CN108893481A - 番茄SlOAS7基因及其应用 - Google Patents
番茄SlOAS7基因及其应用 Download PDFInfo
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- CN108893481A CN108893481A CN201810644816.6A CN201810644816A CN108893481A CN 108893481 A CN108893481 A CN 108893481A CN 201810644816 A CN201810644816 A CN 201810644816A CN 108893481 A CN108893481 A CN 108893481A
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Abstract
本发明属基因工程技术领域，提供一种番茄SlOAS7基因，其核苷酸序列如SEQ ID No.1所示。该基因编码的SlOAS7蛋白，由SEQ ID No.2所示的氨基酸序列组成的蛋白；或为SEQ ID No.2所示的氨基酸序列经取代、缺失和/或增加一个或多个氨基酸且具有同等活性的衍生的蛋白。番茄SlOAS7基因属于番茄O‑乙酰‑L‑丝氨酸(硫醇)裂解酶(OASTL)编码基因家族成员，在番茄中抑制SlOAS7基因的表达能明显影响到番茄植株叶片的叶形和大小，产生叶片大小变小，复杂度降低的复叶。由于植物叶片发育同光合作用效率紧密相关，因而可用于改良番茄植株光合作用效率，具有较好的潜在应用价值。
Description
技术领域
本发明属于基因工程技术领域，具体涉及番茄SlOAS7基因及其应用，该基因属于叶片发育相关基因及其在复叶发育和叶片大小形成过程中的应用。
背景技术
植物叶片是高等植物重要的营养器官，是植物进行光合作用的主体，同时也是植物感知外界环境变化，接受并传递环境信号的重要器官。在显花植物中，叶片形态多种多样，但根据成熟叶的形态，叶片基本上可分为单叶和复叶两种类型。
番茄是一种在全世界范围内种植广泛的重要果菜和经济作物。番茄叶片为羽状复叶，是番茄植株进行光合作用的重要营养器官。
番茄复叶的形成，从发育的角度来讲，其复杂度是由其边缘风暴层（marginalblastozone，MB）所具有的瞬时叶片组织形成能力的时间长短所决定的，时间越长，叶片的复杂度越高，反之叶片越简单。目前关于番茄叶片发育的分子机理，已发现有多个基因参与其中。按照基因的功能可将基因分为提高叶片复杂度和降低叶片复杂度两种。已有研究表明CINNCINATA-TEOSINTE BRANCED1-CYCLOIDEA -PCF (CIN-TCP) 家族转录因子（例如LA）、BELL转录因子家族蛋白BIPINNATE (BIP)、以及BLADE ON PETIOLE a (BOPa) 是叶片复杂度的抑制因子；而MADS-box转录因子APETALA1/FRUIT- FULL (AP1/FUL)、Knotted1-like同源盒因子KNOXI和PTS是叶片复杂度的促进因子。
多种植物激素也参与番茄叶片复杂度的建立过程。已有研究表明赤霉素降低叶片复杂度，而细胞分裂素则是叶片复杂度的促进因子。KNOXI抑制赤霉素的合成，而CIN-TCP类转录因子LA促进赤霉素的合成。同时KNOXI也是细胞分裂素的上游促进因子，而CIN-TCP类转录因子则降低了对细胞分裂素的敏感性。因而KNOXI和CIN-TCP在叶片发育过程中的拮抗作用也体现在赤霉素和细胞分裂素之间的动态平衡上。
