【摘要】：甜菜堿是一種無(wú)毒滲透保護(hù)劑,在非生物脅迫條件下大量積累來(lái)維持細(xì)胞的滲透壓,保證植物的正常生長(zhǎng)。磷酸乙醇胺氮甲基轉(zhuǎn)移酶(phosphoethanolamine methyltransferase,PEAMT)作為甜菜堿合成的關(guān)鍵酶,其基因表達(dá)受上游啟動(dòng)子的調(diào)節(jié)。目前關(guān)于玉米(Zea mays L.)內(nèi)源PEAMT基因啟動(dòng)子的研究較少。本研究以玉米自交系B73(cv.B73)為材料,根據(jù)NCBI數(shù)據(jù)庫(kù)(national center for biotechnology information)中玉米PEAMT基因序列設(shè)計(jì)引物,利用普通聚合酶鏈?zhǔn)椒磻?yīng)(polymerase chain reaction,PCR)克隆其上游5′側(cè)翼序列。通過(guò)構(gòu)建含有玉米PEAMT啟動(dòng)子全長(zhǎng)和缺失片段融合β-葡萄糖苷酸酶報(bào)告基因(GUS)的表達(dá)載體并轉(zhuǎn)化煙草(Nicotiana benthamiana L.),利用組織化學(xué)染色和GUS酶活性分析確定PEAMT基因啟動(dòng)子全長(zhǎng)和不同缺失片段響應(yīng)干旱(15%PEG-6000,m/v)、鹽(100 mmol·L-1 NaCl)、低溫(4℃)和氧化脅迫(100 mmol·L-1 H2O2)等非生物脅迫的能力。主要研究結(jié)果如下:1.根據(jù)NCBI數(shù)據(jù)庫(kù)玉米PEAMT基因序列設(shè)計(jì)引物,克隆了PEAMT啟動(dòng)子序列,序列長(zhǎng)度為1048 bp(序列信息詳見(jiàn)附錄一)。利用在線預(yù)測(cè)軟件Promoter prediciton、PLACE和PlantCare預(yù)測(cè)出該序列中存在17個(gè)非脅迫應(yīng)答順式作用元件、13個(gè)光誘導(dǎo)信號(hào)轉(zhuǎn)導(dǎo)元件、9個(gè)激素應(yīng)答順式作用元件以及5個(gè)花粉發(fā)育特異性激活元件等。2.構(gòu)建了PEAMT基因啟動(dòng)子融合GUS報(bào)告基因表達(dá)載體并轉(zhuǎn)化煙草,發(fā)現(xiàn)克隆得到的PEAMT基因啟動(dòng)子能夠驅(qū)動(dòng)GUS基因的高效表達(dá)。經(jīng)過(guò)GUS酶活性分析,PEAMT基因啟動(dòng)子呈正調(diào)控表達(dá)模式,在干旱、鹽、低溫和氧化脅迫下的活性分別為27.4、39.9、23.5和19.7 nmol 4-MU·min-1·mg-1 protein,與對(duì)照(3.44 nmol 4-MU·min-1·mg-1protein)相比分別提高了1.64倍、2.84倍、1.47倍和3.59倍。3.進(jìn)一步對(duì)該啟動(dòng)子進(jìn)行5′端缺失分析,克隆了4種不同長(zhǎng)度的5′端缺失片段,序列長(zhǎng)度分別為826 bp(P2)、642 bp(P3)、428 bp(P4)和245 bp(P5)。將這些啟動(dòng)子片段與GUS報(bào)告基因融合構(gòu)建表達(dá)載體,獲得含有不同缺失啟動(dòng)子片段的陽(yáng)性轉(zhuǎn)化植株。GUS組織化學(xué)染色結(jié)果表明,4種啟動(dòng)子缺失片段均具有表達(dá)活性。GUS酶活性結(jié)果表明,不同類(lèi)型的非生物脅迫均能誘導(dǎo)不同缺失片段啟動(dòng)子活性的增加。其中,P3片段啟動(dòng)子在干旱和低溫脅迫條件下的表達(dá)量均相對(duì)較高,分別為37.18和19.76 nmol4-MU·min-1·mg-1 protein,與對(duì)照相比分別增加了4.76倍和5.33倍;P4片段啟動(dòng)子在鹽脅迫處理下的表達(dá)量較高,為36.46 nmol 4-MU·min-1·mg-1 protein,與對(duì)照相比增加了6.12倍;P2片段啟動(dòng)子在氧化脅迫下的表達(dá)量較高,為20.50 nmol 4-MU·min-1·mg-1protein,比對(duì)照增加了7.51倍。由此可見(jiàn),不同缺失片段對(duì)以上4種非生物脅迫的響應(yīng)程度存在差異,其中P3片段啟動(dòng)子對(duì)多種非生物脅迫均較為敏感。本文初步揭示了玉米PEAMT基因啟動(dòng)子的序列特征和表達(dá)特點(diǎn),該啟動(dòng)子具有響應(yīng)非生物脅迫的能力,且呈現(xiàn)正調(diào)控的表達(dá)模式,這為進(jìn)一步深入研究PEAMT基因的表達(dá)調(diào)控提供了理論基礎(chǔ)。
[Abstract]:Betaine is a kind of nontoxic osmotic protective agent, which accumulates under abiotic stress to maintain the osmotic pressure of cells and ensure the normal growth of plants. Phosphoethanolamine methyltransferase (PEAMT) is a key enzyme in betaine synthesis, and its gene expression is regulated by upstream promoter. Maize (Zea mays L.) There are few studies on endogenous PEAMT gene promoter. In this study, maize inbred line B73 (cv.B73) was used as material, primer was designed according to the sequence of maize PEAMT gene in NCBI database (national center for biotechnology information), and its upstream 5 'flanking sequence was cloned by ordinary polymerase chain reaction (polymerase chain reaction). The full length and deletion fragment of Maize PEAMT promoter was constructed and transformed into tobacco (Nicotiana benthamiana L.) by fusion of 尾 -glucuronidase reporter gene (Gus). The total promoter of PEAMT gene was determined by histochemical staining and Gus activity analysis. The long and different missing fragments were able to respond to abiotic stresses such as drought (15PEG-6000 m / v), salt (100 mmol L-1 NaCl), low temperature (4 鈩，

本文編號(hào)：2135985