CN113388015A - 一种梨蛋白片段PyDwarf1-462及其编码序列与应用 - Google Patents
一种梨蛋白片段PyDwarf1-462及其编码序列与应用 Download PDFInfo
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- CN113388015A CN113388015A CN202110708551.3A CN202110708551A CN113388015A CN 113388015 A CN113388015 A CN 113388015A CN 202110708551 A CN202110708551 A CN 202110708551A CN 113388015 A CN113388015 A CN 113388015A
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Abstract
本发明公开了一种梨蛋白片段PyDwarf1‑462，具有序列表中SEQ ID NO：2所示的氨基酸序列的蛋白，或者是将SEQ ID NO：2的氨基酸残基序列经过一个以上氨基酸残基的取代、缺失或添加，且具有与SEQ ID NO：2的氨基酸残基序列相同活性的由SEQ ID NO：2衍生的蛋白质；还公开了上述梨蛋白片段PyDwarf1‑462的编码序列PyDwarf1‑1389的应用。通过基因工程的方法将梨蛋白片段PyDwarf1‑462转入植物中，或通过基因诱变或基因编辑对植物中PyDwarf1‑462同源的基因序列进行定点突变，使其突变为编码产物具有PyDwarf1‑462功能的基因序列，可以有效降低植物的株高。由于该基因是园艺作物梨本身存在的内源基因，其同源基因广泛存在于不同植物物种中，该基因或其同源基因的表达不会影响植物的食品安全性，可广泛应用于各种植物的育种改良。
Description
技术领域
本发明涉及植物基因工程技术领域，具体涉及一种梨蛋白片段PyDwarf1-462及其编码序列与应用。
背景技术
因为果树矮化可缩短幼龄期，提高果实品质和产量，便于机械化、设施化栽培管理，矮化栽培在苹果、甜樱桃、桃等果树的现代生产中得到广泛应用。果树的矮化栽培可通过嫁接合适的矮化型砧木或直接培育矮化型品种获得，前者如苹果中使用的矮化砧，后者如‘寿星桃’等。果树中通过传统方法选育矮化型砧木或矮化型品种的周期很长，借助转基因或基因编辑技术可在不改变遗传背景的前提下针对目标性状进行定点编辑，大大缩短育种周期、提高育种效率。
在果树中曾有过关于矮化基因克隆和功能研究的报道，桃树中的GID1c基因突变产生了赤霉素信号不敏感型矮化突变(Hollender等，New Phytologist，2016，210:227–239，doi:10.1111/nph.13772；Cheng等Plant Biotechnol.J.,2019，https://doi.org/10.1111/pbi.13094)；梨树中油菜素内酯合成相关基因PcDWF1的表达变异(下调表达)导致了矮化株型形成(Zheng等，BMC Plant Biology(2020)20:109)。除了赤霉素和油菜素内酯，生长素信号途径在植物株高调控中发挥重要作用。
不同于以往报道的，本申请中要求保护的梨矮化基因PyDwarf1-462在生长素合成途径中发挥作用，此外，对GA和ABA的合成或代谢也产生影响(图1)，PyDwarf1-462通过抑制细胞膨大而影响茎秆伸长，产生矮化表型(图2)。
发明内容
针对现有技术存在的不足，本发明的目的在于提供一种梨蛋白片段PyDwarf1-462，并提供上述蛋白片段的编码序列以及上述基因片段在培育矮化材料或品种中的应用。
为了实现本发明的第一目的，本发明提供如下技术方案：
一种梨蛋白片段PyDwarf1-462，它是具有序列表中SEQ ID NO：2所示的氨基酸序列组成的蛋白质，或者是将SEQ ID NO：2的氨基酸残基序列经过一个或一个以上氨基酸残基的取代、缺失或添加，且具有与SEQ ID NO：2的氨基酸残基序列相同活性的由SEQ ID NO：2衍生的蛋白质。
作为优选，所述的梨蛋白片段PyDwarf1-462具有序列表中SEQ ID NO：2所示的氨基酸序列。
为了实现本发明的第二目的，本发明提供如下技术方案：
一种梨蛋白片段PyDwarf1-462的编码序列，它是下列核苷酸之一：
(1)序列表中SEQ ID NO：1所示的核苷酸序列；
(2)编码序列表中SEQ ID NO：2所示的氨基酸序列的多核苷酸；
(3)在高严谨条件下可与序列表中SEQ ID NO：1限定的核苷酸序列杂交的核苷酸序列。
作为优选，梨蛋白片段PyDwarf1-462的编码序列是序列表中SEQ ID NO：1所示的核苷酸序列；梨蛋白片段PyDwarf1-462的编码序列含有1,389bp的核苷酸。
