CN109750048A - 苹果果实糖转运蛋白基因MdERDL6及其应用 - Google Patents
苹果果实糖转运蛋白基因MdERDL6及其应用 Download PDFInfo
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- CN109750048A CN109750048A CN201910198437.3A CN201910198437A CN109750048A CN 109750048 A CN109750048 A CN 109750048A CN 201910198437 A CN201910198437 A CN 201910198437A CN 109750048 A CN109750048 A CN 109750048A
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Abstract
本发明公开了一种苹果果实糖转运蛋白基因MdERDL6,核苷酸序列如序列表1。本发明还公开了含有苹果果实糖转运蛋白基因MdERDL6的重组表达载体、表达盒、转基因细胞系、转基因植物组织或者重组菌,本发明还公开了苹果果实糖转运蛋白基因MdERDL6编码的蛋白,氨基酸序列如序列2所示,以及苹果果实糖转运蛋白基因MdERDL6的应用。本发明的苹果果实糖转运蛋白基因MdERDL6,能够合成苹果果实糖转运蛋白;苹果果实糖转运蛋白基因MdERDL6,能够促进葡萄糖、果糖等进入细胞质的运输,提高果实品质;苹果果实糖转运蛋白应用到提早开花的转基因植物中和提高植物果实糖含量等领域均有显著的效果。
Description
技术领域
本发明属于生物基因工程技术领域,涉及苹果果实糖转运蛋白基因MdERDL6,本发明还涉及该苹果果实糖转运蛋白基因MdERDL6的蛋白,同时本发明还公开了苹果果实糖转运蛋白基因MdERDL6的应用。
背景技术
在大多数植物中,源器官叶片通过光合作用产生光合同化物,然后通过韧皮部装载、长距离运输和韧皮部卸载等一系列途径运输到不能进行光合作用的库组织和器官,比如种子、果实和花等。在大多数植物中,植物从源到库的运输以蔗糖为主(蔷薇科植物主要以山梨醇为主),到达库组织后,蔗糖会被转化酶和蔗糖合酶分解成己糖,山梨醇被山梨醇脱氢酶转化为果糖,后被己糖激酶磷酸化进入植物代谢而利用,这整个过程离不开各类糖转运蛋白的协助。
植物中的糖转运蛋白根据运输物质不同,分为单糖转运蛋白和多糖转运蛋白,单糖转运蛋白主要运输葡萄糖和果糖,而多糖转运蛋白主要转运蔗糖。植物中的糖转运蛋白属于协助扩散超家族,且都具有12个跨膜结构域,主要包括己糖转运蛋白(HT)、液泡膜糖转运蛋白(TST)、早期响应脱水类似蛋白(ERD6-like)、液泡葡萄糖转运蛋白(VGT)和质体葡萄糖转运蛋白(pGlcT)等,此外植物还包含糖转运蛋白(STP)、蔗糖转运蛋白(SUT),肌醇转运蛋白(INT),糖醇/单糖转运蛋白(PMT)。
ERD6家族几乎存在于所有的高等植物中,是液泡膜糖转运蛋白家族重要的一员,参与植物体响应干旱胁迫、高温胁迫、低温胁迫、黑暗、创伤、种子萌发、种子营养物质储藏以及植物体内糖的转运、稳态和积累。
1996年,伊利诺伊大学的教授Chiou和Bush首次在甜菜中克隆到了此类蛋白称作cDNA-1,其编码490个氨基酸,蛋白大小54kDa,包含12个跨膜域结构,表达分析发现cDNA-1在甜菜的下胚轴和真根表达较高,幼叶向成熟叶发育过程中表达量也逐渐升高,定位于甜菜液泡膜,转到烟草中对蔗糖有较高的转运活性。
Kiyosue等人在1994年从拟南芥中首次克隆到此类蛋白,并命名为ERD,ERD6属于早期响应干旱家族分类的第6类,克隆后发现ERD6含有1741bp的cDNA序列,编码496个氨基酸,蛋白分子大小为54.35kDa,与甜菜U43629序列同源性大70%多,分析氨基酸序列发现同时含有12个跨膜结构域和中央亲水环,表达分析AtERD6不只能被干旱诱导,还能被低温所诱导。
