CN116334101B - 一种玉米甾醇含量调控基因ZmSCYL2及其应用 - Google Patents
一种玉米甾醇含量调控基因ZmSCYL2及其应用 Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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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
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
Abstract
本发明涉及一种玉米甾醇含量调控基因ZmSCYL2及其应用,涉及植物基因工程技术领域,该基因具有如SEQ ID NO.1所示的核苷酸序列,本发明通过构建过量表达载体并将ZmSCYL2基因作为目的基因导入玉米基因组中,发现ZmSCYL2基因过量表达提高了玉米种子中的甾醇含量,表明ZmSCYL2基因参与了玉米种子中甾醇含量调控,可为作物育种提供基因资源,将ZmSCYL2基因应用于玉米育种中,为快速培育高甾醇含量的玉米品种提供依据,对植物育种与应用具有重要的理论和实践意义。
Description
技术领域
本发明属于植物基因工程技术领域,具体涉及一种玉米甾醇含量调控基因ZmSCYL2及其应用。
背景技术
植物甾醇是植物中的活性成分,存在于植物的根、茎、叶、花、果实、种子等器官,种类繁多,是一类化合物的总称,主要代表物质有β-谷甾醇(β-sitosterol)、菜油甾醇(campesterol)、豆甾醇(stigmasterol)等,植物甾醇在植物细胞中发挥着重要作用,比如维持细胞膜的稳定性、参与光合作用、清除自由基等,值得注意的是,植物甾醇是植物细胞的组成部分,广泛存在于自然界中的植物体,人体自身没有合成植物甾醇的能力,唯一获取的途径就是通过膳食,现代研究显示,植物甾醇在降血脂、抗氧化、抗肿瘤、抗炎与免疫调节等方面都有着重要作用,摄入高甾醇含量植物性食品的人群能从日常饮食中获得更多的植物甾醇,从膳食补充、疾病预防、营养添加等多个角度来说,合理选择甾醇含量较丰富的膳食(如玉米油等)显得尤为重要,因此,提高玉米中的甾醇含量是对玉米营养性状的改良,其具有广阔的应用前景。
ZmSCYL2基因是SCYL基因家族的一个成员,SCYL蛋白家族是一类进化保守的、广泛表达的蛋白,可以调节细胞内物质的转运,参与囊泡运输,在动植物的生长发育过程中发挥着重要的作用,但是对植物种子甾醇含量相关作用却未见报道。
发明内容
本发明的目的就在于为了解决上述问题而提供一种玉米甾醇含量调控基因ZmSCYL 2及其应用。
本发明通过以下技术方案来实现上述目的:
一种玉米甾醇含量调控基因ZmSCYL2,该基因具有如SEQ ID NO.1所示的核苷酸序列。
作为本发明的进一步优化方案,所述玉米品种为玉米自交系KN5585。
一种玉米甾醇含量调控基因ZmSCYL2在调控玉米组织的甾醇积累中的应用。
作为本发明的进一步优化方案,所述基因ZmSCYL2过量表达使玉米叶片、玉米种子中甾醇含量上升。
一种玉米甾醇含量调控基因ZmSCYL2的编码蛋白,该编码蛋白具有如SEQ ID NO.2所示的氨基酸序列。
作为本发明的进一步优化方案,该编码蛋白定位于玉米高尔基体中。
一种过量表达载体,通过在玉米表达载体pZZ-EYFP(购买于未米公司)上转入所述玉米甾醇含量调控基因ZmSCYL2获得,具体为pZZ-EYFP-ZmSCYL2基因过量表达载体。
一种高甾醇型转基因玉米品种的获得方法,将所述基因ZmSCYL2作为目的基因导入玉米基因组中进行过量表达,培育获得高甾醇型转基因玉米品种。
本发明的有益效果在于:
本发明通过构建过量表达载体并将ZmSCYL2基因作为目的基因导入玉米基因组中,发现ZmSCYL2基因过量表达提高了玉米种子中的甾醇含量,表明ZmSCYL2基因参与了玉米种子中甾醇含量调控,可为作物育种提供基因资源,将ZmSCYL2基因应用于玉米育种中,为快速培育高甾醇含量的玉米品种提供依据,对植物育种与应用具有重要的理论和实践意义。
