本文選題：油菜 + LPAT4/5基因��； 參考：《湖南農業(yè)大學》2015年碩士論文

【摘要】：油菜(Brassica napus)——全球重要經濟作物,食用植物油及優(yōu)質蛋白的主要來源。數(shù)據(jù)表明,就單位面積產油量而論,油菜油脂含量每提高1個百分點,相當于增產菜籽2.5個百分點,故而提高菜籽含油量是提升生產效益的有效途徑。LPAT為三酰甘油組裝過程中的一個關鍵酶,能影響脂質生物合成。提高LPAT酶活性能夠降低合成抑制作用。因此,通過基因工程手段克隆LPAT基因,并分析其功能,對研究菜籽含油量具有一定的實踐價值。主要實驗結論匯總如下：1.采用同源克隆方法,獲得2條LPAT4基因全長CDS序列,長度依次為1143 bp和1140 bp。生物信息學分析顯示,它們都包含LPLAT_LCLAT1樣結構域,是LPLAT超基因家族中的成員,命名為BnLPAT4-1和BnLPAT4-2。2.同源克隆得到BnLPAT4啟動子序列,啟動子元件分析顯示其可能參與光、缺氧、逆境脅迫、真菌、脫落酸、赤霉素、水楊酸、乙烯等誘導和莖尖特異性表達及晝夜節(jié)律調控。pBnLPAT4啟動子介導GUSA基因擬南芥異源表達顯示,該啟動子介導的基因主要表達部位為幼苗、根、莖、葉、花瓣、花萼、花托及果莢,而花藥及種子中未檢測到啟動子激活。3. BnLPAT4-1和BnLPAT4-2為普遍型表達基因,BnPAT4-1和BnPAT4-2的最高表達分別在葉和胚中。逆境分析顯示,NaCl、PEG4000、水漬、6-BA和ABA脅迫下,BnLPAT4-1和BnLPAT4-2呈現(xiàn)不同的表達模式。極差分析表明,ABA對BnLPAT4-1影響較大,而BnLPAT4-2卻對PEG4000敏感。4. BnLPAT4-1和BnLPAT4-2酵母異源表達顯示,該酶具備轉移酶活性。轉BnLPAT4-1菌株,66.88%為飽和脂肪酸,是不飽和脂肪酸的2.89倍,較YES2.0轉化子飽和脂肪酸25.62%,凈增長1.61倍。其中硬脂酸比例最高,為50.60%。BnLPAT4-2轉化子顯著提高了C16脂肪酸(棕櫚酸及棕櫚油酸)的比例。較YES2.0轉化子C16脂肪酸25.62%凈增長22.40%,提高70%。5. napin啟動子介導BnLPAT4-1在擬南芥T3代種子中表達結果顯示,BnLPAT4-1能夠降低芥酸、飽和脂肪酸以及長鏈多不飽和脂肪酸含量,且特異性地提高花生一烯酸含量。6.同源克隆湘油15 LPAT5全長CDs序列,其長度是1041bp,命名BnLPAT5。序列分析表明它擁有LPLAT超基因家族的特征,是此基因家族中的一員。7. BnLPAT5為普遍型表達基因,水漬、NaCl及PEG4000處理下,BnLPAT5呈“驟降緩升”的表達模式,3h時,達到最低表達量。對于ABA和6BA的處理,BnLPAT5呈“緩降緩升”的表達模式,12h時,表達量最低。8. BnLPAT5酵母表達顯示其表達產物具備轉移酶活性。轉BnLPAT5菌株,含不飽和脂肪酸85.73%,是飽和脂肪酸的5.82倍。亞油酸和油酸的比例之和約占總比例的85%,符合健康油標準。BnLPAT5對油酸和亞油酸具有偏好性,底物選擇的順序依次為：亞油酸油酸。9. napin啟動子介導BnLPAT5在擬南芥T3代種子中表達表明,BnLPAT5能夠抑制擬南芥LPAT對芥酸、花生酸、花生二烯酸及花生三烯酸的選擇,且不同株系BnLPAT5選擇性呈現(xiàn)出差異,但均能提高長鏈低不飽和脂肪酸的比例。
[Abstract]:Brassica napusus is the main source of edible vegetable oil and high quality protein in the world. In terms of oil production per unit area, the oil content of rape increased by 1 percentage point, which was equivalent to 2.5 percentage points of rapeseed yield. Therefore, increasing the oil content of rapeseed is an effective way to improve the efficiency of production. LPAT is a key enzyme in triglyceride assembly, which can affect lipid biosynthesis. Increasing the activity of LPAT could reduce the inhibition of synthesis. Therefore, cloning and analyzing the function of LPAT gene by genetic engineering has certain practical value for the study of rapeseed oil content. The main experimental conclusions are summarized as follows: 1: 1. By using homologous cloning method, two full-length CDS sequences of LPAT4 gene were obtained, the length of which was 1143 BP and 1140 BP, respectively. Bioinformatics analysis shows that they all contain LPLAT_LCLAT1 like domains and are members of LPLAT supergene family named BnLPAT4-1 and BnLPAT4-2.2. Homologous cloning of BnLPAT4 promoter sequence showed that the promoter might be involved in light, hypoxia, stress, fungi, abscisic acid, gibberellin and salicylic acid. Ethylene induction and stem tip specific expression and circadian regulation. PBnLPAT4 promoter mediated the heterologous expression of GUSA gene in Arabidopsis thaliana. The main sites of the promoter gene expression were seedlings, roots, stems, leaves, petals, calyx, receptacle and fruit pods. However, no promoter activation. 