在拟南芥中克隆到AtERD6家族另一个基因,命名为AtESL1。AtESL1主要在中柱鞘和木质部薄壁细胞表达,能被干旱、高盐、脱落酸(ABA)、黑暗、高温和创伤诱导,显示对葡萄糖有较强的吸收能力,且是一类低亲和转运蛋白。利用膜片钳技术,证明了AtERDL6是质子驱动的葡萄糖外运蛋白。在外源糖供应条件下,拟南芥AtERDL6表达量下调,同时甜菜中的同源基因BvIMP在甜菜中的表达量也下调。在拟南芥Aterdl6突变体中,葡萄糖含量上升,种子重量增加了10%。在拟南芥中过表达BvIMP,可以使葡萄糖含量降低,且表现为不抗冻。
Antony等在2008年从菠萝中克隆到AcMST1,编码496个氨基酸,蛋白分子大小为53.6kDa,含有12个跨膜结构域,其与甜菜U43629和拟南芥AtERD6的同源序列相似性达到74%和81%。亚细胞定位显示其定位于液泡膜。表达分析表明,AcMST1主要在菠萝的果实中表达,与果实糖的积累密切相关。
近年来,对果树中的糖转运蛋白做了详细的进化分析和表达分析,发现AtERD6在苹果中的同源基因MdERDL6、在梨中的同源基因PbSFP、在柑橘中的同源基因CsERD6L和在葡萄中的同源基因VvERD6-like主要在果实中表达,并随着果实的发育表达量不断升高,说明其与果实中糖的积累密切相关。
我国苹果的总生产量和总消费量均排在世界首位,因此苹果对我国参与农产品国际贸易具有非常重要的作用。21世纪苹果产业发展的一个重要方向是提高果实品质,而果实的糖含量与糖酸比是衡量果实品质的重要指标,主要取决于碳水化合物在果实内的分布和积累。在植物体内,糖的运输、储藏和分配主要依靠糖转运蛋白的表达,才使得植物体内的糖始终处于一个动态平衡的状态。
MdERDL6属于单糖转运蛋白家族,其表达量与果实发育阶段的糖含量密切相关,但是MdERDL6是如何影响苹果果实的糖含量尚不明确,本发明通过分子技术手段,解析了MdERDL6在苹果果实发育过程中的生物学功能。
发明内容
本发明提供了一种苹果果实糖转运蛋白基因MdERDL6,能够合成苹果果实糖转运蛋白。
本发明提供的苹果果实糖转运蛋白MdERDL6,能够促进葡萄糖、果糖和蔗糖的积累,在提高苹果果实品质方面发挥重要的作用。
本发明还提供了该苹果果实糖转运蛋白基因MdERDL6的应用。
本发明所采用的第一种技术方案是,苹果果实糖转运蛋白基因MdERDL6,核苷酸序列如序列表1所示。
本发明采用的第二种技术方案是,含有苹果果实糖转运蛋白基因MdERDL6的重组过量表达载体。
重组过量表达载体将MdERDL6基因通过同源重组的方法构建到pGWB402载体中。
本发明采用的第三种技术方案是,含有苹果果实糖转运蛋白基因MdERDL6的重组菌和转基因植物组织。
本发明采用的第四种技术方案是,苹果果实糖转运蛋白基因MdERDL6编码的蛋白,氨基酸序列如序列2所示。
本发明采用的第五种技术方案是,苹果果实糖转运蛋白基因MdERDL6在构建延迟开花结果的转基因植物中的应用。
本发明采用的第六种技术方案是,苹果果实糖转运蛋白基因MdERDL6在构建促进茎增粗增高的转基因植物中的应用。
本发明采用的最后一种技术方案是,苹果果实糖转运蛋白基MdERDL6在提高植物果实糖含量中的应用。
本发明的有益效果是:
本发明的苹果果实糖转运蛋白基因MdERDL6,能够合成苹果果实糖转运蛋白;
本发明的苹果果实糖转运蛋白基因MdERDL6,能够明显促进主茎的增粗和植株增高,为以茎干为利用价值的植物提供了重要的基因资源;
本发明的MdERDL6过表达植株,能促进葡萄糖、果糖和蔗糖的大量积累,在提高苹果果实品质方面发挥重要的作用。
附图说明
图1是本发明苹果果实糖转运蛋白基因MdERDL6的研究技术路线图;
图2是苹果果实发育的不同时期葡萄糖、果糖、蔗糖、山梨醇及总糖含量折线图;
