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人參bZIP基因家族生物信息學分析

2022-05-06 07:26王思嘉孫嘉瑩劉美琦任偉超于欣欣劉秀波
中草藥 2022年9期
關鍵詞:細胞核擬南芥人參

王思嘉,孫嘉瑩,劉美琦,任偉超,于欣欣,劉秀波,馬 偉, 3*

人參基因家族生物信息學分析

王思嘉1,孫嘉瑩1,劉美琦1,任偉超1,于欣欣1,劉秀波2*,馬 偉1, 3*

1.黑龍江中醫藥大學藥學院,黑龍江哈爾濱 150040 2.黑龍江中醫藥大學佳木斯學院,黑龍江佳木斯 154007 3.教育部北藥基礎與應用研究重點實驗室,黑龍江哈爾濱 150040

通過人參基因家族生物信息學分析,為人參功能基因開發利用提供理論依據。運用PlantTFDB數據庫預測人參基因組中基因;通過ExPASy網站得到人參基因家族信息和特征;使用MEME網站對基因家族保守基因序列進行分析;運用MEGA軟件建立基因家族系統進化關系;利用TBtools軟件進行表達分析。人參基因組中含有157個基因家族成員,其中152個定位在細胞核,其余5個分別定位在葉綠體和內質網,相對分子質量在14 009.93~83 440.38,等電點在4.53~10.05,氨基酸數目在120~760 aa,除以外,均為親水蛋白。在干旱脅迫下的表達量為83.47;在根和鹽脅迫條件下表達量均最高,分別為119.82和117.86;在葉中表達量為48.54;花中表達量最高為79.55;成熟果實中表達量最高是,為119.32;在未成熟果實中的表達量最高,且為65.32。推測與同源基因功能相似,可以調控根的分生組織活性;在干旱脅迫條件下表達量最高,推測對干旱脅迫有重要調控作用;A亞族成員/()、()、/()參與干旱脅迫和鹽脅迫調控,推測與A亞族成員具有同源關系的、、、在干旱脅迫和鹽脅迫條件下具有相似調控作用。

人參;;生物信息學;基因家族;鹽脅迫;干旱脅迫

作為成員眾多的轉錄因子家族,在植物領域研究較為透徹[1]。該基因家族對植物的生長發育有重要調節作用,參與植物次生代謝和非生物脅迫的調控[2-3]。通過前人研究成果發現,在擬南芥、陸地棉、花生、小麥、番茄等植物中分別含有75、151、112、156、70個基因成員[4-8]。前期研究將基因家族分為10個亞族[9],分為A、B、C、D、E、F、G、H、I和S。其中,A亞族的和參與應激響應脫落酸(ABA)信號[10],可激活多個晚期胚胎發育[11];C亞族中的、與共同表達可調控種子特異性[12];在D亞族中,確定花器官數目,并參與莖部和花分生組織的表達[13];E亞族中和通過其他基因家族成員相互作用參與植株發育[14]。除此之外,在芹菜中發現基因在干旱、低溫、鹽處理條件下的表達量不同,并隨時間不同發生變化[15];蒺藜苜蓿中基因參與鹽脅迫,和均參與干旱脅迫[16]。

人參C.A.Meyer是五加科、人參屬多年生草本植物[17],在明代《理虛元鑒》中提到其具有大補元氣、復脈固脫之效[18],所以對人體機能恢復有一定作用。人參用于治療神經衰弱和抑郁型精神病,也可用于治療冠心病[19],且人參皂苷Rg1有保護糖尿病大鼠腎臟的作用[20],人參皂苷Rg3與紫杉醇結合可抑制腫瘤細胞增長[21],如Luminal型乳腺癌[22]。同時,人參的一些活性成分在美容領域有一定研究[23]。近期研究表明影響人參皂苷的合成[24],可見,人參在臨床上具有較高的藥用價值且應用廣泛。

本研究利用了生物信息學技術,對人參基因家族成員進行鑒定,分析基因家族信息、系統進化、保守基序以及基因表達等,為深入研究人參轉錄因子在干旱、鹽、低溫等協迫條件下的調控作用提供理論基礎。