附图说明
图1为ZmSCYL2基因组织表达模式分析;
图2玉米GFP-ZmSCYL2基因亚细胞定位表达载体示意图;
图3为ZmSCYL2基因在玉米原生质体中的亚细胞定位;
图4为玉米ZmSCYL2基因过量表达载体示意图;
图5为转基因玉米ZmSCYL2基因的遗传转化过程图(A.侵染后的玉米幼胚;B.长出愈伤组织;C.愈伤组织分化出抗性小苗;D.大试管中壮苗培养);
图6为ZmSCYL2基因过量表达玉米株系的荧光定量表达;
图7为野生型植株(WT)和ZmSCYL2转基因植株的表型与甾醇含量图;
图8为野生型植株(WT)和ZmSCYL2转基因玉米种胚的甾醇含量图。
具体实施方式
下面结合附图对本申请作进一步详细描述,有必要在此指出的是,以下具体实施方式只用于对本申请进行进一步的说明,不能理解为对本申请保护范围的限制,该领域的技术人员可以根据上述申请内容对本申请作出一些非本质的改进和调整。
1、材料
本实施例所用方法如无特别说明均为本领域的技术人员所知晓的常规方法,所用的试剂等材料,如无特别说明,均为市售购买产品。
2、方法
2.1ZmSCYL2基因不同组织表达模式分析
对三叶期玉米自交系B73的根、茎、叶以及成熟的种子进行采集,在RNA的提取以及反转录结束后,以cDNA为模板,使用诺唯赞的SYBR染料和Thermo Scientific PikoR ealCycler荧光定量PCR仪进行荧光定量PCR反应,qRT-PCR反应体系如表1:
表1qRT-PCR反应体系
2×AceQ qPCR SYBR Green Master Mix | 10μL |
SEQ ID NO.3:Primer F(10μM) | 0.4μL |
SEQ ID NO.4:Primer R(10μM) | 0.4μL |
RNase free water | 7.2μL |
稀释10倍的反转录产物 | 2μL |
Total | 20μL |
点样完成后,使用离心机将混合液体混匀并防止有气泡的产生,随后使用封口膜将PCR平板封闭盖严,放入Thermo Scientific PikoReal Cycler荧光定量PCR仪进行PCR反应,反应结束后,使用2–ΔΔCT算法对结果进行分析,计算ZmSCYL2基因的相对表达量,P CR反应程序如表2:
表2PCR反应程序
实验结论:通过组织表达模式分析发现ZmSCYL2基因在种子(Seed)的表达量最高,叶片(Leaf)次之,茎(Stem)的表达量最低,经显著性比较可知根(Root)、茎(St em)和叶片(Leaf)中的ZmSCYL2基因的表达量无显著性差异,种子(Seed)中的表达量差异与其他组织相比较为显著,具体见图1。
2.2ZmSCYL2基因的亚细胞定位分析
为了研究ZmSCYL2基因(核苷酸序列如SEQ ID NO.1所示)的亚细胞定位,使用pCAMBIA1305载体,构建pCAMBIA1305-GFP-ZmSCYL2融合载体(如图2),该载体使用35S启动子用以调控基因的表达,在获取ZmSCYL2基因转录本后,将转录本中的终止密码子去掉,以SpeI和XbaI为酶切位点设计引物:
SEQ ID NO.5:GFP-ZmSCYL2-F:ACTAGTATGGCGCTCAACATGAAGACC;
SEQ ID NO.6:GFP-ZmSCYL2-R:TCTAGAAAGTAAATCCAGGATAGGTTGTTGT CCT;
引物设计完成后,用从玉米自交系B73提取的RNA反转录后的cDNA为模板,根据NCBI提供的关于用高保真酶进行PCR扩增,PCR反应体系和反应程序同2.1一致。
将构建好的pCAMBIA1305-GFP-ZmSCYL2融合载体以及高尔基体定位载体RFP-Man49(玉米高尔基体定位载体RFP-Man49,载体发表于Control of secondary cell wall patterning involves xylan deacetylation by a GDSL esterase.Nature Plants,2017,3,17017.由中国农业科学院惠赠)共转化原生质体中,培养36h后使用激光扫描共聚焦显微镜观察蛋白的定位。