图3是苹果的不同组织中转运蛋白基因MdERDL6的表达量图;其中,F表示花,YF表示幼果,MF表示成熟果,Pe表示果皮,Fl表示果肉,YL表示幼叶,ML表示成熟叶,OL表示老叶,R表示根,FP表示果柄,LP表示叶柄,P表示韧皮部,X表示木质部;
图4是苹果果实发育不同时期糖转运蛋白基因MdERDL6表达量图;
图5是本发明糖转运蛋白基因MdERDL6结构示意图;
图6是本发明糖转运蛋白MdERDL6的跨膜结构域分析图;
图7是本发明的糖转运蛋白基因MdERDL6亚细胞定位图;其中,图7A共聚焦显微镜观察到的液泡膜定位marker的蓝色荧光糖转运蛋白,图7B是观察到的目的基因MdERDL6-GFP绿色荧光蛋白;图7C是叶绿体自发荧光,图7D是明场下的细胞视野,图7E是合并图像,图7F是空载体在荧光显微镜下的视野;图7G是空载体在荧光显微镜下叶绿体自发荧光;图7H是空载体明场下的细胞视野;图7I是合并图像;
图8是本发明实施例5中得到的MdERDL6的糖转运特性的结果图,图8A是以葡萄糖为唯一糖源的酵母生产情况,图8B是以果糖为唯一糖源的酵母生长情况,图8C是以蔗糖为唯一糖源的酵母生长情况,图8D是以半乳糖为唯一糖源的酵母生长情况,其中CK1和CK2是两个空载体对照,LI和L2是将MdERDL6转入酵母突变体EBY.VW-4000的两个菌株;
图9为本发明实施例克隆的MdERDL6连接到过表达载体的鉴定图;
图10为本发明克隆的MdERDL6转基因番茄的鉴定,图10A是在DNA水平的鉴定,图10B是在mRNA水平的鉴定,图10C是在蛋白水平的鉴定;
图11为番茄的生长状况图,图11A为转基因番茄与野生番茄开花时的对比图;图11B为转基因番茄与野生番茄结果时的对比图。
图12为番茄植株的茎粗统计图;
图13为番茄植株的茎高统计图;
图14为番茄植株叶片糖含量图;其中,图14A为番茄植株叶片葡萄糖含量图;图14B为番茄植株叶片果糖含量图;图14C为番茄植株叶片蔗糖含量图;
图15为番茄果实糖含量图;其中,图15A为番茄果实葡萄糖含量图;图15B为番茄果实果糖含量图;图15C为番茄果实蔗糖含量图,图15D为番茄果实可溶性固形物的含量图。
具体实施方式
下面结合附图和具体实施方式对本发明进行详细说明。
实施例1,苹果糖转运蛋白基因MdERDL6家族遗传进化分析
如图1所示,以拟南芥AtERDL6(AT1G75220)的蛋白序列为模板在蔷薇科基因组数据库(https://www.rosaceae.org/)的苹果基因组数据库GDDH13v1.1版本中进行Blastp,得到了同源性最高的一个基因,命名其为MdERDL6(MDP0000341008MD15G1026400)。其与拟南芥AtERDL6基因的蛋白序列一致性约为83.10%。
实施例2,苹果糖转运蛋白基因MdERDL6表达模式及与果实糖积累的关系
取“嘎拉”苹果花后0天、15天、30天、45天、60天、75天、90天、105天、120天果实样品进行糖含量测定,每个样品设三个生物学重复,苹果果实糖含量测定方法如下,
(1)将样品液氮研磨成粉末,每个样品称重0.1g左右于2ml硅化离心管并记录具体重量;
(2)加入1.4ml的75%甲醇抑制酶活,并加入100μl Ribitol(400ppm)作为内参对照,立即涡旋振荡混匀;
(3)950rpm,70℃金属浴震荡30min,11000g离心10min,取上清液至烘干10ml玻璃管;
(4)加入750μl三氯甲烷和1400μL ddH2O,涡旋振荡,2200g离心15min;
(5)取2μl上清液至1.5ml离心管中,真空干燥30min,其余上清液-50℃备样;
(6)加入40μl甲氧基胺盐酸盐,950rpm,37℃金属浴震荡2h,并做空白对照;
(7)加入60μl MSTFA进行衍生,终体积为100μl,300rpm,37℃金属浴震荡30min,将全部液体装入测糖专用玻璃管;
(8)将样品上样,用气相色谱和质谱联用仪测定;
