CN105037500B - 红掌myb转录因子AaMYB1及其载体、工程菌与应用 - Google Patents
红掌myb转录因子AaMYB1及其载体、工程菌与应用 Download PDFInfo
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Abstract
本发明公开了一种红掌myb转录因子AaMYB1及其载体、工程菌与应用。所述转录因子AaMYB1的完整预测氨基酸序列如SEQ ID NO：1所示；编码基因如SEQ ID NO：2所示。首次从红掌中克隆MYB类调控花色苷合成的转录因子，通过过量表达，提高目标植物的合成花色苷的能力。
Description
技术领域
本发明涉及一种红掌myb转录因子AaMYB1及其载体、工程菌与应用。
背景技术
红掌（Anthurium andraeanum）是世界性盆栽和鲜切花花卉。在我国高档盆花种植中占有相当重要的地位。然而，同其他大宗花卉一样，我国在红掌育种上也是处于较为落后的阶段。传统杂交育种必须要求有足够的杂交亲本和长期的经验积累，但是随着生物技术的迅速发展，利用基因改良选择性、定向培育红掌新品种则成为可能。但是由于缺乏可选基因，所以通过转基因技术使红掌获得优良性状目前尚未实现，而其重要基因的发掘和功能验证是通过转基因手段改良红掌性状的前提。
发明内容
本发明目的是提供一种红掌myb转录因子AaMYB1及其载体、工程菌与应用，通过超量表达证明该转录因子具有提高植物合成花色苷的能力，可为培育花色、叶色更为丰富的新品种提供基因资源。
本发明采用的技术方案是：
一种红掌myb转录因子AaMYB1，所述转录因子AaMYB1的完整预测氨基酸序列如SEQID NO：1所示；编码基因如SEQ ID NO：2所示。
进一步包括至少90%同源且具有相同功能的所述转录因子AaMYB1的衍生物，所述的衍生物包括氨基酸序列或核酸序列。由于氨基酸序列的特殊性，任何含有SEQ ID NO：1所示氨基酸序列的肽蛋白的片段或其变体，如其保守性变体、生物活性片段或衍生物，只要该肽蛋白的片段或肽蛋白变体与前述氨基酸序列同源性在90%以上，均属于本发明保护范围之列。具体的所述改变可包括氨基酸序列中氨基酸的缺失、插入或替换；其中，对于变体的保守性改变，所替换的氨基酸具有与原氨基酸相似的结构或化学性质，如用亮氨酸替换异亮氨酸，变体也可具有非保守性改变，如用色氨酸替换甘氨酸。由于核苷酸序列的特殊性，任何如SEQ ID NO：2所示多核苷酸的变体，只要其与该多核苷酸具有90%以上同源性，均属于本发明保护范围之列。所述多核苷酸的变体是指一种具有一个或多个核苷酸改变的多核苷酸序列。此多核苷酸的变体可以使生的变位变异体或非生的变异体，包括取代变异体、缺失变异体和插入变异体。如本领域所知的，等位变异体是一个多核苷酸的替换形式，它可能是一个多核苷酸的取代、缺失或插入，但不会从实质上改变其编码的肽蛋白的功能。
一种含有所述的红掌myb转录因子AaMYB1的编码基因的重组载体。
一种含有所述的重组载体的重组基因工程菌。
一种根据所述的重组载体的重组基因工程菌的构建方法，构建含有转录因子AaMYB1的编码基因的重组载体，将所述重组载体转化至农杆菌菌株EHA105中，获得的重组基因工程菌进行诱导培养，培养液分离纯化获得含有转录因子AaMYB1的编码基因的重组基因工程菌的菌体细胞。
一种根据所述的红掌myb转录因子AaMYB1的应用，通过过量表达，提高目标植物的合成花色苷的能力。
与现有技术相比，本发明的有益效果主要体现在：本发明首次从红掌中克隆MYB类调控花色苷合成的转录因子，即AaMYB1；本发明通过与拟南芥等不同物种同类MYB转录因子序列比对，发现其属于第R2R3类MYB；通过转基因实验，发现该基因具有明显的提高植物合成花色苷的能力，证明其是一个非常有应用价值的转录因子。
附图说明
图1 是来源于红掌的转录因子AaMYB1与其他物种同源基因的多序列比对结果；
图2是转基因烟草在不同生长阶段表现；
图3是转基因和野生型烟草花器官比较，上部分示转基因器官，下部分示野生型器官；
