CN105936914A - 芦笋查尔酮合成酶基因及其编码的蛋白与应用 - Google Patents
芦笋查尔酮合成酶基因及其编码的蛋白与应用 Download PDFInfo
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- CN105936914A CN105936914A CN201610464151.1A CN201610464151A CN105936914A CN 105936914 A CN105936914 A CN 105936914A CN 201610464151 A CN201610464151 A CN 201610464151A CN 105936914 A CN105936914 A CN 105936914A
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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Abstract
本发明提供芦笋查尔酮合成酶基因及其编码的蛋白与应用。芦笋查尔酮合成酶基因AoCHS1的CDS序列如SEQ ID NO:1所示,其编码蛋白的氨基酸序列如SEQ ID NO:2所示。本发明首次从芦笋中克隆得到查尔酮合成酶基因AoCHS1,该基因为植物类黄酮合成路径中的关键基因之一,采用基因工程的方法,将基因AoCHS1转化到目标植株中,可促进转基因植株中总黄酮含量的增加,为今后利用基因工程技术改良植物品质,获得具有高抗氧化性的药物或食物提供了重要的理论依据,具有广阔的应用前景和极大的经济价值。
Description
技术领域
本发明涉及植物基因工程技术领域,具体地说,涉及芦笋查尔酮合成酶基因及其编码的蛋白与应用。
背景技术
植物活性次生代谢产物是其代谢途径中特有基因群的产物。随着植物功能基因组研究的广泛深入,独具特色又有广阔应用前景的植物次生代谢合成相关功能基因的研究逐渐成为研究的热点。类黄酮是植物中重要的次生代谢产物之一,具有重要的抗氧化和清除自由基的功能,对于提高人体免疫力具有重要作用。研究发现,在植物类黄酮生物合成途径中,查尔酮合成酶是第一个关键酶,又称限速酶,它催化丙二酰CoA的3个乙酸基和β-香豆酰CoA的1个乙酸基发生缩合反应,产生查尔酮,形成类黄酮物质的基本碳架结构,查尔酮合成酶是植物次生代谢途径中的关键酶之一,对植物具有非常重要的生理意义。
芦笋(Asparagus officinalis L.)为天门冬科天门冬属多年生草本植物,以嫩茎供食,具有极高的营养和保健价值,富含黄酮、皂苷、天冬酰胺、硒和植物多糖等多种活性成分,能抗肿瘤、抗氧化和降血脂,被誉为“蔬菜之王”、“世界十大名菜”之一(Jaiswal et al.,2014;Nishimura et al.,2013)。同时,芦笋加工产业链长,可生产出芦笋抗癌药、芦笋茶、酒和饮料等高附加值产品,在食品、医药等领域应用开发前景广阔。目前,我国芦笋种植及加工发展迅速,已成为世界第一大生产和出口国,种植面积超过95,000公顷,约占全球的43%(陈光宇,2013;张岳平等,2013)。
目前,已从很多植物中克隆得到查尔酮合成酶基因(CHS),如拟南芥、水稻、矮牵牛、葡萄、大豆等。然而,芦笋作为富含黄酮的 重要保健蔬菜作物之一,目前未有任何与芦笋CHS基因及其编码蛋白的相关文献报道,对于芦笋CHS基因及其编码的蛋白序列尚不清楚。
发明内容
本发明的目的是提供芦笋查尔酮合成酶基因及其编码的蛋白。
本发明的另一目的是提供芦笋查尔酮合成酶基因在调控植物类黄酮生物合成中的应用。
本发明针对芦笋中类黄酮活性物质生物合成途径基础研究薄弱的现状,首次克隆得到芦笋查尔酮合成酶基因AoCHS1,并进一步分析了该基因在促进类黄酮生物合成中的作用。