(9)手动积峰,并计算各种糖酸的含量。
注:(1)三氯甲烷和75%甲醇需放置在-20℃,其余试剂放置在4℃;
(2)干燥2μl或5μl样品,使用5mg/ml的甲氧基胺盐酸盐,干燥20μl或50μl样品,使用20mg/ml的甲氧基胺盐酸盐。
检测结果如图2所示,葡萄糖、果糖和蔗糖含量随着果实发育成熟不断升高,山梨醇含量不断降低,其中果实中果糖含量最高,蔗糖、葡萄糖次之,山梨醇含量最低。
取苹果不同组织,包括花、幼果、成熟果、果皮、果肉、幼叶、成熟叶、老叶、根、果柄、叶柄、韧皮部、木质部,在液氮中研磨并称取0.1g左右,用天根生化科技(北京)有限公司生产的RNAprep Pure多糖多酚植物总RNA提取试剂盒(DP441)提取植物总RNA,并使用宝日医生物科技有限公司生产的PrimeScriptTM 1st Strand cDNA Synthesis Kit合成cDNA,具体操作步骤详见试剂盒操作步骤。基因表达量使用Applied Biosystems ABI 7500型定量PCR仪,定量引物为qMdERDL6-F:GGCATCATATCAGTGGTTGG,qMdERDL6-R:AGGTTCCTCCACTGCTCCAT。分析糖转运蛋白基因MdERDL6在这些组织中的表达量如图3所示,结果显示MdERDL6在成熟果、果皮和果肉中的表达量最高,其次是花和果柄,表明MdERDL6具有果实定位特异性的特点
同时,对苹果花后0天、15天、30天、45天、60天、75天、90天、105天、120天果实样品,分析糖转运蛋白基因MdERDL6的表达量,植物总RNA提取,cDNA合成以及实时荧光定量分析方法与上一步一致,结果如图4所示,随着果实的发育成熟,MdERDL6的表达量逐渐增大,这与果实发育过程中糖含量的变化呈正相关,表明MdERDL6参与了果实发育过程中糖的积累。
实施例3,苹果糖转运蛋白基因MdERDL6基本信息
苹果糖转运蛋白MdERDL6在苹果数据库中注释为MFS超家族转运蛋白,该基因的ORF全长为1464bp,编码487个氨基酸,位于苹果基因组第12条染色体上。预估MdERDL6的蛋白质大小为52.8kDa,等电点为8.95。
分析MdERDL6的结构,如图5所示,含有18个extron(外显子)和17个intron(内含子),DNA序列全长为4355bp。该基因相比于其他基因含有较多的内含子和外显子,说明其基因结构和进化状态更为复杂。其中5’UTR长度为275bp,3’UTR长度为372bp。
MdERDL6作为一个糖转运蛋白,如图6所示,其跨膜结构域发现含有12个跨膜区域,中间区域为中央环,且中央环以及氨基酸序列的N端和C端都在胞质内,两边各有6个跨膜区域,符合糖转运蛋白跨膜结构域的基本特征。
实施例4苹果糖转运蛋白基因MdERDL6亚细胞定位
MdERDL6的亚细胞定位使用pmdc83载体,GFP在目的基因的C端,选用载体上的酶切位点Asc I(Sgs I)和Pac I对载体进行双酶切,注意基因中不能包含这两个酶的酶切位点,具体步骤如下,
(1)设计引物时,加酶切位点和保护碱基查看载体图谱和载体序列,排除移码突变的可能,设计得到的引物为:
PacI-S-pMDC83CCTTAATTAATGGGTTCCAGGCAATCC;
AscI-A-pMDC83TTGGCGCGCCATCTGAAGGACCATTGGAT;
(2)用合成的引物扩增DNA序列,并跑琼脂胶检测目的基因条带的亮度,若目的基因的条带较亮,证明其质量较好;
(3)对pMDC83载体在37℃的温度下进行双酶切1h,然后80℃热失活5min,随后降至4℃待用,pMDC83载体双酶切体系如表1所示;
表1 pMDC83载体双酶切体系
对DNA片段在37℃的温度下进行双酶切1h,然后80℃热失活5min,随后降至4℃待用,DNA片段双酶切体系如表2所示,
表2 DNA片段双酶切体系