植物O-乙酰丝氨酸（硫醇）裂解酶（O-acetylserine(thiol)lyase, OASTL）家族蛋白是一类具有多种重要催化功能的酶类，其发现来源于半胱氨酸的合成过程。第一个被发现的OASTL家族蛋白催化半胱氨酸合成的最后一步，即在以5’-磷酸吡哆醛（PLP）为辅基的情况下，催化O-乙酰丝氨酸和硫化物的结合进而形成半胱氨酸。近来发现，OASTL家族成员还具有多种其它重要催化活性。比如，拟南芥OASTL家族蛋白DES1具有半胱氨酸脱巯基酶的活性，可以半胱氨酸为底物，催化生成硫化氢、丙酮酸盐和氨；CYS-C1具有解氰毒的作用，可以半胱氨酸和氰化物为底物，催化生成β-氰丙氨酸和硫化氢；而SCS则是一个硫代半胱氨酸合成酶，可以O-乙酰丝氨酸和硫代硫酸盐为底物生成硫代半胱氨酸。这些经OASTL催化产生的代谢物多为重要信号分子，在植物生长发育和抵御胁迫过程中具有重要作用。
但目前尚未有OASTL参与植物叶型发育的报道，以及关于OASTL具有除催化功能以外的其它功能的详细报道。
发明内容
本发明的目的在于提供一种番茄SlOAS7基因，本发明的另一目的在于提供该基因编码的SlOAS7蛋白，本发明的目的还在于提供含有所述基因或其片段的载体及其宿主细胞。
为了实现上述目的，本发明由如下技术方案实现的：番茄SlOAS7基因，其核苷酸序列如SEQ ID No.1所示。
番茄SlOAS7基因编码的SlOAS7蛋白，其为由SEQ ID No.2所示的氨基酸序列组成的蛋白；或为SEQ ID No.2所示的氨基酸序列经取代、缺失和/或增加一个或多个氨基酸且具有同等活性的衍生的蛋白。
该基因是从番茄Ailsa Craig（以下简称AC）中克隆得到的、属于OASTL基因家族的番茄离区发育基因，具有如SEQ ID No.1所示的核苷酸序列。
序列分析结果显示，番茄SlOAS7基因属于OASTL基因家族，由该基因所编码的蛋白同其它许多植物中的OASTL蛋白一样也具有保守的PLP结合区和底物结合区。系统进化分析显示，番茄SlOAS7基因属于CysC亚家族，由该基因所编码的蛋白同拟南芥中CYS-C1的相似性最高，为84%；在番茄中，同SlOAS8的相似性最高，为91%。番茄SlOAS7基因在幼苗和心皮中的表达较强；在叶片中，随着叶片年龄的增加，该基因的表达量逐渐升高。
应当理解，本领域技术人员可根据本发明公开的氨基酸序列，在不影响其活性的前提下，取代、缺失和/或增加一个或几个氨基酸，得到所述蛋白的突变序列。例如在非活性区段，将第（58）位的（T）替换为（V），或是将第（159）位的（G）缺失，或是在（283位后面）增加（一个L）。因此，本发明的番茄SlOAS7蛋白还包括SEQ ID No.2所示氨基酸序列经取代、替换和/或增加一个或几个氨基酸，具有番茄SlOAS7蛋白同等活性的由番茄SlOAS7蛋白衍生得到的蛋白质。本发明基因包括编码所述蛋白的核酸序列。此外，应理解，考虑到密码子的简并性以及不同物种密码子的偏爱性，本领域技术人员可以根据需要使用适合特定物种表达的密码子。
本发明还提供含有上述番茄SlOAS7基因或其片段的载体，以及含有该载体的宿主细胞；所述载体为所述番茄SlOAS7基因或其片段的克隆载体或各类表达载体；所述片段是指番茄SlOAS7基因cDNA的一段5’端序列，其核苷酸序列如SEQ ID No.9所示。
具体地说，本发明将番茄SlOAS7基因cDNA的一段5’端序列（246 bp，其核苷酸序列如SEQ ID No.9所示）反向互补构建到双元载体pART27中，并在大肠杆菌DH5α中扩繁。
本发明还通过农杆菌介导转化方法，将pART27携带的由两段SlOAS7基因 cDNA 5’端序列构成的茎环结构转入番茄，获得番茄转化植株。
本发明的番茄SlOAS7基因在调节番茄复叶发育中的应用。所述应用是指通过抑制番茄SlOAS7基因的表达，产生叶片复杂度降低、叶片变小叶型简单的转基因植株。
本发明的优点在于，本发明的番茄SlOAS7基因，其属于OASTL基因家族的番茄离区发育基因，在番茄中抑制SlOAS7基因的表达能够明显影响到番茄叶片的发育，产生叶片变小叶型简单的转基因植株；具有影响番茄植株光合作用的潜在应用价值。