图4是转基因和野生型烟草果实比较，A部分为去掉部分萼片的果实，B部分为去掉果皮的果实。
具体实施方式
本发明的要点在于提供了如SEQ ID NO：1所示的氨基酸序列和SEQ ID NO：2所示的核苷酸序列，在已知该氨基酸序列和核苷酸序列的情况下，该氨基酸序列和核苷酸序列的获得，以及相关载体、宿主细胞的获得，对于本领域技术人员来说均是显而易见的。
本发明所提供的转录因子AaMYB1是从红掌中获得的，它属于具有显著提高花色苷含量功能的众多MYB转录因子家族中的一员。通过反转录RT-PCR，获得了该基因的完整编码框，随后构建超量表达载体并异源转化烟草，发现该转录因子具有明显的提高烟草体内花色苷含量的能力。
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此。
实施例1来源于红掌的转录因子AaMYB1的获得
于2013年5月取6年生红掌品种“阿拉巴马”的幼嫩苞片和叶片为材料，采用Invitrogen公司生产的Trizol试剂提取总RNA，然后使用Promega公司生产的反转录试剂盒对总RNA进行处理、反转录合成cDNA，以此为模板。采用同源克隆法，设计兼并引物对P1-GATCTCATCATCAGGCTGCATG（SEQ ID NO：3），P2-GATTTCCTGGGACAACAGT（SEQ ID NO：4）,用上述模板进行扩增，该反应程序为95 ℃ 1 min, 94 ℃50 s, 56 ℃1 min, 72 ℃ 1 min,36个循环，72 ℃5 min，所用试剂（LATaq酶）均购自大连宝生物公司，扩增体系：LAtaq酶（5U/µl）0.5 µl，10×LAtaq buffer Ⅱ5 µl，dNTP mixture 8 µl，template 1 µl，PI 1 µl，P2 1 µl，灭菌蒸馏水32.5 µl。经过PCR扩增获得336 bp的中间片段。
bp的中间片段为参考，设计3’ RACE和5’RACE引物;以红掌幼嫩花芽为组织材料，按照述方法提取总RNA，参照CLONTECH公司RACE试剂盒说明书（Cat. No(s). 634858）分别进行进行3’RACE和5’RACE模板合成，备用。具体是，3’RACE用到的引物对：3’f—GGGGAACCGGTGGTCTCTCATAG（SEQ ID NO：5）， 3’r—CTAATAC GACTCAC TATAG GGC（SEQ IDNO：6），扩增体系：LAtaq酶（5 U/µl）0.5 µl，10×LAtaq buffer Ⅱ5 µl，dNTP mixture 8 µl，template 1 µl，3’f 1 µl，3’r 1 µl，灭菌蒸馏水32.5 µl。反应程序为95 ℃1 min, 94℃25 s, 68 ℃1 min, 72 ℃1 min，36个循环，72 ℃5 min，通过PCR扩增获得中间序列及下游片段。
5’RACE用到的引物对：5’f—CTAATAC GACTCAC TATAG GGC（SEQ ID NO：6）, 5’r—GGGA CCA CCACTGAGGCCCTC（SEQ ID NO：7），反应体系参照3’RACE，扩增程序为95 ℃1 min,94 ℃25 s, 66 ℃1 min, 72 ℃1 min, 36个循环，72 ℃5 min，经过PCR扩增获得中间序列及上游片段。
将AaMYB1基因的上游片段和下游片段测序后进行序列拼接，拼接方法为手动拼接，如 “123, 234拼接变成1234”一样，即可获得完整编码框（CDS），经过http://www.ncbi.nlm.nih.gov/gorf/gorf.html在线分析，该编码框有303个氨基酸，具有明显的起始和终止密码子，说明该基因的完整性。分析推测的氨基酸序列，该基因编码303个氨基酸残基，与单子叶植物R2R3-MYB类转录因子存在较高相似度，初步认定其为R2R3-MYB类转录因子。Expasy在线软件分析，其15-71号氨基酸属于R2 motif，72-122号氨基酸属于R3mitif。综合单双子叶植物同类蛋白与AaMYB1进行多序列比对，结果发现，该基因与水稻C1基因有52%的一致序列，与拟南芥PAP1基因有75%的一致性，与苹果MdMYB10基因有72%的一致性，DNAMAN比对结果显示所有参试基因都含有保守的R2R3结构域，该区段序列相似度较高，而其余部分则变异较大，在所有比对的序列中，都含有一段较为保守的(N/S/G)ND(V/I)区段，该区段被认为在单双子叶植物花色苷合成中发挥关键性作用，