(4)将酶切产物在22℃的温度下连接20min,即可将MdERDL6成功连到亚细胞定位载体pMDC83上,GFP连在MdERDL6的C端,酶切产物连接体系如表3所示;
表3酶切产物连接体系
PCR检测目的基因是否连接到载体上,MdERDL6的CDS大小为1464bp,条带大小与目的基因大小一致,证明载体构建成功。
提取拟南芥原生质体,采用PEG介导法,同时转入MdERDL6-GFP质粒和液泡膜定位marker质粒,另一组原生质体转入GFP空载体pMDC83作为对照,在共聚焦显微镜下观察。如图7F所示,空载体的绿色荧光充满着整个原生质体,说明空载体可以使用。观察目的蛋白的绿色荧光时,如图7B和图7E所示,看到红色叶绿体被绿色荧光包裹到外面,同时与液泡膜marker完全吻合,说明MdERDL6定位在液泡膜。
实施例5,苹果糖转运蛋白基因MdERDL6糖转运特性
为了鉴定苹果MdERDL6对不同糖的转运能力,将苹果糖转运蛋白基因MdERDL6去除终止密码子的ORF片段连接到表达载体pYES2.0,构建载体具体步骤如实施例4中构建载体的方法,并转化到酵母突变体EBY.VW4000,该酵母突变体仅对麦芽糖有转运活性,转化酵母使用宝日医生物科技有限公司生产的Quick&Easy Yeast Transformation Mix试剂盒。选取载体上的AgeI和Pme I两个酶切位点,引物序列为:
AgeI-MdERDL6-F:ACGCACCGGTATGAGTTTCCGGGAAGACAG,
PmeI-MdERDL6-R:
AGCTTTGTTTAAACTCATCTGAAGGACCATTGGATCT。
利用酵母YPD培养基培养上述转化酵母株系,检测培养基中添加果糖、葡萄糖、蔗糖、半乳糖,判断不同糖类对酵母生长的影响及其对己糖的吸收能力。每个酵母菌斑用移液器滴入8μL的酵母菌液(酵母菌液培养时间一致)。YPD培养基配置方法为:
将下列试剂溶于100m L水中:1g酵母提取物、2g蛋白胨、2g糖源;固体培养基:加入2g Agar;高压灭菌20min;室温保存或降温后倒板。
2天后观察酵母生长情况,如图8所示,因转化空载体的酵母株系只能在含有麦芽糖的培养基上生长,所以酵母CK1和CK2呈现透明菌斑。如图8A所示,在葡萄糖为唯一糖源的培养基上,带有目的基因的酵母菌斑长势很旺,几乎完全不透明;而如图8B、8C和8D所示,在其他几个糖源的培养基上,酵母只在菌斑边缘生长,中间几乎为透明的。结果表明,MdERDL6对葡萄糖有最强的转运活性。
实施例6苹果糖转运蛋白基因MdERDL6过表达分析
(1)载体构建
过表达载体使用Gateway系统的pGWB402载体,该载体含有attb1和attb2位点,该载体有35S启动子作为强启动子,分别通过BP反应和LR反应将目的基因连到载体上,引物序列为:
MdERDL6-402-F:
GGGGACAAGTTTGTACAAAAAAGCAGGCTATGAGTTTCCGGGAAGACAG,
MdERDL6-402-R:
GGGGACCACTTTGTACAAGAAAGCTGGGTTCTAAAGGACCATTGAATCT。
具体按照下述步骤进行:
a.经过测序的目的基因片段,通过添加attb酶切位点的引物PCR,产生带有attb位点的基因片段。
b.BP反应:用donor 222载体与attb-PCR产物反应,25℃反应过夜,反应产物转化TOP10感受态大肠杆菌,涂板,挑取阳性克隆。
c.用M13通用引物检测阳性克隆。
d.提取质粒,进行LR反应。用402植物表达载体与BP产物反应。25℃温育过夜。反应产物转化TOP10(大肠杆菌感受态细胞),涂板,挑取阳性克隆。
e.用attb通用引物PCR检测阳性克隆。
将MdERDL6成功连到过表达载体pGWB402上,PCR检测图片如图9所示,左侧条带是2000bp DNA marker,右侧为目的基因PCR条带。MdERDL6的CDS大小为1464bp,条带大小与目的基因大小一致,证明载体构建成功。