1 材料

人參基因組序列文件下載自人參基因組數據庫(ginseng genome database,http://ginsengdb.snu.ac.kr/data.php),擬南芥基因家族來自TAIR數據庫(http://www.arabidopsis.org/index.jsp)。

2 方法

2.1 人參bZIP基因家族的鑒定

使用生信軟件TBtools(v1.086)提取人參全部基因序列,將得到的蛋白序列文件提交plantTFDB數據庫(http://planttfdb.cbi.pku.edu.cn/)進行預測,得到人參基因家族成員,用TBtools軟件去除重復轉錄本,并利用Expasy網站(https://web.expasy.org/ protparam/)獲取人參的等點量、相對分子質量、蛋白質疏水性、不穩定系數、脂肪族系數等信息,利用WoLFPSORT (https://wolfpsort.hgc.jp/)對人參基因家族成員進行亞細胞定位。

2.2 人參bZIP保守基序分析

使用MEME網站(http://meme-suite.org/tools/ meme),將人參bZIP家族成員蛋白序列的文件進行保守基序預測,最大保守基序數目設為10,其余條件默認,并使用TBtools軟件進行可視化。

2.3 人參bZIP基因家族的系統進化

把人參bZIP蛋白序列與擬南芥bZIP蛋白序列整合,將所得文件使用MUSCLE程序進行多序列比對,利用Mega-X軟件采用鄰接法(neighbor-joining,NJ),Bootstrap值設置為1000次重復,模型選擇Poisson model,構建系統發育樹。

2.4 人參bZIP基因表達分析

根據人參的等基因數據,在TBtools中繪制熱圖,進一步對人參基因家族進行表達分析。

3 結果與分析

3.1 人參bZIP基因家族信息和特征

根據人參基因家族的信息(表1)可知,氨基酸數目在120 aa(PgbZIP19)~760 aa(PgbZIP22),相對分子質量的變化范圍為14 009.93(PgbZIP19)~83 440.38(PgbZIP22),等電點的范圍介于4.53~10.05(PgbZIP76~PgbZIP4),亞細胞定位顯示除PgbZIP5、PgbZIP11、PgbZIP79、PgbZIP116、PgbZIP130以外均定位在細胞核,PgbZIP5、PgbZIP116定位在葉綠體,PgbZIP11、PgbZIP79、PgbZIP130定位在內質網;蛋白質疏水性(GRAVY)除PgbZIP157外均為負值;PgbZIP5、PgbZIP115、PgbZIP126的不穩定系數小于40,為穩定蛋白,其余為不穩定蛋白。

