CN105949293A - 植物源高效转录激活功能域sac3及应用 - Google Patents
植物源高效转录激活功能域sac3及应用 Download PDFInfo
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Abstract
本发明属于生物技术领域，提供一种植物源高效转录激活功能域SAC3及应用，其氨基酸序列为DEEGCGTCFDFAENTEQKEFLDNWHFEDDFVDYAQFAVFDDTADSAWDVVNLEAFIDGDYSTSWELETIC。本发明从杉木的cDNA文库中分离得到一段高效转录激活序列SAC3，CPRG结果显示其转录激活功能在酵母中为VP16的19倍左右；以Gal4‑UAS为基础的双荧光素酶系统结果显示：在拟南芥中SAC3的转录激活活性为VP16的两倍以上、EDLL的50倍。SAC3可以应用于植物的基因工程技术中，用以精确激活植物体内特定基因的表达，改善植物与该特定基因相关的重要性状，大幅提高作物的经济价值。
Description
技术领域
本发明属于生物技术领域，具体涉及一种植物源高效转录激活功能域SAC3及应用。
背景技术
全球人口的日益膨胀和生存环境的改变，给农作物的经营和发展提出了极大的挑战。兴起于上世纪80年代的基因工程技术，是大幅改善农作物的产量及提高其对环境改变的适应性的强有力手段。基因技术在植物中有大规模的应用，常将外源基因直接随机整合入目标植物基因组中，并以此改善植物一些重要性状，如抗除草剂和抗虫性。然而这种方法可能会产生一些副作用，而且无法达到精确控制特定基因表达的效果。
拟转录激活因子效应器，Transcription activator-like effectors (TALEs)，是实现精确控制特定基因表达最有效和应用最广泛的技术；以及近年来CRISPR-Cas9（clustered regularly interspaced short palindromic repeat - CRISPR-associated9）系统的开发和应用，生物体的研究和改造带来巨大潜力，这类人工合成的转录调节因子为精确控制植物內源基因的表达提供了可靠的工具。然而由于其转录激活活性的发挥完全取决于转录激活功能域。迄今为止应用在基因工程中的效果显著的转录激活功能域基本来源于动物病毒（VP16），在植物中缺乏完全匹配的工作环境，使其功能发挥受到限制。已知的植物中的转录激活因子主要是对环境因素响应的蛋白，其中EDLL motif为已报道的最强植物源转录激活因子，该功能域的发现和运用让人们有理由相信植物中存在强大的转录激活功能域，但其转录激活功能与VP16相比依然很低，以酵母为筛选载体，本发明从杉木的cDNA文库中分离得到一段高效转录激活序列SAC3(Strong Activation domain from Chinafir-3)，CPRG结果显示其转录激活功能在酵母中为VP16的19倍左右；以Gal4-UAS为基础的双荧光素酶（LUC和REN）系统结果显示：在拟南芥中SAC3的转录激活活性为VP16的两倍以上、EDLL的50倍。SAC3可以应用于植物的基因工程技术中，用以精确激活植物体内特定基因的表达，改善植物与该特定基因相关的重要性状，大幅提高作物的经济价值。
发明内容
本发明的目的在于提供一种植物源高效转录激活功能域SAC3及应用，可以应用于植物的基因工程技术中，用以精确激活植物体内特定基因的表达，改善植物与该特定基因相关的重要性状，大幅提高作物的经济价值。
为实现上述目的，本发明采用如下技术方案：
植物源高效转录激活功能域SAC3，所述的SAC3的氨基酸序列为DEEGCGTCFDFAENTEQKEFLDNWHFEDDFVDYAQFAVFDDTADSAWDVVNLEAFIDGDYSTSWELETIC。
所述的植物源高效转录激活功能域SAC3在改善植物基因表达中的应用。
本发明的优点在于：本发明从杉木的cDNA文库中分离得到一段高效转录激活序列SAC3(Strong Activation domain from China fir-3)，CPRG结果显示其转录激活功能在酵母中为VP16的19倍左右；以Gal4-UAS为基础的双荧光素酶（LUC和REN）系统结果显示：在拟南芥中SAC3的转录激活活性为VP16的两倍以上、EDLL的50倍。SAC3可以应用于植物的基因工程技术中，用以精确激活植物体内特定基因的表达，改善植物与该特定基因相关的重要性状，大幅提高作物的经济价值。
附图说明
图1 TUP1转录激活因子筛选系统简图。
图2 杉木文库在酵母TUP1系统中筛选结果的自激活能力。
图3 SAC3转录激活功能域的确定。
图4植物中SAC3的转录激活能力。
具体实施方式
实施例1
1.杉木中转录激活因子的筛选
1.1载体的构建
本系统筛选载体以pGBKT7(Clontech)为背景，首先将酵母转录抑制因子构建入pGBKT7中GBD的C端，采用的构建方式为将线性化的pGBKT7（引物1：5’-TATGGCCATGGAGGCCGAATTCC-3’; 引物2：5’- TGCAGGTCCTCCTCTGAGATCAGC-3’）与TUP1(引物3：5’-TCAGAGGAGGACCTGCATATG atgactgccagcgtttcgaatacgcag-3’; 引物4：5’-CTCCATGGCCATATG TGCCACGGAAACCTGGGGAGG-3’)通过In-fusion融合成GBD-TUP1。