(2)MdERDL6过表达转化番茄及鉴定
为了探索MdERDL6在糖转运和积累中的具体功能,将MdERDL6通过农杆菌转化法异源表达到“小汤姆”番茄中。番茄作为探究基因功能的模式植物,生长周期快,能产生大量的花和果实,尤其适合探究在果树果实中大量表达的基因功能。
在卡那霉素培养基筛选之后,对转基因MdERDL6番茄叶片取样,同时使用野生的番茄株系作为对照,用质粒做模板的阳性对照,用水做模板的阴性对照,分别将其DNA水平、RNA水平和蛋白水平分别做了鉴定,得到了如图10A所示的DNA水平的鉴定图、如图10B所示的mRNA水平图和如图10C所示的蛋白水平鉴定图,其中,M是2000bp DNA marker,WT是野生对照株系,OE-1和OE-2是两个过量表达MdERDL6的转基因番茄株系,P是用质粒做模板的阳性对照,Control是用水做模板的阴性对照。从图10A、图10B和图10C可以看到在野生番茄中检测不到MdERDL6的表达,转基因株系在DNA水平、mRNA水平和蛋白水平明显过量表达,确定了转基因株系的可靠性。
(3)MdERDL6过表达番茄植株对生长的影响
如图11A和图11B所示,将MdERDL6过表达到番茄中后,观察到植株生长发生了明显的变化,转基因植株与野生对照型植株相比,开花结果明显延迟,大约延迟20-30天。
对转基因株系的主茎的茎粗和野生株系的茎粗进行测量,如图12所示,WT为野生株系,OE-1和OE-2均为转基因株系,转基因株系能明显使主茎增粗增长,茎粗由野生型的4.25mm增加到6.37mm和6.45mm,增加了50%和52%;
对转基因株系的主茎的茎高和野生株系的茎高进行测量,如图13所示,WT为野生株系,OE-1和OE-2均为转基因株系,株高由野生型的18.06cm增加到28.06cm和31.00cm,增加了55%和72%,这表明本发明克隆的苹果MdERDL6基因参与调控植株的生长发育,具有能够延迟植株开花结果的应用价值和促进植株增高增粗的应用价值。
(4)MdERDL6过表达番茄植株对叶片和果实糖含量的影响
MdERDL6是一个糖转运蛋白,与苹果果实中糖的积累密切相关。将该基因异源表达到番茄中,观察糖含量是否会发生变化。取叶片磨样,通过气质联用系统GC-MS测得了糖含量的变化,具体方法如实施例2。检测结果如图14A所示,葡萄糖由原来的2.6mg/g增加到5.3mg/g和5.6mg/g左右,增加到野生番茄的2倍左右,;如图14B所示,果糖由原来的0.5mg/g增加到2.6mg/g和3.0mg/g左右,增加到野生番茄的5倍左右;如图14C所示,蔗糖由原来的3.4mg/g增加到7.4mg/g和7.8mg/g左右,增加到2倍多,转基因株系叶片中糖含量明显增加。
MdERDL6是在苹果果实中高度表达,并与糖积累高度相关的糖转运蛋白。由于苹果是多年生乔本植物,童期较长,通过转基因苹果技术得到过量表达MdERDL6的转基因果实过程较长,而番茄的果实便成为研究这一类基因的模式植物。前面测定转基因番茄叶片中糖含量明显上升,并发现液泡膜糖转运蛋白SlTST表达明显上调,于是进一步测定了转基因番茄果实中的糖含量。
通过GC-MS分析,果实中的葡萄糖、果糖和蔗糖含量也发生了明显的上升。如图15A所示,葡萄糖由原来的14.9mg/g增加到28.0mg/g和27.3mg/g,增长了88%和83%;如图15B所示,果糖由原来的18.1mg/g增加到31.0mg/g和29.8mg/g,分别增长了70%和63%;如图15C所示,蔗糖由原来的2.3mg/g增加到4.9mg/g和4.7mg/g,分别增长了115%和109%;如图15D所示,可溶性固形物的含量由原来的5.1mg/g增加到7.8mg/g和7.5mg/g,分别增长了52%和48%,这也充分说明MdERDL6异源表达可以引起番茄果实糖含量的大量积累。比较番茄叶片和果实中的糖含量,发现果实中蔗糖含量低于叶片中的含量,而积累了较多的葡萄糖和果糖,这可能是果实风味较浓的原因。