3 was detected in anthers and seeds. BnLPAT4-1 and BnLPAT4-2 were the highest expression of BnPAT4-1 and BnPAT4-2 in leaves and embryos, respectively. Stress analysis showed that NaClPAT4000. BnLPAT4-1 and BnLPAT4-2 showed different expression patterns under the stress of 6-BA and ABA. Range analysis showed that ABA had a great effect on BnLPAT4-1, while BnLPAT4-2 was sensitive to PEG4000. 4. The heterologous expression of BnLPAT4-1 and BnLPAT4-2 showed that the enzyme had the activity of transferase. 66.88% of BnLPAT4-1 strain was saturated fatty acid, 2.89 times of unsaturated fatty acid, which was 1.61 times higher than that of YES2.0 transformant 25.62%. The ratio of stearic acid was the highest, and the ratio of C 16 fatty acid (palmitic acid and palmitic oil acid) was significantly increased by 50.60%.BnLPAT4-2 transformant. Compared with the YES2.0 transformant C16 fatty acid 25.62%, the net increase was 22.4040%, and increased 70%. 5. The expression of BnLPAT4-1 mediated by napin promoter in T3 generation seeds of Arabidopsis thaliana showed that BnLPAT4-1 could reduce the contents of erucic acid, saturated fatty acid and long chain polyunsaturated fatty acids. The content of arachidonic acid. The homologous clone Xiangyou 15 LPAT5 was named BnLPAT5 with a length of 1041bp. Sequence analysis shows that it has the characteristics of LPLAT supergene family and is a member of this gene family. BnLPAT5 is a universal expression gene. The lowest expression level of BnLPAT5 can be reached at 3 h after treatment with PEG4000 and WBnLPAT5. For the treatment of ABA and 6BA, the expression level of BnLPAT5 was the lowest when the expression pattern of BnLPAT5 was "slowly descending and rising slowly" for 12h. The expression of BnLPAT5 yeast showed that its expression product had the activity of transferase. The BnLPAT5 strain contained 85. 73% unsaturated fatty acids, 5.82 times of saturated fatty acids. The ratio of linoleic acid to oleic acid is about 85% of the total, which meets the healthy oil standard. BnLPAT5 has a preference for oleic acid and linoleic acid. The order of substrate selection was linoleic acid .9.The expression of BnLPAT5 in Arabidopsis thaliana T3 seeds mediated by napin promoter showed that BnLPAT5 could inhibit the selection of erucic acid, arachidonic acid and arachidonic acid by LPAT in Arabidopsis thaliana. The BnLPAT5 selectivity of different lines was different, but all of them could increase the proportion of long chain low unsaturated fatty acids.
【學位授予單位】：湖南農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S565.4

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