表1 人參bZIP基因家族信息和特征

Table 1 Information and characteristics of ginseng bZIP gene family

基因名稱基因序列不穩定系數氨基酸數量相對分子質量等電點蛋白質疏水性亞細胞定位脂肪族系數 PgbZIP1Pg_S0015.258.5142047 358.916.49?0.838細胞核67.62 PgbZIP2Pg_S0054.554.5340746 070.726.10?0.814細胞核71.43 PgbZIP3Pg_S0157.158.8332636 432.058.65?0.489細胞核86.01 PgbZIP4Pg_S0161.2961.5320421 865.7510.05?0.750細胞核61.23 PgbZIP5Pg_S0185.134.0616718 520.479.85?0.278葉綠體87.07 PgbZIP6Pg_S0227.3247.5136340 718.146.94?0.886細胞核57.36 PgbZIP7Pg_S0266.1766.6237040 533.445.57?0.791細胞核61.03 PgbZIP8Pg_S0270.157.8445549 459.836.20?0.753細胞核64.15 PgbZIP9Pg_S0292.4145.3833737 695.697.75?0.736細胞核74.07 PgbZIP10Pg_S0431.2552.7333036 872.618.34?0.814細胞核69.73 PgbZIP11Pg_S0431.545.9475682 233.396.04?0.632內質網63.43 PgbZIP12Pg_S0447.753.3338341 604.956.24?0.840細胞核58.36 PgbZIP13Pg_S0500.3441.0020123 151.389.52?0.040細胞核97.01 PgbZIP14Pg_S0602.2541.5736140 739.216.09?0.419細胞核83.88 PgbZIP15Pg_S0624.1961.2415517 986.097.91?1.008細胞核64.84 PgbZIP16Pg_S0659.1055.9946051 724.917.06?0.482細胞核75.57 PgbZIP17Pg_S0662.3860.8150455 852.426.88?0.586細胞核74.42 PgbZIP18Pg_S0662.4051.2933137 878.636.37?0.888細胞核64.20 PgbZIP19Pg_S0662.4355.7712014 009.939.64?0.828細胞核70.75 PgbZIP20Pg_S0684.2852.3331635 490.074.63?0.456細胞核88.83 PgbZIP21Pg_S0687.150.7016918 223.945.94?0.691細胞核64.67 PgbZIP22Pg_S0741.1645.6376383 440.386.28?0.487細胞核75.48 PgbZIP23Pg_S0741.3858.4643047 802.186.85?0.658細胞核69.70 PgbZIP24Pg_S0745.2167.2937642 193.557.82?0.891細胞核63.40 PgbZIP25Pg_S0762.1964.2937640 738.815.72?0.649細胞核65.48 PgbZIP26Pg_S0788.342.2213315 153.859.57?0.377細胞核87.29 PgbZIP27Pg_S0872.672.6418320 453.586.59?0.997細胞核51.86 PgbZIP28Pg_S0884.151.7628731 741.486.67?0.206細胞核79.16 PgbZIP29Pg_S0889.6139.6525527 585.928.95?0.634細胞核76.94 PgbZIP30Pg_S0905.134.6127529 893.275.86?0.683細胞核64.18 PgbZIP31Pg_S0938.951.1029533 135.474.72?0.467細胞核86.88 PgbZIP32Pg_S0967.2773.7025228 878.759.91?0.616細胞核73.45 PgbZIP33Pg_S0981.1549.6640142 806.986.04?0.818細胞核51.17 PgbZIP34Pg_S0992.2254.1859765 001.448.66?0.727細胞核61.68 PgbZIP35Pg_S1022.244.7219921 669.626.15?0.778細胞核65.68 PgbZIP36Pg_S1055.2752.2729033 092.188.72?0.890細胞核63.55 PgbZIP37Pg_S1084.852.5445449 502.749.53?0.649細胞核72.80 PgbZIP38Pg_S1097.3269.3914316 053.836.96?0.694細胞核55.24 PgbZIP39Pg_S1144.256.8543546 958.299.11?0.694細胞核65.49 PgbZIP40Pg_S1164.663.1117220 021.326.16?0.836細胞核77.15 PgbZIP41Pg_S1176.1459.0241243 666.805.86?0.942細胞核46.04 PgbZIP42Pg_S1222.150.0333738 200.436.03?0.433細胞核83.18 PgbZIP43Pg_S1241.249.2813515 270.995.91?0.696細胞核57.78 PgbZIP44Pg_S1242.2368.0833837 650.935.89?0.715細胞核67.87 PgbZIP45Pg_S1290.3562.9144148 732.357.15?0.655細胞核68.82 PgbZIP46Pg_S1338.2056.0458965 153.926.35?0.527細胞核76.53 PgbZIP47Pg_S1348.1064.9336339 935.995.82?0.796細胞核64.10 PgbZIP48Pg_S1349.160.0013215 826.019.46?0.883細胞核74.55 PgbZIP49Pg_S1389.3351.3040843 739.046.19?0.884細胞核49.80