将杉木文库通过In-fusion插入到GBD-TUP1的BamHI 位点（引物5：5’-TATGGCCATGGAGGCCGAATTCC-3’; 引物6：5’- TGCAGGTCCTCCTCTGAGATCAGC-3’），文库大小为2×105。
1.2 酵母的转化与筛选
酵母的转化参照Matchmaker Gold Yeast Two-Hybrid System User Manual(PT4084-1)_092413, Clontech. 宿主菌株为Gold, 筛选文库大小为2×106。
本研究采用的TUP1筛选系统能最大程度分离出转录激活活性极强的基因片段，通过在SD筛选培养基（SD/-His-Ade-Trp, 3-AT, AbA, X-alpha-gal）上的显色反应，鉴定出目的克隆，通过载体引物（T7: 5’-TAATACGACTCACTATAGGGCG-3’; 3’BD: 5’-TTTTCGTTTTAAAACCTAAGAGTC-3’）扩增出目的基因片段，并测序。
2.SAC3转录激活功能的鉴定
SAC3的转录激活功能用CPRG方法鉴定，将目的片段克隆入pGBKT7中（BamHI位点），并转入酵母Y187中（方法参照1.1），用SD/-Trp培养基筛选出阳性克隆，检测并比较转化菌株的β-galactosidase units （具体方法参照 Yeast Protocols handbook，PT3024-1，Clontech）。
3.SAC3转录激活功能域的确定
转录激活因子SAC3的功能片段的鉴定同样借助CPRG的方法。首先通过生物信息学手段，预测SAC3可能的功能域，而后分别克隆这些功能域到pGBKT7中（BamHI位点），转入酵母Y187中（方法参照1.1），用SD/-Trp培养基筛选出阳性克隆，检测并比较转化菌株的β-galactosidase units （具体方法参照 Yeast Protocols handbook，PT3024-1，Clontech）。
4.植物中SAC3的转录激活功能
4.1载体的构建
本实验采用GAL4-UAS系统，首先分别构建报告载体和效应载体。报告载体以pGreenII0800为背景在LUC基因序列的5’端插入UAS及迷你35S启动子序列（5’-CGGAGTACTGTCCTCCGCGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACACGCTGGGATCC-3’），构成UAS-mini35S:: LUC (以35S::REN为内参)；效应载体为35S::GAL4 (DNA-BD) – AD，以pEarlyGate104为背景, 其中GAL4 (DNA-BD)为酵母GAL4的DNA binding domain（1-147aa），AD 分别为SAC3(DEEGCGTCFDFAENTEQKEFLDNWHFEDDFVDYAQFAVFDDTADSAWDVVNLEAFIDGDYSTSWELETIC) / EDLL(EVFEFEYLDDKVLEELLDSEERKR) / VP16(413-490 aa)。 所有质粒均由无内毒素质粒大提试剂盒制备（OMEGA，D6922-02）。
4.2 瞬时表达
拟南芥叶肉细胞原生质体的制备以及原生质体转化参照Tiwari 等描述的方法（PubMed ID:16739582），其中效应载体的使用量为10微克，报告载体的使用量为5微克，共转入原生质体中。转化后的原生质体在黑暗中培养12h后，用双荧光报告系统检测试剂盒（Promega, E2920）检测样品中的LUC表达量，并以REN表达量为内参比较各样品中LUC/REN的值。每个样品均作3个生物学重复，每个生物学重复作3个技术重复。
实验结果：
1.酵母中SAC3转录激活功能
通过在酵母TUP1系统中对杉木的文库进行筛选，得到4个自激活能力大于等于VP16的基因片段，分别命名为SAC1、SAC2、SAC3和SAC4。通过CPRG检测发现，SAC3（179aa）在酵母中的自激活能力极显著高于VP16(19倍以上)。
2.SAC3的转录激活功能域
通过序列分析，将SAC3的转录激活功能域假定为1-60aa、61-110aa、110-179aa和60-179aa，分别检测这些功能域的转录激活功能发现：SAC3的转录激活能力可由C端（110-179aa）完全替代，其氨基酸序列为，DEEGCGTCFDFAENTEQKEFLDNWHFEDDFVDYAQFAVFDDTADSAWDVVNLEAFIDGDYSTSWELETIC。
3.植物中SAC3的转录激活功能
双萤光素酶报告系统检测结果显示，SAC3在植物中有显著较高的转录激活活性，大概是VP16的2倍和EDLL的8-10倍。
以上所述仅为本发明的较佳实施例，凡依本发明申请专利范围所做的均等变化与修饰，皆应属本发明的涵盖范围。