因此，将获得的编码框（CDS）命名为转录因子AaMYB1是可行的。通过公共平台NCBI网站中ORFinder软件的应用，获得了转录因子AaMYB1的编码框区域，并得到了预测的氨基酸组成信息。该基因编码区的核苷酸序列为SEQ ID NO.2所示，氨基酸序列为SEQ ID NO.1所示，该基因编码氨基酸与不同物种同源物多序列比对结果见图1。
实施例2含转录因子AaMYB1编码基因的重组基因工程菌的制备
利用特异引物对：GSPf-- ctggtaccATGGAAGACGTGCCCAACCA (SEQ ID NO：8，含Kpn1酶切位点)，GSPr—gctctagaTTAGTTTTCTGGGAATGTTAG（SEQ ID NO：9，含Xba1酶切位点），以实施例1中获得的高质量反转录第一链产物cDNA为模板进行PCR反应，体系参照上述RACE反应体系，反应程序为：95 ℃1 min，94 ℃50 s，66 ℃1 min，72 ℃1.5 min，36个循环，72 ℃5 min。将获得的PCR产物回收后进行Kpn1、Xba1双酶切，酶切体系按照TAKARA公司内切酶说明书进行。酶切后的基因片段与同样酶切回收的表达载体pCAMBIA2300（M）中，测序验证，测序结果需与SEQ ID NO.2完全一致。然后将阳性质粒电击导入农杆菌菌株EHA105中，获得含转录因子AaMYB1编码基因的重组基因工程菌，即含有超量表达载体的农杆菌菌株EHA105。
实施例3含转录因子AaMYB1编码基因在制备花色苷富集植物中的应用
用实施例2制备的含有超量表达载体的农杆菌菌株EHA105侵染普通烟草无菌苗叶片，采用通用叶盘法转化（材料与方法严格参照Horsch RB, Fry JE, Hoffman NL,Eicholtz D, Rogers SG, Fraley RT (1985) A simple and general method fortransferring genes into plants. Science 227:1229–1231）。经过分化、再生、筛选，获得含有转录因子AaMYB1编码基因的抗性烟草植株。生根后移栽入温室，进行性状鉴定和分析。
转基因抗性植株在移栽后，均出现不同程度的颜色变化，主要体现在叶片、生殖器官变成紫红色，光照越强，颜色越深，无法接受光照的部位，肉眼难以观察到颜色变化。与野生型植株的比较分析见图2-4。
SEQUENCE LISTING
<110> 浙江省萧山棉麻研究所
<120> 红掌myb转录因子AaMYB1及其载体、工程菌与应用
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Claims (5)
1.一种红掌myb转录因子AaMYB1，其特征在于，所述转录因子AaMYB1的完整预测氨基酸序列如SEQ ID NO：1所示；编码基因如SEQ ID NO：2所示。
2.一种含有所述权利要求1的红掌myb转录因子AaMYB1的编码基因的重组载体。
3.一种含有权利要求2所述的重组载体的重组基因工程菌。
4.一种根据权利要求3所述的重组载体的重组基因工程菌的构建方法，其特征在于，构建含有转录因子AaMYB1的编码基因的重组载体，将所述重组载体转化至农杆菌菌株EHA105中，获得的重组基因工程菌进行诱导培养，培养液分离纯化获得含有转录因子AaMYB1的编码基因的重组基因工程菌的菌体细胞。
5.一种根据权利要求1所述的红掌myb转录因子AaMYB1的应用，其特征在于，通过过量表达，提高目标植物的合成花色苷的能力。
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