含有上述梨蛋白片段PyDwarf1-462编码序列的表达载体、转基因细胞系和基因编辑材料也在本发明的保护范围之内，利用现有分子生物学的方法可以得到不同的表达载体、转基因细胞系和基因编辑材料。
为了实现本发明的第三目的，本发明提供如下技术方案：
该梨蛋白片段PyDwarf1-462的编码序列在培育矮化材料和品种中的应用，梨蛋白片段PyDwarf1-462的编码序列可降低植物的节间长度、实现植物的矮化。
利用任何一种可以引导外源基因在植物中超量表达的载体，将本发明所提供的梨蛋白片段PyDwarf1-462编码序列PyDwarf1-1389导入植物细胞，或对植物同源基因进行定点编辑可获得改变植物株高的转基因细胞系、转基因植株及基因编辑植株。使用载体时，其转录起始核苷酸前可加上任何一种增强型启动子或诱导型启动子或组织特异表达启动子。
为了便于对转基因植物或转基因细胞或基因编辑材料进行筛选，可以对载体进行加工，如加入抗生素标记基因(如潮霉素、卡那霉素和庆大霉素等)，加入抗生素标记基因之后的载体用于转化，可以在转化后的植物培养基中加入抗生素，抑制非转基因细胞系和植株的生长，有助于快速和有效的获得转基因材料或基因编辑材料。
为了便于观察外源基因的表达，可以在载体中启动子和外源基因之间或是外源基因与终止子之间加入报告基因(GUS基因、GFP和萤火虫荧光素报告基因)，构建外源基因和报告基因融合表达的载体，该载体用于遗传转化，可以观察报告基因的表达与否和表达量高低，推测外源基因在植物体内的表达情况。
为了转基因植物释放的安全性，也可以在构建载体时不携带任何筛选标记基因或非抗生素筛选标记基因，直接通过PCR鉴定或表型筛选鉴定。
含有本发明的PyDwarf1-1389片段的表达载体或基因编辑载体可通过使用基因枪、农杆菌介导、发粉管通道、电击、显微注射、Ti质粒、Ri质粒或植物病毒等常规生物学方法转化植物细胞或组织，并将转化后的植物细胞培育成完整的植株。被转化的植株既可以是双子叶植物，也可以是单子叶植物，如：梨、苹果、桃、李、杏、水稻、棉花、小麦、大豆、油菜、烟草、拟南芥、大麦、高粱、玉米、黄瓜、番茄、白菜、萝卜、杨树、药材和花卉等。
与现有技术相比，本发明的有益效果是：
通过基因工程的方法将本发明提供的梨蛋白片段PyDwarf1-462转入植物中，或对种物中的同源基因进行基因定点编辑，可以降低植物的株高，培育矮化材料或品种。由于该基因是果树作物梨本身存在的内源基因，因此，该基因的超量表达或同源序列的基因编辑不会影响植物的食品安全性，可广泛应用于各种植物的育种应用。
附图说明
图1为梨PyDwarf1矮化基因型与野生型对照在伸长区茎秆中的激素含量比较柱状图。
图2为梨树矮化基因型PyDwarf1与野生型对照的表型比较图。
图3为梨树矮化基因型PyDwarf1与野生型对照的一年生枝条比较图。
图4为梨树矮化基因型PyDwarf1与野生型对照的节间长度比较柱状图。
图5为梨树矮化基因型PyDwarf1与野生型对照细胞形态比较图，其D1～D3为矮化基因型PyDwarf1茎秆伸长区皮层细胞纵切、髓细胞纵切与髓细胞横切的细胞形态图；E1～E3为野生型对照茎秆伸长区皮层细胞纵切、髓细胞纵切与髓细胞横切的细胞形态图。
图6为PyDwarf1转基因拟南芥较野生型株高明显矮化。
具体实施方式
根据下述实施例，可以更好地理解本发明。然而，本领域的技术人员容易理解，实施例所描述的仅用于说明本发明，而不应当也不会限制权利要求书中所详细描述的本发明。
实施例1：梨蛋白片段PyDwarf1-462编码序列的获得。
以砂梨(Pyrus pyrifolia)矮化资源‘Aicui’为材料，在生长季节取700mg伸长区幼茎，用英俊公司的Plant Trizol试剂盒提取样品总RNA，用甲醛变性胶电泳鉴定总RNA质量，然后在分光光度计上测定RNA含量。参照梨公布的基因组序列，用Macvector软件设计引物P1。
P1引物如下：
F-5’-ATGCAGAGCTTCAAAGCAGC’-3’(SEQ ID NO:3)；
R-5’-TTAGTCCCTAGGACGCGCTTGC-3’(SEQ ID NO:4)。
采用Promega的反转录试剂盒进行反转录，合成单链cDNA为模板，用引物P1扩增目标片段。PCR反应体系为25μL，包含5ng模板，F和R引物各5pmol、2.5μl 10x PCR buffer、37.3nmol MgCl2、5nmol dNTP、0.5U的rTaq聚合酶。
扩增程序为：94℃预变性3min，94℃20s，60℃30s，72℃延伸90s，30个循环后，72℃反应5min。通过PCR扩增，获得1389bp的核苷酸序列，将PCR产物克隆至pMD18-T载体，测序获得的核苷酸序列如SEQ ID NO:1所示。该核苷酸序列编码462个氨基酸，该氨基酸序列如SEQID NO:2所示。
实施例2：梨蛋白片段PyDwarf1-462的获得。
