CN116042653B - 一种玉米ZmMYB155基因在调控玉米籽粒形态和淀粉合成过程中的应用 - Google Patents
一种玉米ZmMYB155基因在调控玉米籽粒形态和淀粉合成过程中的应用 Download PDFInfo
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Abstract
本发明公开了一种玉米ZmMYB155基因在调控玉米籽粒形态和淀粉合成过程中的应用,涉及基因工程技术领域。该基因具有如SEQ ID NO.1所示的核苷酸序列。该基因调控玉米籽粒形态和淀粉含量的合成,ZmMYB155通过与ZmGBSSⅠa、ZmBT2、ZmPTST1等基因启动子结合从而调控基因的表达,进而影响到胚乳发育和淀粉含量以及籽粒大小,调控玉米籽粒淀粉的合成。该基因的发现,对创建优良的高产玉米品种,具有重要的理论和实践意义。
Description
技术领域
本发明涉及基因工程技术领域,特别涉及一种玉米ZmMYB155基因及其应用。
背景技术
如何创建优良的高产玉米品种,归其根本是提高玉米籽粒胚乳的发育,因此研究玉米的胚乳生物合成的调控过程并进行应用是人们研究的重要方向。研究表明,玉米胚乳的发育就是淀粉、蛋白质合成和贮存的过程,而淀粉的的合成需要经过多种酶的催化和转运。根据分子生物学研究和转录组测序方法发现了许多调控玉米淀粉合成的功能基因和调节它们转录因子。淀粉通常在叶绿体和淀粉体中合成的,在这个过程中需要经过一系列的酶催化和调控。在叶绿体中,通过呼吸作用、卡尔文循环和其他代谢途径合成了1-磷酸葡萄糖,后在ADPase的催化下生成淀粉合成前体(ADPG),经过一系列的酶促反应最终合成直链淀粉和支链淀粉。
在玉米淀粉合成的过程中,不同的淀粉合成相关酶在授粉后到成熟期的酶活不尽相同,经过荧光定量PCR测定,发现各种酶基因的表达量呈抛物线形式。并且许多淀粉合成酶的基因表达并不是自发的,是受各种转录因子调控的。研究较多的是DOF家族的ZmDOF1、ZmDOF2、ZmDOF3、ZmDOF36等转录因子,还有NAC家族的ZmNAC34、ZmNAC36、ZmNAC128和ZmNAC130等,以及bZIP家族中的ZmbZIP91、ZmbZIP22等基因都可以通过自身表达或者调控下游靶基因来调控淀粉合成。而MYB家族中在胚乳中高表达的转录因子鲜有报道,仅有ZmMYB14作为调节转运子ZmBT1的关键调节基因,参与到淀粉的生物合成。
发明内容
本发明的主要目的在于提供一种玉米ZmMYB155基因及其应用,以提供一种属于MYB家族的调控玉米淀粉合成的新基因及其在玉米胚乳发育和淀粉合成过程中的应用。
为实现上述目的,本发明采取的技术方案为:
本发明提供一种玉米ZmMYB155基因,该基因具有如SEQ ID NO.1所示的核苷酸序列,全长1296bp。
本发明还提供一种上述玉米ZmMYB155基因在调控玉米籽粒形态和淀粉合成过程中的应用。
进一步改进在于,所述玉米ZmMYB155基因的缺失对玉米籽粒形态和淀粉含量合成具有负调控作用,包括:籽粒形体和胚的形态缩小,直支链淀粉、蛋白质、油脂含量降低,淀粉合成速度降低,淀粉粒数量减少,淀粉颗粒形体变小。
进一步改进在于,所述玉米ZmMYB155基因的缺失对玉米可溶性糖含量具有正调控作用。
本发明还提供一种质粒载体,所述质粒载体为通过在pGBKT7载体上插入如上述玉米ZmMYB155基因获得。
本发明还提供一种遗传工程化的宿主细胞,所述宿主细胞为含有如上述质粒载体的大肠杆菌感受态TransT1细胞或农杆菌感受态GV3101细胞。
本发明还提供一种玉米ZmMYB155基因的获得方法,以玉米ZmMYB155基因的核苷酸序列为模板设计引物,以玉米B73自交系品种为材料,采用Trizol法提取RNA,提取的RNA反转录成cDNA,以其为模板进行RT-PCR扩增获得玉米ZmMYB155基因。
进一步改进在于,所述RT-PCR扩增的引物为:
qPCR-ZmMYB155-F:AACAATGACGACCTTGACCTAG;
