本文選題：萬壽菊 + 番茄紅素β-環(huán)化酶基因�。� 參考：《中國(guó)農(nóng)業(yè)科學(xué)》2017年24期

【摘要】：【目的】克隆萬壽菊番茄紅素β-環(huán)化酶基因和其啟動(dòng)子,進(jìn)行生物學(xué)信息分析,并預(yù)測(cè)啟動(dòng)子功能,為萬壽菊類胡蘿卜素的代謝機(jī)制和葉黃素含量的調(diào)控提供參考依據(jù)。【方法】通過RT-PCR技術(shù)從萬壽菊總RNA中克隆得到TeLCYb的cDNA序列;應(yīng)用生物學(xué)方法分析其DNA序列及其編碼蛋白質(zhì)序列特征,使用DNAMAN和在線軟件Box Shade對(duì)其氨基酸序列同源性比對(duì)分析,并利用MEGA6.0構(gòu)建進(jìn)化樹,分析其親緣關(guān)系;根據(jù)其cDNA序列利用FPNI-PCR法克隆其啟動(dòng)子序列,利用Plant Care在線數(shù)據(jù)庫(kù)分析萬壽菊TeLCYb啟動(dòng)子調(diào)控元件,構(gòu)建啟動(dòng)子缺失表達(dá)載體pTeLCYb(-1969)∷GUS和pTeLCYb(-1140)∷GUS,利用農(nóng)桿菌介導(dǎo)法侵染煙草,以GUS為報(bào)告基因研究不同調(diào)控元件的活性。【結(jié)果】從萬壽菊中成功克隆TeLCYb,生物信息學(xué)分析發(fā)現(xiàn),其全長(zhǎng)共1 865 bp,開放閱讀框?yàn)? 527 bp,編碼508個(gè)氨基酸,氨基酸序列同源比對(duì)結(jié)果表明其與西洋蒲公英和菊花的同源性最高,且具有LCYb蛋白質(zhì)特有的保守功能位點(diǎn)和特征多肽序列,在進(jìn)化上與菊科單獨(dú)聚為一枝。在已知基因序列的基礎(chǔ)上獲得長(zhǎng)度為1 806 bp的TeLCYb啟動(dòng)子序列,生物學(xué)信息分析表明:該啟動(dòng)子除包含核心啟動(dòng)子元件TATA-box、CAAT-box外,還含有多種光應(yīng)答元件和激素響應(yīng)元件,其中光響應(yīng)元件有13個(gè),激素響應(yīng)元件有5個(gè),此外還具有MYBHv1結(jié)合位點(diǎn)元件、耐熱響應(yīng)元件、生理調(diào)控作用元件等順式調(diào)控元件。GUS化學(xué)組織染色結(jié)果顯示不同長(zhǎng)度啟動(dòng)子均能夠驅(qū)動(dòng)GUS在莖、葉、花藥及柱頭中表達(dá),但不同組織GUS染色藍(lán)色斑點(diǎn)深度不同,其中花器官中花藥和柱頭中GUS酶活性最強(qiáng);相對(duì)于啟動(dòng)子pTeLCYb(-1969),pTeLCYb(-1140)啟動(dòng)子還能夠驅(qū)動(dòng)GUS在根、葉和花萼中特異性表達(dá),且各組織中GUS酶活性明顯強(qiáng)于啟動(dòng)子pTeLCYb(-1969)的GUS化學(xué)組織染色結(jié)果�！窘Y(jié)論】不同長(zhǎng)度TeLCYb啟動(dòng)子均能驅(qū)動(dòng)下游GUS的表達(dá),但啟動(dòng)子的作用部位和作用強(qiáng)度存在差異,推測(cè)在ATG上游1 140 bp和164 bp區(qū)間的光響應(yīng)元件可能具有增強(qiáng)子的功能,而全長(zhǎng)啟動(dòng)子特有的激素響應(yīng)元件和熱響應(yīng)元件可能具有抑制或降低啟動(dòng)子功能的作用。
[Abstract]:[objective] to clone lycopene 尾 -cyclase gene and its promoter of Tagetes marigold, analyze the biological information and predict the function of the promoter. [methods] the cDNA sequence of TeLCYb was cloned from total RNA of marigold by RT-PCR. The DNA sequence and its coding protein sequence were analyzed by biological method. The amino acid sequence homology was analyzed by DNAMAN and box Shade, and the phylogenetic relationship was analyzed by using MEGA 6.0 to construct an evolutionary tree. According to its cDNA sequence, the promoter sequence was cloned by FPNI-PCR, and the promoter regulator element of TeLCYb promoter was analyzed by Plant Care online database. The promoter-deletion expression vectors pTeLCYb-1969) -GUS and pTeLCYb-1140) -GUSwere constructed, and Agrobacterium tumefaciens were used to infect tobacco. The activity of different regulatory elements was studied by using Gus as a reporter gene. [results] TeLCYb was cloned successfully from marigold. Bioinformatics analysis showed that the total length of TeLCYb was 1 865 BP, and the open reading frame was 1 527 BP, encoding 508 amino acids. The results of amino acid sequence homology alignment showed that it had the highest homology with dandelion and chrysanthemum, and had the unique conserved functional site and characteristic polypeptide sequence of LCYb protein. A 1 806 BP TeLCYb promoter sequence was obtained on the basis of the known gene sequence. The biological information analysis showed that the promoter contained not only the core promoter TATA-box CAAT-box, but also a variety of light responders and hormone response elements. Among them, there are 13 light response elements, 5 hormone response elements, and MYBHv1 binding site element, heat resistant response element. The expression of Gus in stem, leaf, anther and stigma were all driven by different length promoters, but the depth of blue spots in different tissues was different. The activity of Gus in anther and stigma was the highest in floral organs, and the specific expression of Gus in root, leaf and calyx was also driven by the promoter pTeLCYb-1969pTeLCYb-1140). The activity of Gus was stronger than that of pTeLCYb-1969. [conclusion] different lengths of TeLCYb promoter can drive the expression of Gus downstream, but the site and intensity of the promoter are different. It is inferred that the photoresponse elements in the upper reaches of ATG (1 140 BP and 164 BP) may have the function of enhancer, while the hormone response element and the thermal response element which are specific to the full-length promoter may inhibit or decrease the function of the promoter.
【作者單位】： 華中農(nóng)業(yè)大學(xué)園藝林學(xué)學(xué)院/園藝植物生物學(xué)教育部重點(diǎn)實(shí)驗(yàn)室/農(nóng)業(yè)部華中都市農(nóng)業(yè)重點(diǎn)實(shí)驗(yàn)室(試運(yùn)行);
【基金】：國(guó)家自然科學(xué)基金(31672181)
【分類號(hào)】：S681.9

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