SEQUENCE LISTING
<110> 福建农林大学
<120> 植物源高效转录激活功能域SAC3及应用
<130> 11
<160> 11
<170> PatentIn version 3.3
<210> 1
<211> 70
<212> PRT
<213> SAC3的氨基酸序列
<400> 1
Asp Glu Glu Gly Cys Gly Thr Cys Phe Asp Phe Ala Glu Asn Thr Glu
1 5 10 15
Gln Lys Glu Phe Leu Asp Asn Trp His Phe Glu Asp Asp Phe Val Asp
20 25 30
Tyr Ala Gln Phe Ala Val Phe Asp Asp Thr Ala Asp Ser Ala Trp Asp
35 40 45
Val Val Asn Leu Glu Ala Phe Ile Asp Gly Asp Tyr Ser Thr Ser Trp
50 55 60
Glu Leu Glu Thr Ile Cys
65 70
<210> 2
<211> 23
<212> DNA
<213> 人工序列
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tatggccatg gaggccgaat tcc 23
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<212> DNA
<213> 人工序列
<400> 3
tgcaggtcct cctctgagat cagc 24
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<211> 48
<212> DNA
<213> 人工序列
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tcagaggagg acctgcatat gatgactgcc agcgtttcga atacgcag 48
<210> 5
<211> 36
<212> DNA
<213> 人工序列
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ctccatggcc atatgtgcca cggaaacctg gggagg 36
<210> 6
<211> 23
<212> DNA
<213> 人工序列
<400> 6
tatggccatg gaggccgaat tcc 23
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<211> 24
<212> DNA
<213> 人工序列
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tgcaggtcct cctctgagat cagc 24
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<211> 22
<212> DNA
<213> 人工序列
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taatacgact cactataggg cg 22
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<212> DNA
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ttttcgtttt aaaacctaag agtc 24
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<212> DNA
<213> 人工序列
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cggagtactg tcctccgcgc acaatcccac tatccttcgc aagacccttc ctctatataa 60
ggaagttcat ttcatttgga gaggacacgc tgggatcc 98
<210> 11
<211> 24
<212> PRT
<213> EDLL
<400> 11
Glu Val Phe Glu Phe Glu Tyr Leu Asp Asp Lys Val Leu Glu Glu Leu
1 5 10 15
Leu Asp Ser Glu Glu Arg Lys Arg
20
Claims (2)
1.植物源高效转录激活功能域SAC3，其特征在于：所述的SAC3的氨基酸序列为DEEGCGTCFDFAENTEQKEFLDNWHFEDDFVDYAQFAVFDDTADSAWDVVNLEAFIDGDYSTSWELETIC。
2.如权利要求1所述的植物源高效转录激活功能域SAC3在改善植物基因表达中的应用。
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