将梨蛋白片段PyDwarf1-462的编码序列PyDwarf1-1389克隆到表达载体PET-32a的EcoR I和BamH I酶切位点中，转化E.coli。挑取单菌落于1mL的LB(Amp 100μg/mL)中摇菌过夜，转至200mL新鲜的LB培养基中摇至菌液浓度A600≈0.6；加入IPTG至终浓度为1.0mM，37℃培养诱导表达3小时。菌液12,000g离心5min，沉淀悬浮于提取缓冲液中(3M NaCl、1mMPMSF、50mM pH8.0磷酸缓冲液)超声破碎细胞，12,000g离心20min，收集上清。用10mM咪唑、50mM pH8.0磷酸缓冲液平衡Ni-Sepharose凝胶；加入细胞裂解液室温结合20min、用5倍凝胶体积的平衡缓冲液洗涤3次；然后用含300mM咪唑、50mM pH8.0磷酸缓冲液进行洗脱，收集洗脱液即为纯化后的Trx-PyDwarf1-462蛋白。纯化后的表达蛋白经透析除盐后按每毫克蛋白样品加入0.1mg肠激酶，于40mM的琥珀酸缓冲液(pH＝5.6)中25℃温育2小时切去组氨酸标签，透析过夜即得到纯化的梨蛋白PyDwarf1的蛋白质片段PyDwarf1-462。
实施例3：梨蛋白片段PyDwarf1-462的编码序列的应用。
以翠冠果皮的总cDNA为模板，用引物P2进行PCR扩增。
P2引物对如下：
F-5’-CAGCTCTAGAATGCAGAGCTTCAAAGCAGC-3’(SEQID NO:5)；
R-5’-GAAGGGATCCTTAGTCCCTAGGACGCGCTTGC-3’(SEQID NO:6)。
将扩增得到的PCR产物用限制性内切酶XbaI和BamHI进行酶切后，与经过同样限制性内切酶进行酶切的双元表达载体pBI121进行连接，获得由CaMV35S启动子启动的PyDwarf1基因片段PyDwarf1-1389超量表达载体。用冻融法将获得的表达载体转入农杆菌菌株LBA4404，通过含有50μg/ml的卡那霉素和50μg/ml利福平的LB培养基进行筛选，获得阳性克隆。原生质体的制备与转化参照Wang等(Bio-protocol,2013,3(22):e979)，将携带有PyDwarf1基因片段PyDwarf1-1389超量表达载体通过农杆菌转化到拟南芥Columbia类型，我们首先提取基因组DNA，用与扩增PyDwarf1-1389基因片段相同的引物和PCR程序进行PCR鉴定。对于PCR阳性的转基因植株，提取总RNA，经过DNAse I处理后，进行反转录合成cDNA第一链，以cDNA第一链为模板，用与检测DNA相同的PCR引物和程序进行转录水平的鉴定。
随机选取7株PCR检测阳性T0代自交繁殖至T3代，每代根据表型特征结合PCR检测进行后代选择，将T3代无株高性状分离转基因材料和非转基因对照材料种子播种在装有营养土的生长箱中，于正常生长季节自然条件生长，结合日常病虫害防治和肥水管理；其中，营养土为包含15％蛭石的泥炭，购自虹越花卉网上商城。
表型观察显示，转基因拟南芥与野生型相比抽薹减缓、株高明显矮化，叶片发育无明显差异(图6)。
SEQUENCE LISTING
<110> 浙江省农业科学院
<120> 一种梨蛋白片段PyDwarf1-462及其编码序列与应用
<130> 2021.06.20
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Claims (6)
1.一种梨蛋白片段PyDwarf1-462，其特征在于，它是具有序列表中SEQ ID NO：2所示的氨基酸序列组成的蛋白质；或者是将SEQ ID NO：2的氨基酸残基序列经过一个或一个以上氨基酸残基的取代、缺失或添加，且具有与SEQ ID NO：2的氨基酸残基序列相同活性的由SEQ ID NO：2衍生的蛋白质。
2.根据权利要求1所述的梨蛋白片段PyDwarf1-462，其特征在于，所述的梨蛋白片段PyDwarf1-462具有序列表中SEQ ID NO：2所示的氨基酸序列。
3.一种如权利要求1所述的梨蛋白片段PyDwarf1-462的编码序列，其特征在于：它是下列核苷酸之一：
(1)序列表中SEQ ID NO：1所示的核苷酸序列；
(2)编码序列表中SEQ ID NO：2所示的氨基酸序列的多核苷酸；
(3)在高严谨条件下可与序列表中SEQ ID NO：1限定的核苷酸序列杂交的核苷酸序列。
4.根据权利要求3所述的梨蛋白片段PyDwarf1-462的编码序列，其特征在于，梨蛋白片段PyDwarf1-462的编码序列是序列表中SEQ ID NO：1所示的核苷酸序列。
5.一种如权利要求3所述的梨蛋白片段PyDwarf1-462的编码序列在培育植物矮化材料、品种中的应用。
6.根据权利要求5所述的梨蛋白片段PyDwarf1-462的编码序列在培育植物矮化材料、品种中的应用，其特征在于：梨蛋白片段PyDwarf1-462的编码序列可降低植物的节间长度、实现植物的矮化。
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