实验结论:如图3所示,ZmSCYL2基因以及RFP-Man49只在原生质体的细胞高尔基体中表达,由此表明,ZmSCYL2基因编码蛋白(氨基酸序列如SEQ ID NO.2所示)是一个高尔基体定位蛋白。
2.3ZmSCYL2基因过量表达株系的鉴定
2.3.1、pZZ-EYFP-ZmSCYL2基因过量表达载体构建
采集玉米自交系KN5585的成熟的种子RNA,将RNA反转录成cDNA,以cDNA为模板,PCR扩增出ZmSCYL2基因序列,使用同源重组克隆(重组克隆试剂盒ClonExpressII)将PCR产物转入表达载体(pZZ-EYFP,购买于未米公司),测序无误后即获得过量表达载体pZZ-EYFP-ZmSCYL2(如图4),其中,PCR扩增ZmSCYL2基因的引物为(含有B amHI和BstEII酶切位点):
SEQ ID NO.7:pZZ-ZmSCYL2-F:GTGTTACTTCTGCAGGGATCCATGGCGCTCAACATGAAGACC;
SEQ ID NO.8:pZZ-ZmSCYL2-R:GGGGAAATTCGAGCTGGTACCCTAAAGTAAATCCAGGATAGGTTGTTG;
采用热击方法将阳性过量表达载体pZZ-EYFP-ZmSCYL2转化至农杆菌感受态细胞中,得到阳性农杆菌,利用SEQ ID NO.9、SEQ ID NO.10引物对农杆菌进行PCR检测,确保用于转化的农杆菌包含目的基因。
2.3.2、ZmSCYL2基因过量表达植株的遗传传化
1)、取玉米自交系KN5585授粉10天左右的玉米穗,保证胚的大小在1.2-2.0mm左右,去掉苞叶和花丝,削去穗子两端,浸泡在含有5%的84消毒液及0.02%的Tween-20的溶液中30min,取出后用无菌水冲洗3次;
2)、在超净工作台上,用无菌的镊子,剥出幼胚,剥好的幼胚放入装有侵染培养基的2mL EP管内,每管50-60个幼胚(如图5A);
3)、将单菌落农杆菌接种在含有卡那霉素和利福平的YEB固体培养基上,28℃黑暗培养24~36h,用接种环刮菌,并将菌混匀于含有10mL侵染培养基的离心管中,200rpm,摇床震荡培养至对数期(OD550为0.3~0.5);
4)、将长出的愈伤组织(如图5B)用侵染培养基清洗2次后吸干液体,使其与活化好的农杆菌侵染液混合,上下颠倒混匀,室温下遮光孵育5min;
5)、将有幼胚的农杆菌侵染液倒入含有无菌滤纸的平皿中,待菌液吸干后,将幼胚转入共培养基上,此时,应使幼胚的盾片朝上,20℃暗培养箱内培养3-4天;
6)、将幼胚转移至恢复培养基上,28℃暗培养箱内培养7-9天;
7)、将幼胚转移至筛选培养基上,28℃暗培养箱内培养,每2周转接一次;
8)、待出现抗性胚状体以后,挑选状态良好的个体将其转移至分化培养基上(如图5C);
9)、将小苗长到1-2cm时,将小苗切下转移至生根培养基(如图5D);
10)、当根系发育好之后,将幼苗转移至温室中土培生长,培养获得过量表达玉米株系的T0代种子。
上述培养过程所涉及到的培养基配方具体如下:
侵染培养基:N6基本培养基、68.4g/L蔗糖、36g/L葡萄糖、0.7g/L L-脯氨酸、0.5mg/L VB1、1.96g/L乙酰丁香酮;pH 5.2。
共培养基:N6基本培养基、30g/L蔗糖、3g/L植物凝胶、0.7g/L L-脯氨酸、0.3g/LL-半胱氨酸、2mg/L 2,4-二氯苯氧乙酸、0.85mg/L硝酸银、0.5mg/L VB1;p H 5.8。
恢复培养基:N6基本培养基、30g/L蔗糖、3g/L植物凝胶、0.5g/L MES、0.7g/L L-脯氨酸、2mg/L 2,4-二氯苯氧乙酸、0.85mg/L硝酸银、0.5mg/L VB1、100mg/L羧苄青霉素、200mg/L特美汀;pH 5.8。
筛选培养基:恢复培养基、10μL双丙氨膦,pH 5.8。