本发明通过对芦笋的全基因组高通量测序结果进行分析,设计一对特异引物(SEQ ID NO:3-4),对芦笋品种‘井冈111’嫩茎样品cDNA进行PCR扩增,获得了芦笋查尔酮合成酶基因AoCHS1的CDS序列(全长1191bp),基因AoCHS1的CDS序列为:
i)SEQ ID NO:1所示的核苷酸序列;或
ii)SEQ ID NO:1所示的核苷酸序列经取代、缺失和/或增加一个或多个核苷酸且表达相同功能蛋白质的核苷酸序列;或
iii)在严格条件下与SEQ ID NO:1所示序列杂交且表达相同功能蛋白质的核苷酸序列,所述严格条件为在含0.1%SDS的0.1×SSPE或含0.1%SDS的0.1×SSC溶液中,在65℃下杂交,并用该溶液洗膜;或
iv)与i)、ii)或iii)的核苷酸序列具有90%以上同源性且表达相同功能蛋白质的核苷酸序列。
本发明还提供芦笋查尔酮合成酶基因AoCHS1编码的蛋白,所述蛋白的氨基酸序列如SEQ ID NO:2所示,或该序列经替换、缺失或添加一个或几个氨基酸形成的具有同等功能的氨基酸序列。
本发明还提供含有芦笋查尔酮合成酶基因AoCHS1的表达盒、载体、工程菌及转基因细胞系。
携带有所述目的基因的表达载体可通过使用Ti质粒、植物病毒 载体、直接DNA转化、微注射、电穿孔等常规生物技术方法导入植物细胞中(Weissbach,1998,Method for Plant Molecular Biology VIII,Academy Press,New York,第411-463页;Geiserson和Corey,1998,Plant Molecular Biology,2nd Edition),并将转化的植物组织培育成植株。
使用本发明的基因片段构建到植物表达载体中时,在其转录起始核苷酸的前端可添加任意一种增强启动子或诱导型启动子。为了便于对转基因植物细胞或者植株进行鉴定及筛选,可对所使用的载体进行加工,如加入具有抗性的抗生素标记物(例如卡那霉素或潮霉素等)。被转化的宿主是包括烟草在内的多种植物,培育不同黄酮含量的植物种类。
本发明还提供芦笋查尔酮合成酶基因AoCHS1在调控植物类黄酮生物合成中的应用。
本发明还提供芦笋查尔酮合成酶基因AoCHS1在提高转基因植物总黄酮含量中的应用。
在本发明的一个具体实施方式中,将基因AoCHS1构建到载体pCAMBIA2301上,用所得重组载体转化烟草,筛选阳性转基因植株。
优选地,采用农杆菌介导的遗传转化法转化烟草,转化所用农杆菌为EHA105。
本发明进一步提供利用上述基因工程技术获得的改良植物(总黄酮含量增加)在食品、保健品和生物医药领域中的应用。
本发明首次从芦笋中克隆得到查尔酮合成酶基因AoCHS1,该基因为植物类黄酮合成路径中的关键基因之一,采用基因工程的方法,将基因AoCHS1转化到目标植株中,可促进转基因植株中总黄酮含量的增加,为今后利用基因工程技术改良植物品质,获得具有高抗氧化性的药物或食物提供了重要的理论依据,具有广阔的应用前景和极大的经济价值,
附图说明
图1为本发明实施例3中AoCHS1基因在转基因烟草中表达量的检测结果;其中图1A是烟草内参基因Actin的跑胶图,1-7为7个转基因株系,WT为对照野生型烟草,M为分子量大小;图1B是转AoCHS基因的跑胶图,1-7为7个转基因株系,H2O为阴性对照,WT为对照野生型烟草,P为重组质粒pCAMBIA2301-AoCHS1,M为分子量大小。
图2为本发明实施例4中总黄酮标准曲线图。
图3为本发明实施例4中对照野生型烟草与转化AoCHS1基因的烟草植株总黄酮含量图。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。若未特别指明,实施例均按照常规实验条件,如Sambrook等分子克隆实验手册(Sambrook J&Russell DW,Molecular Cloning:a Laboratory Manual,2001),或按照制造厂商说明书建议的条件。