附图说明
图1是本发明实施例1的克隆载体pEASY-T1；图2是本发明实施例2的番茄SlOAS7编码蛋白的结构分析；图3是本发明实施例3的SlOAS7蛋白的系统进化分析；黑色圆圈指示SlOAS7蛋白；图4是本发明实施例4的植物表达载体pART27；图5是本发明实施例5的SlOAS7-RNAi转基因植株分子水平阳性鉴定图；图中：(a) SlOAS7-RNAi转基因植株DNA水平阳性鉴定，GSP-F：基因特异上游引物，Intron-F：载体间序上游引物，GSP-R：基因特异下游引物，Intron-R：载体间序下游引物，数字代表不同转基因株系；(b) SlOAS7-RNAi转基因植株RNA水平阳性鉴定，ACTIN被用作内参照；(c) 转基因植株中SlOAS7和SlOAS8转录水平分析；图6是本发明实施例6的番茄SlOAS7基因影响番茄复叶叶型复杂度和大小图；图7是本发明实施例7的番茄SlOAS7基因编码蛋白的亚细胞定位分析；GFP：绿色荧光蛋白，AHL-RFP：核阳性对照，BF：明场，Merge：重叠，g为显示绿色，y为显示红色；图8是本发明实施例6的番茄SlOAS7基因的表达谱；其中，R，根；SD，幼苗；S，茎；YL，幼叶；OL，老叶；IN，花序；Se，萼片；Pe，花瓣；St，雄蕊；Ca，心皮；PD，花柄；IMG，未成熟果实；BF，露白期果实；YF，黄果期；RF，红果期；图9是本发明实施例9的蛋白纯化与酶活分析图；(a) SlOAS7-His融合蛋白的诱导与纯化，其中，M，蛋白质分子量标准；1，未诱导菌液蛋白；2，诱导后的菌液蛋白；3，纯化的蛋白；(b-d)SlOAS7-His融合蛋白的酶活分析；图10是本发明实施例10的SlOAS7蛋白转录激活活性分析图，图中黑色框线内显示为蓝色。
具体实施方式
以下结合附图和实施例，进一步详细说明本发明。以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例1：番茄SlOAS7基因编码区的克隆
利用核苷酸序列如SEQ ID No.3所示的正向引物和核苷酸序列如SEQ ID No.4所示的反向引物，从番茄Ailsa Craig的叶片cDNA中克隆SlOAS7基因的编码区序列；
PCR程序：94℃，5分钟；94℃，30秒；55℃，30秒；72℃，45秒；重复35次；72℃，10分钟。
PCR体系：2×EasyTaqPCR SuperMix（全式金公司） 25μl；
正向引物（10μM） 2μl；
反向引物（10μM） 2μl；
DNA模板 5μl；
双蒸水 补足50μl。
将上述PCR产物直接按照TA克隆方法克隆连接到pEASY-T1 Simple上（如图1所示）；连接产物转化大肠杆菌Top10，并在其中扩繁，阳性克隆经过测序筛选获得该序列；其核苷酸序列如SEQ ID No.1所示，由其编码的蛋白质的氨基酸序列如SEQ ID No.2所示。
实施例2：番茄SlOAS7蛋白的序列结构分析
番茄SlOAS7具有OASTL家族蛋白的特征序列区：PLP结合区（PXXSVKDR），同拟南芥中Cys-C1蛋白序列的相似性为84%，同番茄中SlOAS8蛋白序列的相似性为91%。结果如图2所示。
实施例3：番茄SlOAS7蛋白的系统进化分析
系统进化分析结果显示，SlOAS7属于CysC亚家族，同拟南芥中的Cys-C1同处于进化树中的一个分支。番茄中属于该亚家族的还有SlOAS8。结果如图3所示。
实施例4：番茄SlOAS7基因RNA干扰载体及转基因植株
