. Differentially expressed genes are widely involved in metabolic pathways and biosynthesis of secondary metabolites. Important salt-tolerant genes, such as Ms GST,Ms L-APXN, GPX,Ms RLK,Ms SIK,Ms Sn RK,Ms CMLs,Ms CMO,Ms MIP,Ms P5CS1, Ms P5CS3, ABCB,Ms WBC,Ms LEA3 and Ms PP2C, were selected as candidates. Transcription factor analysis showed that the largest number of responsive genes was MYB gene family, followed by WRKY,NAC,b HLH,AP2-EREBP and GRAS gene families. Candidate transcription factors such as Ms Alfin-like,Ms ERF110,Ms ANT,Msb HLH36,Ms NAI1,Msb ZIP,Ms C _ 3H _ 2 Ms GRAS,Ms NAD,Ms MYB,Ms MYB85,Ms NAC,Ms RWP-RK,Ms Trihelix and Ms WRKY were selected.
【學(xué)位授予單位】：浙江農(nóng)林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S541.9

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