qPCR-ZmMYB155-R:TCATGCACACCAAGATACTCTT。
本发明还提供一种亚细胞定位载体,所述亚细胞定位载体为将上述玉米ZmMYB155基因和pCAMBIA1305载体同时用SpeI和BamHI切割后采用同源重组的方法连接获得,具体为35S::ZmMYB155-GFP载体,连接时用上下游引物如下:
P1305-ZmMYB155-F:
GGACAGCCCAGATTCAACTAGTATGGAATCCGATGAGTCTGCC;
P1305-ZmMYB155-R:
GCCCTTGCTCACCATGGATCCTCACTTGAACCTGGCCAAGC。
与现有技术相比,本发明具有如下有益效果:本发明提供了一种玉米ZmMYB155基因及其应用,ZmMYB155基因调控玉米籽粒的发育和淀粉的合成,ZmMYB155通过与ZmGBSSⅠa、ZmBT2、ZmPTST1等基因启动子结合从而调控基因的表达,进而影响到胚乳发育和淀粉含量以及籽粒大小,调控玉米籽粒淀粉的合成。该基因的发现,对创建优良的高产玉米品种,具有重要的理论和实践意义。
附图说明
图1为本发明的ZmMYB155基因组织表达模式图,其中A:ZmMYB155基因组织表达模式分析图,B:授粉后胚乳表达模式分析图;
图2为本发明的ZmMYB155基因亚细胞定位分析图,其中A:玉米原生质体亚细胞定位图,B:烟草亚细胞定位图;
图3为本发明的ZmMYB155基因转录自激活分析图;
图4为本发明的myb155突变体验证图,其中A:myb155突变体表型图,B:ZmMYB155基因突变位点及验证图,C:myb155突变体籽粒图,D:ZmMYB155基因胚乳表达模式图;
图5为本发明的myb155突变体表型分析图,其中A:myb155突变体籽粒表型图,B:myb155突变体籽粒粒长图,C:myb155突变体籽粒粒宽图,D:myb155突变体籽粒粒厚图,E:myb155突变体百粒重图;
图6为本发明的myb155突变体内含物分析图,其中A:myb155突变体籽粒横切图,B:myb155突变体籽粒纵切图,C:myb155突变体籽粒γ衍射图,D:myb155突变总链淀粉含量图,E:myb155突变体支链淀粉含量图,F:myb155突变体直链淀粉含量图,G:myb155突变体可溶性糖含量图,H:myb155突变体脂肪含量图,I:myb155突变体蛋白质含量图,J:myb155突变体含水率测定图,K:myb155突变体纤维测定图;
图7为本发明的myb155突变体淀粉粒表型分析图,其中A:myb155突变体淀粉粒数量分布图,B:myb155突变体淀粉粒体积分布图,C:myb155突变体表面积分布图,D:myb155突变体脱支淀粉分子量分布图,E:myb155突变体淀粉长度分布图;
图8为本发的myb155突变体淀粉粒扫描电镜图;
图9为本发明的myb155突变体籽粒切片扫描电镜图;
图10为本发明的myb155突变体淀粉粒透射电镜图;
图11为本发明的myb155突变体转录组分析图,其中A:淀粉和蔗糖代谢途径基因表达变化图,B:淀粉合成途径中有关淀粉相关合成基因表达量变化图,C:转录组数据的结果;
图12为本发明的淀粉合成相关酶及糖代谢基因表达量分析图;
图13为本发明的酵母单杂实验图;
图14为本发明的LUC烟草叶片活体荧光成像图;
图15为本发明的p1305载体图谱。
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、材料与试剂
玉米品种为B73自交系,玉米品种的培养条件:玉米材料每年4月到11月种植于种植园,11月到3月在海南繁种基地种植,种植环境依当地气候条件决定。
玉米ZmMYB155基因,核苷酸序列如SEQ ID NO.1所示。
烟草为品种为本氏烟草,烟草材料的培养条件:烟草种植在温室中,光照强度0.350mmols-1m-2,温度22±1℃,空气湿度在60%-80%,16h光照/8h黑暗。