实施例1 AoCHS1基因的克隆
通过前期对芦笋新品种‘井冈111’(JK111)的全基因组高通量测序结果进行分析,设计特异引物P1正向引物:5’-ATGGCTGCAACATCAATG-3’和P2反向引物:5’-CTATATAGGCACACTGTGAAG-3’(SEQ ID NO:3-4),采用常用的CTAB法(参照《植物基因工程》,王关林,方宏筠主编)从芦笋品种‘井冈111’中提取嫩茎总RNA,并反转录合成cDNA,利用上述引物P1和P2从RNA反转录得到的cDNA中扩增出如SEQ ID NO:1所示的芦笋查尔酮合成酶基因AoCHS1的CDS序列,基因AoCHS1的CDS序列全长1191bp。
具体步骤如下:
(1)向离心管中加入CTAB(十六烷基三甲基溴化铵)提取缓冲液[2%(W/V)CTAB,NaCl 1.4mol/L,EDTA(乙二胺四乙酸)20mmol/L, Tris·HCl 100mmol/L,2%(W/V)PVP]和10%β-巯基乙醇,在水浴锅中预热;
(2)将芦笋嫩茎用液氮冷却研磨,加入提取液中,混匀,65℃水浴10分钟;
(3)加入等体积的氯仿:异戊醇(体积比24:1)混合液,颠倒混匀,静置10min,4℃12000g离心10min;
(4)取上清,重复步骤(3);
(5)取上清,加入终浓度为2mol/L的LiCl,冰浴10-12小时,11000rpm,4℃离心15min,弃上清,用75%乙醇清洗沉淀两次,溶于适量的DEPC(焦碳酸二乙酯)处理水中待用;
(6)从芦笋品种‘井冈111’中提取嫩茎总RNA为模板,利用反转录酶(购自Thermo Fisher Scientific公司)将其反转录合成cDNA第一条链,反应条件为:65℃5min,42℃50min,70℃10min;
(7)利用上述引物P1和P2从RNA反转录得到的cDNA中扩增出芦笋查尔酮合成酶基因AoCHS1的CDS序列;
反应条件:94℃预变性4min;94℃30sec,55℃30sec,72℃1.5min,33个循环;72℃延伸10min。将扩增获得的PCR产物连入pMD18-T载体(购自宝生物工程大连有限公司),转化大肠杆菌感受态细胞,筛选阳性克隆并测序,获得所需的全长基因。从阳性克隆中提取携带有基因AoCHS1CDS序列的质粒,命名为pMD18-AoCHS质粒。
实施例2 AoCHS1基因超量表达载体的构建与遗传转化
为了能更好地分析基因AoCHS1的生物学功能,进一步将该基因在烟草中实现超量表达,从转基因植株总黄酮含量的表型特征来验证基因AoCHS1的生物学功能。具体步骤如下:
首先将实施例1中得到的pMD18-AoCHS质粒用BamH I和Knp I双酶切,回收目的片段;同时,用同样的方法酶切携带双烟草花叶病 毒启动子35S的遗传转化载体pCAMBIA2301。酶切完毕,用包含AoCHS1基因的酶切片段和酶切的pCAMBIA2301载体做连接反应,转化大肠杆菌DH5α(菌株购自宝生物工程大连有限公司)。通过酶切筛选阳性克隆,获得重组载体,命名为pCAMBIA2301-AoCHS1。
通过农杆菌介导的烟草遗传转化方法将其导入到烟草中,经过侵染、共培养、筛选同时具有卡那霉素抗性和潮霉素抗性的转化苗,再通过生根、练苗移栽等常规步骤(参照:J.萨姆布鲁克,EF弗里奇,T曼尼阿蒂斯著;黄培堂,王嘉玺等译;分子克隆实验指南(第三版);北京,科学出版社;2002版),得到转基因植株。
本实施例中使用的主要试剂和遗传转化方法如下:
(1)主要试剂