續表1

基因名稱基因序列不穩定系數氨基酸數量相對分子質量等電點蛋白質疏水性亞細胞定位脂肪族系數 PgbZIP50Pg_S1420.156.6046050 405.539.41?0.707細胞核71.17 PgbZIP51Pg_S1456.247.4926728 784.606.24?0.675細胞核64.31 PgbZIP52Pg_S1457.1359.9629433 033.726.27?0.906細胞核61.77 PgbZIP53Pg_S1511.260.7139744 477.497.71?0.577細胞核80.83 PgbZIP54Pg_S1514.651.1429532 949.184.66?0.474細胞核86.54 PgbZIP55Pg_S1544.549.3135039 660.196.68?0.405細胞核88.69 PgbZIP56Pg_S1552.2561.4134537 802.025.59?0.731細胞核67.39 PgbZIP57Pg_S1563.1855.9418821 978.335.88?0.973細胞核64.84 PgbZIP58Pg_S1565.159.0034338 577.779.00?0.678細胞核79.91 PgbZIP59Pg_S1583.1460.7416818 710.599.69?1.162細胞核52.92 PgbZIP60Pg_S1622.461.6929232 892.646.27?0.887細胞核62.53 PgbZIP61Pg_S1649.248.0935039 141.396.57?0.847細胞核60.60 PgbZIP62Pg_S1650.555.0020823 519.519.54?0.853細胞核69.47 PgbZIP63Pg_S1658.2046.0748052 700.005.62?0.471細胞核74.83 PgbZIP64Pg_S1668.1455.7217220 028.355.62?0.734細胞核79.42 PgbZIP65Pg_S1678.943.5416819 224.617.99?0.878細胞核58.86 PgbZIP66Pg_S1679.4154.5615517 984.116.52?0.954細胞核66.71 PgbZIP67Pg_S1693.2669.1521024 377.308.54?0.851細胞核67.24 PgbZIP68Pg_S1717.661.7142446 545.546.13?0.793細胞核64.25 PgbZIP69Pg_S1721.958.4451256 857.386.81?0.516細胞核73.01 PgbZIP70Pg_S1800.161.3735739 424.489.07?0.437細胞核84.31 PgbZIP71Pg_S1864.159.7149855 340.856.86?0.524細胞核73.45 PgbZIP72Pg_S1869.757.8350855 745.978.39?0.507細胞核76.22 PgbZIP73Pg_S1900.367.6815817 294.219.64?1.116細胞核64.24 PgbZIP74Pg_S1915.1751.3544148 531.729.72?0.723細胞核67.71 PgbZIP75Pg_S1933.569.9235540 525.187.32?0.885細胞核68.73 PgbZIP76Pg_S1993.2851.7021824 386.154.53?0.621細胞核88.01 PgbZIP77Pg_S2000.1168.2646950 995.106.43?1.158細胞核45.20 PgbZIP78Pg_S2014.560.4917520 042.415.72?0.786細胞核61.43 PgbZIP79Pg_S2057.2546.0975281 850.906.36?0.657內質網63.51 PgbZIP80Pg_S2057.353.6533036 880.537.10?0.808細胞核70.61 PgbZIP81Pg_S2086.353.4237041 530.805.96?0.751細胞核79.95 PgbZIP82Pg_S2109.144.6326728 731.586.71?0.633細胞核64.31 PgbZIP83Pg_S2176.163.2024827 929.969.20?1.026細胞核54.84 PgbZIP84Pg_S2176.266.7623726 141.146.35?0.705細胞核62.32 PgbZIP85Pg_S2207.2453.1616218 637.776.29?0.916細胞核61.48 PgbZIP86Pg_S2245.4137.0529732 562.615.74?0.513細胞核71.85 PgbZIP87Pg_S2306.247.7047151 983.506.40?0.490細胞核75.20 PgbZIP88Pg_S2466.160.0217719 957.388.38?0.802細胞核72.66 PgbZIP89Pg_S2496.2052.9849753 511.616.40?0.710細胞核63.08 PgbZIP90Pg_S2533.261.0315817 289.108.96?1.124細胞核59.94 PgbZIP91Pg_S2631.1035.4927830 366.925.39?0.564細胞核73.67 PgbZIP92Pg_S2754.461.2535639 287.319.07?0.423細胞核86.74 PgbZIP93Pg_S2757.562.8116117 636.529.68?1.115細胞核59.44 PgbZIP94Pg_S2874.474.0135540 591.358.48?0.872細胞核71.21 PgbZIP95Pg_S2936.2468.6837541 992.327.81?0.910細胞核62.53 PgbZIP96Pg_S2971.3358.3114016 833.149.89?1.040細胞核58.57 PgbZIP97Pg_S2990.265.3419822 967.385.64?0.968細胞核69.13 PgbZIP98Pg_S3098.2156.6242344 644.976.24?0.891細胞核45.56 PgbZIP99Pg_S3109.1757.2518721 425.135.72?0.710細胞核61.66 PgbZIP100Pg_S3138.2158.0025928 290.966.38?0.568細胞核73.40 PgbZIP101Pg_S3210.142.6430033 871.757.72?0.858細胞核64.07 PgbZIP102Pg_S3210.252.1033137 937.737.02?0.883細胞核64.17 PgbZIP103Pg_S3210.559.9457063 743.438.27?0.639細胞核73.18 PgbZIP104Pg_S3356.1451.3941043 461.267.82?0.768細胞核60.05 PgbZIP105Pg_S3402.152.1236140 840.446.62?0.445細胞核81.16 PgbZIP106Pg_S3475.156.4815417 766.859.00?0.882細胞核67.79 PgbZIP107Pg_S3530.959.4219822 184.806.12?0.586細胞核81.87 PgbZIP108Pg_S3584.373.9914316 078.926.96?0.677細胞核57.97