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
2.1 ZmMYB155基因组织表达模式分析
为验证ZmMYB155基因是否为胚乳中特异性表达,根据Trizol法提取了玉米B73自交系的初生根、气生根、茎杆、叶片、雄穗、花丝、棒心以及在授粉后不同时期的种子和胚乳的RNA,将其反转录成cDNA,利用RT-PCR技术,分析ZmMYB155基因的组织表达结果,如图1A、1B。
从图中可以很明显的观察出ZmMYB155基因在授粉后的胚乳中表达量很高,并且在第9d时表达量达最高,但是在其他组织中表达量很低,因此说明ZmMYB155基因的胚乳高表达特性。
2.2 ZmMYB155基因的亚细胞定位
为了进一步了解玉米ZmMYB155基因亚细胞定位特征,构建了亚细胞融合载体,将ZmMYB155基因和pCAMBIA1305载体同时用SpeI和BamHI切割后采用同源重组的方法连接,具体为35S::ZmMYB155-GFP载体,如图15所示,连接时用上下游引物如下:
P1305-ZmMYB155-F:
GGACAGCCCAGATTCAACTAGTATGGAATCCGATGAGTCTGCC;
P1305-ZmMYB155-R:
GCCCTTGCTCACCATGGATCCTCACTTGAACCTGGCCAAGC。
将构建好的35S::ZmMYB155-GFP载体转入提纯后的玉米原生质体细胞中,经两天的暗培养,在激光共聚焦显微镜下观察,发现转入p1305载体在GFP信号通路下,玉米原生质体细胞的细胞膜和细胞核中都有绿色荧光信号,并且转入35S::ZmMYB155-GFP载体和NLS载体的细胞中,在GFP信号通路下细胞膜和细胞核同样出现了绿色荧光,但是在NLS信号通路下,只有细胞核发出红色荧光,如图2A,因此推测ZmMYB155是一个细胞质表达蛋白。为了确保玉米亚细胞定位实验的正确性,又将35S::ZmMYB155-GFP载体转入到了农杆菌感受态GV3101中,将配制好的菌液注射入烟草叶片中暗培养两天,转入p1305载体的烟草叶片在GFP信号通路下,细胞膜和细胞核中都有绿色荧光,转入35S::ZmMYB155-GFP载体的烟草叶片在GFP信号通路下细胞膜和细胞核同样出现了绿色荧光。
如图2B,结果表明ZmMYB155是一个细胞质表达的蛋白,并且可能在胞质中发挥着一定的作用。
2.4 ZmMYB155基因的转录活性分析
2.4.1ZmMYB155基因的转录活性实验
ZmMYB155属于MYB家族转录因子,对ZmMYB155基因的转录自激活活性和转录活性区域进行验证。将ZmMYB155与pGBKT7载体相连接转化到TransT1,将测序正确得质粒转入Y2HGold,涂布在SD/Trp固体培养基,培养3-5d。挑取阳性单克隆稀释点在SD/Trp/His/Ade平板中,共转pGBKT7-53、pGADT7-T为阳性对照,单独转化pGBKT7空载为阴性对照,培养3-5d。
结果如图3,观察记录,发现ZmMYB155在酵母三缺板上能够正常生长,说明ZmMYB155基因在酵母细胞中具有转录自激活活性。
2.4.2 ZmMYB155基因的转录活性区段分析
每个转录因子都具有自己的转录活性区域,为确定ZmMYB155的具体转录活性区域,将ZmMYB155基因分成了4段,并且设计了相对的引物与pGBKT7载体连接后,转入Y2HGold中,涂布在SD/Trp固体培养基中培养3-5d。挑取阳性单菌落稀释点在SD/Trp/His/Ade平板中。
实验结果如图3,最终确定ZmMYB155基因转录活性区域在第一个MYB结构域上。
2.5 myb155突变体的验证
