本文選題：陸地棉 + 鹽脅迫應(yīng)答　； 參考：《中國(guó)農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：棉花是鹽堿地先鋒作物。研究耐鹽機(jī)理、提高棉花耐鹽性具有重要理論與實(shí)踐意義。基于實(shí)驗(yàn)室前期研究工作,從鹽脅迫下陸地棉根系抑制性雜交差減文庫篩選得到5個(gè)候選基因GhSnRK2.6, GhCIPK6, GhCPK5、GhC2H2和GhWRKY41。通過克隆全長(zhǎng)基因,構(gòu)建植物表達(dá)載體,分別轉(zhuǎn)化野生型擬南芥及棉花,分析其功能；通過構(gòu)建瞬時(shí)表達(dá)載體,進(jìn)行亞細(xì)胞定位；通過調(diào)查轉(zhuǎn)基因材料耐鹽相關(guān)性狀,分析這5個(gè)鹽脅迫應(yīng)答基因的功能；篩選耐鹽品系,為棉花耐鹽育種提供材料來源。(1)克隆了鹽脅迫應(yīng)答基因GhSnRK2.6, GenBank登錄號(hào)為JN872373, ORF全長(zhǎng)1,086bp,編碼361個(gè)氨基酸。該基因包含9個(gè)外顯子,8個(gè)內(nèi)含子。構(gòu)建瞬時(shí)表達(dá)載體p35S-GhSnRK2.6:GFP,基因槍法轉(zhuǎn)化洋蔥表皮細(xì)胞進(jìn)行瞬時(shí)表達(dá)分析,該蛋白定位于細(xì)胞核。構(gòu)建植物表達(dá)載體p2301M-GhSnRK2.6,分別轉(zhuǎn)化野生型擬南芥和棉花,分析轉(zhuǎn)基因材料耐鹽性。結(jié)果GhSnRK2.6過表達(dá)擬南芥苗期及成熟期的耐鹽性提高；轉(zhuǎn)基因棉花后代材料鹽脅迫下發(fā)芽率顯著高于受體株系,耐鹽性提高；另外,鹽脅迫下轉(zhuǎn)基因棉花的產(chǎn)量性狀和纖維品質(zhì)也優(yōu)于受體對(duì)照。(2)構(gòu)建瞬時(shí)表達(dá)載體p35S-GhCIPK6:GFP,基因槍轟擊法轉(zhuǎn)化洋蔥表皮細(xì)胞進(jìn)行瞬時(shí)表達(dá)分析,證明GhCIPK6定位于細(xì)胞質(zhì)。BiFC分析確定GhCIPK6與GhCBLl和GhCBL8均存在互作關(guān)系,GhCBLl-GhCIPK6復(fù)合體定位于細(xì)胞核,GhCBL8-GhCIPK6定位于細(xì)胞核及細(xì)胞膜。將植物表達(dá)載體pBI-GhCIPK6轉(zhuǎn)化棉花,分析耐鹽性。證明鹽脅迫下,GhCIPK6通過參與不同調(diào)控路徑,調(diào)控細(xì)胞K+平衡,維持細(xì)胞膜穩(wěn)定性,降低鹽脅迫對(duì)質(zhì)膜的傷害,保證植株正常生長(zhǎng),提高轉(zhuǎn)基因棉花后代的耐鹽性。(3)構(gòu)建瞬時(shí)表達(dá)載體進(jìn)行亞細(xì)胞定位,結(jié)果轉(zhuǎn)錄因子GhC2H2定位于細(xì)胞核。將前期構(gòu)建的植物表達(dá)載體pBI-GhC2H2轉(zhuǎn)化棉花,分析其耐鹽性。結(jié)果證明過表達(dá)GhC2H2可提高轉(zhuǎn)基因株系的衣分和單鈴重,促進(jìn)纖維增長(zhǎng),保證轉(zhuǎn)基因棉花產(chǎn)量,提高轉(zhuǎn)基因棉花后代的耐鹽性。(4)構(gòu)建瞬時(shí)表達(dá)載體進(jìn)行亞細(xì)胞定位,結(jié)果GhCPK5定位于細(xì)胞核。將前期構(gòu)建的植物表達(dá)載體pBI-GhCPK5轉(zhuǎn)化棉花,分析相關(guān)性狀,結(jié)果在鹽脅迫下,GhCPK5轉(zhuǎn)基因株系的單鈴重和衣分高于受體,纖維長(zhǎng)度增長(zhǎng),馬克隆值下降,斷裂比強(qiáng)度提高,纖維品質(zhì)優(yōu)于受體對(duì)照。(5)構(gòu)建瞬時(shí)表達(dá)載體進(jìn)行亞細(xì)胞定位,結(jié)果轉(zhuǎn)錄因子GhWRKY41定位于細(xì)胞核。將前期構(gòu)建的植物表達(dá)載體p2301M-GhWRKY41轉(zhuǎn)化棉花并分析耐鹽性。結(jié)果表明,過表達(dá)GhWRKY41可提高轉(zhuǎn)基因棉花脅迫下發(fā)芽率,鹽脅迫下轉(zhuǎn)基因株系的鈴重和衣分變化不大,但對(duì)纖維品質(zhì)存在不利影響。
[Abstract]:Cotton is a pioneer crop of saline - alkali land . It is of great theoretical and practical significance to study the salt tolerance mechanism and improve the salt tolerance of cotton . Based on the research work in the early stage of the laboratory , five candidate genes GhSnRK2 . 6 , GhCIP6 , GhCP5 , GhC2H2 and GhWRKY41 were isolated from the root system of upland cotton under salt stress .
carrying out sub - cell positioning by constructing a transient expression vector ;
By investigating the salt tolerance properties of transgenic materials , the function of these five salt stress response genes was analyzed .
The expression vector p35S - GhSnR2.6 : GFP was used to transform wild - type Arabidopsis thaliana and cotton to analyze the salt tolerance of transgenic materials .
Under salt stress , the germination rate of transgenic cotton progeny was higher than that of receptor strains , and the salt tolerance was improved .
The results showed that GhCBL8 - GhCBL1 and GhCBL8 could improve the salt tolerance of transgenic cotton . The results showed that GhCBL8 - GhCBL1 was located in the nucleus . The results showed that GhCBL8 - GhCBL8 was located in nucleus .

【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S562
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本文編號(hào)：1832165