番茄离区发育相关基因SlOAS7的一段5’端序列，246bp，其核苷酸序列如SEQ ID No.9所示，以反向互补的方式构建在图4所示的植物表达载体pART27中，用于利用RNA干扰的原理在番茄中抑制SlOAS7的表达水平，进而研究其功能。载体构建过程中SlOAS7的cDNA 5’端序列的扩增利用核苷酸序列如SEQ ID No.5所示的正向引物和核苷酸序列如SEQ ID No.6所示的反向引物，以及核苷酸序列如SEQ ID No.7所示的正向引物和核苷酸序列如SEQ IDNo.8所示的反向引物来完成。
通过农杆菌介导转化方法，将pART27携带的由SlOAS7的cDNA 5’端序列和pART27载体上的间隔序列共同构成的茎环结构序列插入番茄基因组DNA中，以番茄种子无菌播种后的子叶为受体，获得番茄转化植株，植物中的筛选标记为卡那霉素。农杆菌介导转化方法和步骤如下：
农杆菌转化番茄的方法和步骤如下：
1.种子准备：将1.5 g番茄种子依次经95%乙醇洗和 20%次氯酸钠消毒和无菌水清洗后，将种子均匀放置于1/2 MSO培养基表面，在24℃培养间光照（16h光照，8h黑暗）培养6天。
2.外植体准备：当子叶从种皮中长出后，用锋利的解剖刀将子叶切成25mm2大小，并将子叶近轴面向上放置在盛有滤纸的D1培养基的表面（注意无菌操作）；于24℃培养间长光照（16h光照，8h黑暗）培养2天。
3.农杆菌准备：固体培养基上活化-70℃存放的农杆菌（Agrobacterium tumefaciens，C58C1），挑单克隆至相应抗性的液体培养基中培养至OD600处于0.6~0.7之间，收集菌体，MSO清洗悬浮并加入乙酰丁香酮（AS）制备成侵染液。
4.共培养：用上一步制成的侵染液侵染子叶，侵染完成后将子叶远轴端朝上培养于新的盛有滤纸和D1培养基的新培养皿中，在24℃培养间放置2天（16 h光照，8 h黑暗）。
5.选择转化愈伤：将子叶转移至2Z培养基上（没有滤纸），约10天更换一次培养基，直至有芽出现后转移至1Z选择性培养基上继续培养，约两周更换一次培养基。
6．生根：当再生苗至少有2cm长并包含至少一个生长点的时候，可从外植体上切下再生苗（不包括愈伤组织），将再生苗放入MMSV培养基中进行生根培养。
生根后的植株生长到足够大时，就可转移至装有跖石和营养土的培养钵中，于一般生长箱中生长。这些植株即为T0代植株。
其中，使用到的培养基配方为：
D1培养基：MS 0.44%
蔗糖 3%
琼脂 0.8%
pH 5.8
120℃高压灭菌20分钟后加入反式玉米素核苷 1.0mg/L
2Z 培养基：MS 0.44%
蔗糖 3%
琼脂 0.8%
pH 5.8
120℃高压灭菌20分钟后加入以下成分，
反式玉米素核苷 1.5mg/L
特美汀 200mg/L
卡那霉素 50mg/L
1Z 培养基：MS 0.44%
蔗糖 3%
琼脂 0.8%
pH 5.8
120℃高压灭菌20分钟后加入以下成分，
反式玉米素核苷 1.0mg/L
特美汀 200mg/L
卡那霉素 50mg/L
MMSV培养基：MS 0.44%
蔗糖 3%
琼脂 0.8%
pH 6.0
120℃高压灭菌20分钟后加入以下成分，
特美汀 200mg/L
卡那霉素 50mg/L
叶酸 0.5mg/L
实施例5：番茄SlOAS7-RNAi转基因植株分子水平阳性鉴定
首先从实施例4中获得的20株番茄转化植株叶片中提取DNA，并在DNA水平利用PCR检测外源片段的插入情况。结果如图5a所示。在获得的20株转化株中，有16株番茄转化株显示外源片段已插入番茄基因组中，分别为3、4、5、6、7、8、9、10、11、13、14、16、17、18、19、20号。
提取这些阳性转化株的RNA，反转录成cDNA之后，通过半定量PCR（以ACTIN基因为内参）检测SlOAS7基因的表达水平，结果如图5b所示。在上一步得到的16株外源片段插入转化株中，有7株番茄转化株中SlOAS7基因的表达量有明显下调，分别是3、4、6、8、9、11、14。