所用到的培养基配方和植物激素的缩写表示如下:1/2MS,MS培养基的配制参照Murashige T.and F.Skoog.Physiol.Plant,1962,15:473-497公开的方法。6-BA(6-BenzylaminoPurine,6-苄氨基嘌呤);NAA(Naphthalene acetic acid,萘乙酸);Kan(Kanamycin,卡那霉素);Cef(Cefotaxime,头孢霉素)。其中,卡那霉素和头孢霉素采用0.25μm滤膜过滤方法灭菌,在上述除Kan和Cef成分以外的培养基经121℃高压蒸汽灭菌20min后,待培养基冷却至50-60℃时,在超净工作台上加入相应的抗生素。
(2)农杆菌介导的遗传转化
1)农杆菌的培养
首先,在带有对应抗性选择的固体LB培养基(10g/L蛋白胨、5g/L酵母提取物、10g/L氯化钠、Kan 100mg/L、琼脂1.5g/L)上预培养携带有目的基因AoCHS1的农杆菌EHA105 48小时,培养温度28℃;挑取预培养农杆菌单菌落,接种于对应抗性选择的液体LB培养基(10g/L蛋白胨、5g/L酵母提取物、10g/L氯化钠、Kan 100mg/L)中,于28℃200rpm摇床培养过夜,至菌液浓度OD600值约为0.6。
2)叶盘转化法
a.剪取早花烟草无菌苗上部完全展开的幼嫩叶片,将叶片剪成1.5cm×1.5cm大小形状,放入无菌烧杯中;
b.将处于对数生长期的农杆菌稀释到OD600约为0.8,倒入烧杯,浸润叶盘,间或振荡30min;
c.将步骤b中的叶片取出,转移至灭好菌的滤纸上吸干;然后放置在共培养培养基(共培养培养基配方:MS培养基、6-BA 2.25mg/L、NAA 0.3mg/L、蔗糖30.0g/L和琼脂8.0g/L,pH值6.0)上暗培养3天,培养温度为28℃;
d.3天后,将叶片转入抗性筛选诱导培养基,光照和暗培养交替(光照强度1000-1500lx,光照时间:16h/d,黑暗时间:8h/d)下培养,进行Kan抗性芽的筛选分化,培养温度为28℃,约一个月在叶盘边缘诱导出绿色芽点,约15天左右继代一次;
e.抗性芽长大后,从外植体上切下,转入芽伸长培养基,在光照和暗培养交替(光照强度1000-1500lx,光照时间:16h/d,黑暗时间:8h/d)下培养,进行Kan抗性苗的筛选,培养温度为28℃,约一个月后,转入生根培养基,约15天继代一次;
f.将筛选得到的抗性苗转入如上所述的生根选择培养基上(生根选择培养基配方:MS培养基、Kan 100mg/L、Cef 400mg/L、蔗糖30.0g/L和琼脂8.0g/L,pH值6.0)使其生根,在光照和暗培养交替(光照强度1000-1500lx,光照时间:16h/d,黑暗时间:8h/d)下培养,培养温度为28℃。
3)移栽
生根健壮后炼苗2天,洗掉转基因烟草植株根上的残留培养基,将具有良好根系的幼苗转入温室,同时在最初一周内保持水分湿润。
本实施例共获得15个株系的PCR检测结果为阳性的转入重组质粒(或称转化质粒)pCAMBIA2301-AoCHS1的T0代转基因烟草。
实施例3 AoCHS1基因转基因T0代在田间的RT-PCR检测
为了验证转基因烟草总黄酮含量的改变是否与转入的AoCHS1基因有关,采用RT-PCR方法对部分转基因烟草植株中AoCHS1基因表达进行了检测,结果见图1。具体步骤如下:
采用TRIZOL试剂(购自宝生物工程大连有限公司)从转基因烟草1-7号株系中提取植株的总RNA(提取方法参照TRIZOL试剂说明书操作),利用反转录酶(购自Thermo Fisher Scientific公司)将其反转录合成cDNA第一条链,反应条件为65℃5min,42℃50min,70℃10min。先用报道的内参基因Actin对反转录得到的cDNA进行检测和浓度调整,根据内参基因Actin的序列设计一对引物P5正向引物(5’-CTTGAAACAGCAAAGACCAGC-3’)和P6反向引物(5’-CATCCTATCAGCAATGCCCG-3’),进行PCR检测,反应条件为:94℃预变性4min;94℃30sec,55℃30sec,72℃30sec,25个循环;72℃延伸10min。扩增产物的琼脂糖凝胶电泳结果如图1A所示,内参基因Actin在对照野生型烟草和转基因烟草植株中均能扩出,并且亮度一致。然后,根据AoCHS1基因的序列,利用引物P1和P2,进行RT-PCR检测,反应条件为:94℃预变性4min;94℃30sec,55℃30sec,72℃1.5min,33个循环;72℃延伸10min。扩增产物的琼脂糖凝胶电泳结果表明(图1B),7个转基因株系烟草内均检测到AoCHS1基因的表达。