SEQUENCE LISTING
<110> 西北农林科技大学
<120> 苹果果实糖转运蛋白基因MdERDL6及其应用
<130> 2019
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1464
<212> DNA
<213> 苹果
<400> 1
atgagtttcc gggaagacag tgaagagggg aggggcgatc tgcggaagcc cttcctccac 60
actgggagtt ggtaccgcat gggttccagg caatccagca tgatgggctc gtcgcaggtg 120
attcgagata gctccatctc tgtggtcgct tgtgtcatga ttgtcgctct gggtcctatc 180
caattcggct tcacctctgg ctattcttct ccaacccaag cagcaatcgt caaggatcta 240
aagcttacag tatcagagta ttcgcttttc ggttctttgt cgaatgtggg agctatggtt 300
ggagctatag ccagcggtca gatttccgag tatattggac gcaaagggtc tttaatgatc 360
gcggctattc ctaatgttat cggttggctt gtcatatcat ttgccagaga ttcttctttt 420
ctctacatgg gaaggttgtt ggaaggattt ggcgtgggaa taatctccta cacggtgcct 480
gtatatatag ctgagatagc acctcaaaac ttaagaggcg ctttgggttc agtcaatcag 540
ctctctgtaa cgattggaat actgctggct tatctgcttg gactttttgt tcaatggagg 600
atacttgcag ttctgggaat attgccttgt acgatattga tacctgggct ctttttcatt 660
ccagaatctc ctcgatggct ggcaaaaatg ggcatgacag aggattttga agcttctctg 720
caagttctcc gaggatttga tacggatatt tcggttgaag tcaatgaaat caagaggtct 780
gtagcatcaa catccaaaag aacaacaatt cggttttcac agcttaaaca aagaagatat 840
tggcttcctc taatgattgg aattggttta cttgttcttc aacaactaag tggaattaac 900
ggtgttctat tctattccac taccattttt gaatctgcag ggatttcgtc aggtaatgta 960
gccacagttg gtctcggagc tgttcaggtc atagcgactg gggtgactac atggttggca 1020
gacaaagcag gccgtcgtct tttgcttatt atctcttctg ctggaatgac gattttcctc 1080
ctcattgttg cgatatcatt ctacataaag gatcttgtgg acgttgattc aaatatttat 1140
agcatattgg gcatcatatc agtggttgga gttgtggcca tggtaatttc attctctctg 1200
ggtatgggag ctattccatg gcttataatg tctgagattc ttccaattaa tattaaaggc 1260