分化培养基:MS培养基、60g/L蔗糖、3g/L植物凝胶、100mg/L羧苄青霉素、10μL双丙氨膦、50mg/L玉米素、100mg/L吲哚乙酸、26μg/L脱落酸;pH 5.6。
生根培养基:MS培养基、40g/L蔗糖、3g/L植物凝胶;pH 5.8。
2.3.3、ZmSCYL2基因过量表达植株筛选
通过转基因技术培育获得过量表达玉米株系的种子后,对T0代种子进行检测,因构建的载体上带有Bar标记基因,故设计引物进行PCR检测,引物为:
SEQ ID NO.9:BarF:CCAGAAACCCACGTCATGCC;
SEQ ID NO.10:BarR:CAGGAACCGCAGGAGTGGA;
Bar PCR反应条件为:94℃预变性5min,94℃变性10sec,60℃退火30sec,72℃延伸30sec,共32个循环,72℃延伸10min,在检测到含有Bar标记基因的株系中,选取了三个不同株系(OE3、OE1、OE2)并提取每个株系的RNA并进行反转录,然后通过荧光定量PCR检测不同转基因株系中ZmSCYL2基因表达量。
实验结论:如图6所示,三个转基因株系(OE3、OE1、OE2)中ZmSCYL2基因表达量显著高于对照组(野生型WT),其中,ZmSCYL2基因在不同的转基因株系中表达量也有所不同,ZmSCYL2基因表达量最高的为OE3株系,OE1株系次之,OE2株系中基因的表达量最低。
2.4玉米苗表型观察及甾醇含量测定
2.4.1、为了研究ZmSCYL2基因对玉米苗表型的影响,本实验鉴定筛选了来自上述三个转基因株系的T1代种子种植在温室进行表型观察。
实验结论:经观察发现,种子发芽长出T2代幼苗一周时,各转基因株系与对照株系(野生型WT)的表型无明显差异(如图7A);
当植株生长60天时,玉米的表型发生了明显变化,无论是从株高还是从叶片来看,转基因株系更具有优势,其株高明显优于对照组(野生型WT),叶片也普遍较大,植株发育的也更快(如图7B)。
2.4.2、为了研究ZmSCYL2基因对玉米甾醇含量的影响,本实验提取上述三个转基因株系的T2代玉米叶片中的甾醇以及三个转基因株系的T2代玉米种胚中的甾醇,并通过GC-MS进行定量定性检测。
实验结论:如图7(DEFG)所示,转基因株系玉米叶片中的植物甾醇(sterol)主要是由菜油甾醇(Campesterol)、豆甾醇(Stigmasterol)和β-谷甾醇(β-Sitosterol)构成,其中β-谷甾醇含量最高,约占总甾醇的80%左右,ZmSCYL2基因表达量最高的株系是OE3株系,最低的株系则是对照组(野生型WT),OE3株系中甾醇的含量约为对照组的2.12倍,OE1株系中总甾醇含量约为对照组的1.64倍,OE2株系中总甾醇含量对照组的1.44倍,差异达到了极显著水平,ZmSCYL2基因的过量表达使玉米叶片中植物甾醇的含量增加;
如图8(ABCD)所示,转基因株系玉米种胚中的植物甾醇(sterol)主要是由菜油甾醇(Campesterol)、豆甾醇(Stigmasterol)和β-谷甾醇(β-Sitosterol)构成,玉米种胚中总甾醇的含量较玉米叶片中的总甾醇含量高,转基因株系玉米种胚中总甾醇的含量约为对照组(野生型WT)的1.61倍(如图8D),ZmSCYL2基因的过量表达不仅增加了玉米叶片中的甾醇含量还增加了玉米种胚中甾醇的含量。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。
Claims (3)
1.过表达玉米甾醇含量调控基因ZmSCYL2在提高玉米组织的甾醇含量中的应用,其特征在于,所述基因ZmSCYL2的核苷酸序列如SEQ ID NO.1所示,所述甾醇为菜油甾醇、豆甾醇和β-谷甾醇。
2.根据权利要求1所述的应用,其特征在于,所述玉米品种为玉米自交系KN5585。
3.根据权利要求1所述的应用,其特征在于,所述基因ZmSCYL2过量表达使玉米叶片、玉米种子中甾醇含量上升。
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