續表1

3.2 人參bZIP家族保守基序分析

使用MEME網站查找人參bZIP蛋白保守基序,可知PgbZIP的保守基序個數為1~6個。Motif9只在A亞族中存在一小部分;D亞族中普遍含有6個保守基序;Motif5只存在于I、E家族中;PgbZIP中均含有Motif1,且少數PgbZIP成員中只含有Motif1,見圖1。

3.3 人參bZIP基因家族的系統進化關系

將人參的157個bZIP蛋白和擬南芥中74個蛋白進行系統進化樹的構建(圖2),bZIP家族分為10個亞家族,分別為A、B、C、D、E、F、G、H、I和S,其中A亞族中包括33個人參bZIP成員;B亞族中只含有2個成員PgbZIP53、PgbZIP144;C亞族中包括12個PgbZIP成員;D亞族中含有22個PgbZIP成員;E亞族中含有15個PgbZIP成員;F亞族中含有4個PgbZIP成員:PgbZIP151、PgbZIP91、PgbZIP86、PgbZIP30;G亞族中含有8個PgbZIP成員;H亞族中含有10個PgbZIP成員;I亞族中含有16個成員;S亞族含有35個成員,是含有人參bZIP成員數最多的亞族。

圖1 人參bZIP蛋白基序以及Motif分析

圖2 人參和擬南芥bZIP轉錄因子的系統發育樹

3.4 人參bZIP基因在植物不同器官中的表達

通過TBtools軟件制作熱圖,對基因家族的表達進行分析。如圖3,表明、在成熟果實中表達量較高,表達最明顯,在未成熟果實中表達量最高;在花中表達最明顯;、在根中表達量較高;在根和鹽脅迫條件下中的表達量最高,在鹽脅迫下表達量較高;在干旱脅迫下表達量最高。

4 討論

bZIP是一類參與植物生長、生物與非生物脅迫的轉錄因子,通過圖2和圖3分析,人參中在根中的表達量最高,在莖和花中的表達量逐漸降低,與其同源的擬南芥基因具有抑制分生組織活性與根生長的功能[25],推測其在也具有負調控作用。()、()、()、()、()、()為擬南芥中TGA中的成員,相互作用參與植物疾病調控[26-27],推測其同源基因、、、也有相似的功能。

非生物脅迫包括干旱脅迫、鹽脅迫、低溫脅迫等[28],Yoshida等[29]發現bZIP轉錄因子對抗逆性有重要作用。在干旱脅迫條件下表達量最高,屬于S亞族,該亞族對非生物脅迫有較強響應,其中AtbZIP44在干旱脅迫中發揮重要作用[30],因與為同源基因,推測在人參中起到相似作用。