接下来，在3、4、11、14、16这几株SlOAS7的下调程度明显不同的转基因植株以及野生型植株中分析了SlOAS8基因的表达水平，发现SlOAS8的表达水平没有下调，表明在SlOAS7-RNAi转基因植株中仅SlOAS7基因的表达量受到影响。结果如图5c所示。
实施例6：番茄SlOAS7-RNAi转基因植株表型分析
从实施例5中获取的转基因植株中挑取SlOAS7表达量下调最为明显的14（SlOAS7-RNAi-14）号作为进一步表型分析的对象。
我们发现同野生型相比，SlOAS7-RNAi-14的叶片大小明显小于野生型，同时其复叶中每一片小叶同野生型相比复杂性降低，深裂变浅，此外小叶还有轻微内卷。结果如图6所示。
实施例7：SlOAS7的亚细胞定位分析
构建SlOAS7-GFP融合基因，并将载有该基因的质粒转化进农杆菌中，然后利用农杆菌介导的瞬时转化将融合基因转化进烟草叶片中。转化细胞中GFP荧光信号存在的位置指示SlOAS7蛋白在细胞中定位于细胞质的沉积体（Aggresome）中。结果如图7所示。
实施例8：SlOAS7基因的表达模式分析
利用实时定量PCR分析SlOAS7基因在各组织器官的时空表达模式，结果如图8所示。
SlOAS7在番茄的各个组织器官中都有表达，其中在幼苗和心皮中的表达量较高。在叶片发育过程中，随着叶片年龄的增加，SlOAS7的表达量逐渐增高。
实施例9：SlOAS7蛋白的酶活分析
构建SlOAS7-pCold原核表达载体，在大肠杆菌BL21中表达SlOAS7-His融合蛋白并纯化。蛋白纯化后测定其酶活，结果如图9所示。结果显示SlOAS7不具有催化半胱氨酸、硫化氢和硫代半胱氨酸生成的能力。
实施例10：SlOAS7的转录激活活性分析
构建SlOAS7基因的BD载体SlOAS7-pDEST32，将该载体转化进酵母菌株AH109中，然后在选择性培养基上分析酵母细胞的生长情况，结果如图10所示。结果显示含有SlOAS7-pDEST32质粒的酵母菌可在添加了1 mM 3-氨基-1,2,4-三氮唑 (3-AT)的-Leu-His双缺培养基上生长，并可以在含有X-α-gal的培养基上产生蓝色菌落，说明SlOAS7具有转录激活活性，有可能具有转录因子的功能。
虽然，上文中已经用一般性说明及具体实施方式，对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
序列表
<110> 山西大学
<120> 番茄SlOAS7基因及其应用
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Claims (6)
1.番茄SlOAS7基因，其特征在于：所述SlOAS7基因的核苷酸序列如SEQ ID No.1所示。
2.权利要求1所述的番茄SlOAS7基因编码的SlOAS7蛋白，其为由SEQ ID No.2所示的氨基酸序列组成的蛋白；或为SEQ ID No.2所示的氨基酸序列经取代、缺失和/或增加一个或多个氨基酸且具有同等活性的衍生的蛋白。
3.含有权利要求1所述的基因或其片段的载体，其特征在于：所述片段为番茄SlOAS7基因cDNA的一段5’端序列，其核苷酸序列如SEQ ID No.9所示。
4.含有权利要求3所述载体的宿主细胞。
5.权利要求1所述番茄SlOAS7基因或其片段在调节番茄复叶发育中的应用，其特征在于：所述片段为番茄SlOAS7基因cDNA的一段5’端序列，其核苷酸序列如SEQ ID No.9所示。
6.根据权利要求5所述的番茄SlOAS7基因或其片段在调节番茄复叶发育中的应用，其特征在于：所述应用是通过抑制番茄SlOAS7基因的表达，产生叶片复杂度降低、叶片变小的转基因植株。
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