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gcaaacttgc tactggaatg gagcagtgga ggaaccttca ccatttacat gctggtgagt 1380
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Lys Leu Thr Val Ser Glu Tyr Ser Leu Phe Gly Ser Leu Ser Asn Val
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Gly Ala Met Val Gly Ala Ile Ala Ser Gly Gln Ile Ser Glu Tyr Ile
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Gly Arg Lys Gly Ser Leu Met Ile Ala Ala Ile Pro Asn Val Ile Gly
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Trp Leu Val Ile Ser Phe Ala Arg Asp Ser Ser Phe Leu Tyr Met Gly
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Arg Leu Leu Glu Gly Phe Gly Val Gly Ile Ile Ser Tyr Thr Val Pro
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Ser Val Asn Gln Leu Ser Val Thr Ile Gly Ile Leu Leu Ala Tyr Leu
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Arg Trp Leu Ala Lys Met Gly Met Thr Glu Asp Phe Glu Ala Ser Leu
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Claims (8)
1.苹果果实糖转运蛋白基因MdERDL6,其特征在于,核苷酸序列如序列表1所示。
2.含有权利要求1所述苹果果实糖转运蛋白基因MdERDL6的重组过量表达载体。
3.根据权利要求2所述的重组表达载体,其特征在于,所述重组过量表达载体将MdERDL6基因通过同源重组的方法构建到pGWB402载体中。
4.含有权利要求1所述苹果果实糖转运蛋白基因MdERDL6的重组菌和转基因植物组织。
5.权利要求1所述的苹果果实糖转运蛋白基因MdERDL6编码的蛋白,其特征在于,氨基酸序列如序列2所示。
6.权利要求1所述的苹果果实糖转运蛋白基因MdERDL6在构建延迟开花结果的转基因植物中的应用。
7.权利要求1所述的苹果果实糖转运蛋白基因MdERDL6在构建促进茎增粗增高的转基因植物中的应用。
8.权利要求1所述的苹果果实糖转运蛋白基因MdERDL6在提高植物果实糖含量中的应用。
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CN116286869A (zh) * | 2023-03-23 | 2023-06-23 | 石河子大学 | 一种羽毛针禾糖转运蛋白基因SpSWEET14在提高植物抗寒性中的应用 |
CN116286869B (zh) * | 2023-03-23 | 2024-04-05 | 石河子大学 | 一种羽毛针禾糖转运蛋白基因SpSWEET14在提高植物抗寒性中的应用 |
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