实施例4 转AoCHS1烟草植株总黄酮的提取及测定
(1)总黄酮标准曲线的绘制
选用芦丁为标准品(经测试芦丁在510nm处有明显的吸收峰)。精密称取标准品50mg,其纯度为91.7%(精确至0.0001g),用60%乙醇溶解,并40℃水浴加热,室温下配成50mL标准溶液,待用。取标准品溶液0.4,1.5,2.5,5,7.5,10mL分别置于50mL容量瓶中,加10mL 30%乙醇和0.7mL的5%NaNO2摇匀,6min后,加入0.7mL的 10%Al(NO3)3,6min后再加入5mL的4%NaOH混匀,用30%乙醇定容至50mL,30~40℃水浴中显色30min,在510nm处测定吸光值。以标准品溶液的浓度(单位mg/ml)为横坐标,吸光值为纵坐标进行标准曲线的制作(表1,图2)。
表1 总黄酮标准曲线制作
标准品浓度(mg/ml) | 吸光值1 | 吸光值2 | 吸光值3 | 平均值 |
0.008 | 0.009 | 0.008 | 0.015 | 0.010666667 |
0.03 | 0.031 | 0.028 | 0.026 | 0.028333333 |
0.05 | 0.041 | 0.039 | 0.038 | 0.039333333 |
0.1 | 0.065 | 0.066 | 0.067 | 0.066 |
0.15 | 0.084 | 0.102 | 0.091 | 0.092333333 |
0.2 | 0.115 | 0.109 | 0.107 | 0.110333333 |
(2)样品的制备及总黄酮的测定
根据RT-PCR结果,选择对转基因植株AoCHS-3、AoCHS-6、AoCHS-7的总黄酮进行提取和测定,具体方法如下:烟草叶60℃过夜烘干,研磨成粉末。精密称取样品粉末0.4g,置于大试管中,加入5mL30%乙醇,于70℃下超声振荡0.5h后,离心机离心15min(4500r/min),过滤至10mL的容量瓶中,用30%乙醇溶液定容至10mL,将10mL过滤液移至50mL的容量瓶中,加入0.7mL的5%NaNO2摇匀,6min后加入0.7mL的10%Al(NO3)3,6min后再加入5mL的4%NaOH混匀,用30%乙醇定容至50mL,30~40℃水浴中显色30min,在510nm处测定吸光值,结果如图3所示。结果表明,转基因植株AoCHS-3、AoCHS-6和AoCHS-7的总黄酮含量比对照野生型烟草(WT)有大幅提高。
虽然,上文中已经用一般性说明及具体实施方案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。
参考文献
1、Jaiswal Y,Liang Z,Ho A,Chen H,and Zhao Z.A comparative rissue-specific metabolite analysis and determination of protodioscin content in asparagus species used in traditional Chinese medicine and ayurveda by use of laser microdissection,UHPLC-QTOF/MS and LC-MS/MS[J].Phytochem Anal.,2014,25(6):514-528.