ABA在植物的生長發育中對環境脅迫發揮重要作用[31],其中擬南芥成員/()、()、/()主要參與ABA、鹽脅迫、干旱脅迫、熱脅迫等[32],成員、、與其為同源基因,且在鹽脅迫條件下表達較高,推測、、可能參與鹽脅迫調控。對鹽脅迫有較強反應[31],、在鹽脅迫條件下的表達量較高,推測其在人參鹽脅迫條件下發揮重要作用。Fujita等[32]發現擬南芥中的ABRE結合因子家族成員依賴ABA信號,增強植物營養組織的抗旱性。煙草研究在ABA的處理下使A亞族中的成員表達量上升[33],結合圖3發現成員、、、、、表達量在葉、花、干旱脅迫下或鹽脅迫有明顯增加,推測在ABA處理下也有相似功能,參與人參的生長發育。

圖3 人參bZIP基因在植物不同器官和非脅迫條件下的表達分析

通過人參基因家族的生物信息學分析,對可能參與人參非生物脅迫的基因進行功能預測,為深入研究基因提供分子基礎,為進一步探索人參次生代謝調控以及藥用活性成分臨床應用提供理論依據。

利益沖突 所有作者均聲明不存在利益沖突

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Bioinformatics analysis ofgene family of

WANG Si-jia1, SUN Jia-ying1, LIU Mei-qi1, REN Wei-chao1, YU Xin-xin1, LIU Xiu-bo2, MA Wei1, 3

1.College of Pharmaceutical Sciences, Heilongjiang University of Chinese Medicine, Harbin 150040, China 2.College of Jiamusi, Heilongjiang University of Chinese Medicine, Jiamusi 154007, China 3.Key Laboratory of Basic and Application Research of Beiyao (Heilongjiang University of Chinese Medicine), Ministry of Education, Harbin 150040, China

To analyzegene family bioinformatics of Renshen (), so as to provide theoretical basis for the development and utilization of ginseng functional genes.Using PlantTFDB database to predict ginsenggene; The genetic family information and characteristics of ginsengwere obtained through ExPASy website; The conserved gene sequences ofgene family were analyzed by MEME website; The phylogenetic relationship ofgene family were established by using MEGA software;expression analysis were conducted by TBtools software.The ginseng genome contained 157 bZIP gene family members, of which 152 were located in the nucleus, and the remaining five were located in the chloroplast and endoplasmic reticulum, respectively.The relative molecular mass was between 14 009.93 and 83 440.38, and the isoelectric point was in the range of 4.53—10.05, the number of amino acids was between 120 aa—760 aa, except for, all were hydrophilic proteins.The expression level ofunder drought stress was 83.47; the expression level ofwas the highest under root (119.82) and salt stress (117.86) conditions, respectively; the expression level ofin leaves was 48.54; the highest expression level ofin flowers was 79.55; The highest expression level ofin mature fruits was 119.32; the highest expression level ofin immature fruits was 65.32.It is speculated thathas similar functions to the homologous gene, and can regulate root meristem activity;has the highest expression under drought stress conditions, and it is speculated thathas an important regulatory effect on drought stress; A subfamily members,,/is involved in the regulation of drought stress and salt stress.It is speculated that,,and, which have a homologous relationship with members of subfamily A, have similar regulatory effects under drought stress and salt stress.

C.A.Meyer;; bioinformatics; gene family; salt stress; drought stress

R282.12

A

0253 - 2670(2022)09 - 2786 - 09

10.7501/j.issn.0253-2670.2022.09.022

2021-11-09

黑龍江省“頭雁”團隊項目(黑龍江省頭雁行動領導小組文件 [2019] 5號);黑龍江中醫藥大學科研基金項目(中藥健康相關產品研發及產業化專項)(2019BJP06)

王思嘉,女,碩士研究生,研究方向為藥用植物生物工程研究。Tel: (0451)87266988 E-mail: 2077737519@qq.com

通信作者:馬 偉,研究員,博士生導師,主要從事藥用植物生物工程研究。Tel: (0451)87266988 E-mail: 88788891@qq.com

劉秀波,教授,碩士生導師,主要從事中藥資源與中藥化學。Tel: 13796353268 E-mail: 358270831@qq.com

[責任編輯 時圣明]

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