2、Nishimura M,Ohkawara T,Kagami-Katsuyama H,Sato H and Nishihira J.Improvement of blood pressure,glucose metabolism,and lipid profile by the intake of powdered asparagus(Lú)bottom-stems and cladophylls[J].J Trandit.Complement Med.,2013,3;250-255.
3、陈光宇.中国芦笋产业发展现状与趋势[J].世界农业,2013,(10):181-186.
4、张岳平,周劲松,罗绍春,汤泳萍,谢启鑫,黄燕萍,陈光宇*.芦笋全雄新品种‘井冈111’[J].园艺学报,2013,40(12):2541-2542.。
Claims (10)
1.芦笋查尔酮合成酶基因AoCHS1,其特征在于,基因AoCHS1的CDS序列为:
i)SEQ ID NO:1所示的核苷酸序列;或
ii)SEQ ID NO:1所示的核苷酸序列经取代、缺失和/或增加一个或多个核苷酸且表达相同功能蛋白质的核苷酸序列;或
iii)在严格条件下与SEQ ID NO:1所示序列杂交且表达相同功能蛋白质的核苷酸序列,所述严格条件为在含0.1%SDS的0.1×SSPE或含0.1%SDS的0.1×SSC溶液中,在65℃下杂交,并用该溶液洗膜;或
iv)与i)、ii)或iii)的核苷酸序列具有90%以上同源性且表达相同功能蛋白质的核苷酸序列。
2.芦笋查尔酮合成酶基因AoCHS1编码的蛋白,其特征在于,所述蛋白的氨基酸序列如SEQ ID NO:2所示,或该序列经替换、缺失或添加一个或几个氨基酸形成的具有同等功能的氨基酸序列。
3.含有权利要求1所述基因AoCHS1的表达盒。
4.含有权利要求1所述基因AoCHS1或权利要求3所述表达盒的载体。
5.含有权利要求1所述基因AoCHS1、权利要求3所述表达盒或权利要求4所述载体的工程菌。
6.权利要求1所述基因AoCHS1在调控植物类黄酮生物合成中的应用。
7.权利要求1所述基因AoCHS1在提高转基因植物总黄酮含量中的应用。
8.根据权利要求7所述的应用,其特征在于,所述植物包括烟草。
9.根据权利要求8所述的应用,其特征在于,将基因AoCHS1构建到载体pCAMBIA2301上,用所得重组载体转化烟草,筛选阳性转基因植株。
10.根据权利要求9所述的应用,其特征在于,采用农杆菌介导的遗传转化法转化烟草,优选所用农杆菌为EHA105。
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CN108342377A (zh) * | 2018-03-13 | 2018-07-31 | 中国农业科学院植物保护研究所 | 陆地棉芳樟醇合成酶基因及其应用 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101220353A (zh) * | 2008-01-31 | 2008-07-16 | 北京未名凯拓农业生物技术有限公司 | 一种甘草查尔酮合成酶及其编码基因与应用 |
CN104745560A (zh) * | 2015-03-13 | 2015-07-01 | 上海交通大学 | 茄子查尔酮合成酶SmCHS1蛋白及其编码基因 |
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
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CN104745560A (zh) * | 2015-03-13 | 2015-07-01 | 上海交通大学 | 茄子查尔酮合成酶SmCHS1蛋白及其编码基因 |
Non-Patent Citations (1)
Title |
---|
GENBANK: "asparagus officinalis CHS1 mRNA,complete cds", 《GENBANK:KX987996.1》 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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CN108342377A (zh) * | 2018-03-13 | 2018-07-31 | 中国农业科学院植物保护研究所 | 陆地棉芳樟醇合成酶基因及其应用 |
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