从安徽农业大学玉米EMS突变体库(http://www.elabcaas.cn/memd/public/index.html)中选购ZmMYB155基因的EMS突变体,简称myb155。因为经过EMS诱变的突变体具有遗传不稳定性,所以本发明对突变体材料进行了回交,从回交后的玉米材料中挑选杂合材料再自交,经过连续多代的自交,从中挑选出遗传性状稳定的纯合突变体。从玉米EMS突变体库(http://www.elabcaas.cn/memd/public/index.html)中下载了该基因的突变后的基因序列,经过对所得到的基因序列分析,发现myb155突变体是属于碱基替换型突变,在ZmMYB155基因5’端第2865号碱基由胞嘧啶(G)突变成了胸腺嘧啶(T),导致ZmMYB155基因的转录提前终止,完全破坏了ZmMYB155蛋白的序列和结构,如图4B。根据突变体库的得到的基因序列设计了引物,并且以提取的回交后代总DNA为模板,进行了PCR基因克隆,根据测序结果,挑选基因型为杂合的玉米材料进行后续实验。继续种植杂合玉米材料,通过自交获得纯合的myb155突变体。提取了同时期种植的玉米B73自交系和myb155突变体材料的根、茎、叶以及授粉后不同时期的的胚乳的总RNA,并且进行了反转录得到了cDNA,利用RT-PCR技术测定了包含ZmMYB155基因的表达量。
发现与野生型玉米材料相比,ZmMYB155基因在授粉后的胚乳中表达量发生了明显的下降,因此断定ZmMYB155基因的表达被抑制,如图4D。
2.6 myb155突变体种子农艺性状和淀粉含量以及结构分析
2.6.1 myb155突变体种子农艺性状测定
以玉米myb155纯合突变体为研究对象,并且对其进行了包括了玉米籽粒的粒长、粒宽、粒厚和百粒重的统计学分析。
结果如图5中的图A-E所示。myb155突变体籽粒的粒长、粒宽、粒厚以及百粒重都极显著低于野生型,说明ZmMYB155基因的缺失导致了玉米籽粒的形体发生了缩小的改变。
2.6.2 myb155突变体玉米籽粒淀粉含量的测定
对玉米籽粒中的淀粉含量进行测定,其中包括总淀粉含量测定、直链淀粉含量的测定和支链淀粉含量的测定。为了保证实验的严谨性,把突变体种子的胚去掉一部分提取DNA,经PCR、测序验证为纯合突变体再将其混合破碎成粉,过100目筛,过夜烘干,以备后续实验。使用试剂盒测定总淀粉、支链淀粉、直链淀粉和可溶性还原糖的含量,同时还对籽粒的脂肪、蛋白质、含水率以及纤维含量进行了测定,每个实验分别做三个生物学重复以及三次技术重复。
结果如图6所示。首先,对籽粒进行了横切,发现myb155突变体中的粉质胚乳增多,蜡质胚乳几乎看不见,如图6A。然后又对籽粒进行纵切,发现突变体中的胚占比总体变小,如图6B。为了更加明确籽粒中胚乳发生的变化,对籽粒进行了γ衍射,其中红色为蜡质胚乳,黑色为粉质胚乳,如图6C。很明显的看出myb155突变体籽粒蜡质胚乳含量的降低,因此更加确定ZmMYB155基因的缺失对其胚乳的一个性质的改变。其中7D所表示的是总淀粉含量的测定结果,野生型总淀粉含量在75.9%,myb155突变体材料总淀粉含量在66.28%,相差了近10%。如图6E、F,而直链淀粉与支链淀粉也比野生型分别低了4.5%和3%,经过计算,这都是极显著低于野生型中淀粉的含量的,胚乳中的主要成分就是淀粉,而myb155突变体的淀粉含量远小于野生型,这也就导致了其胚乳的大小发生了改变。因此ZmMYB155基因的缺失对玉米籽粒大小和淀粉的含量产生了一定的影响。
对myb155突变体籽粒的可溶性还原糖进行测定,发现突变体中的还原糖含量达到了17%,如图6G,竟然显著高于野生型中还原糖的含量。在淀粉的合成中,糖是淀粉合成的原材料,myb155突变体中的淀粉含量下降,但是糖的含量却发生了上升,这也与上面对突变体淀粉合成相关酶的基因的表达量降低所导致的结果对应上。同时对突变体材料的脂肪含量、蛋白质含量进行了测定,发现它们的含量都发生了下降,如图6H、I,蛋白质和脂肪的减少也证明了籽粒中胚组织的占比较少。但是突变体中的含水率以及纤维含量却没有发生明显的改变,如图6J、K。经过对籽粒多项指标的测定,发现ZmMYB155基因的缺失导致籽粒中多种物质含量的下降,这也与其为胚乳中高表达基因的测定形成了对应。进一步验证了ZmMYB155基因参与到了玉米籽粒胚乳中多种物质的合成,并在其过程中发挥着重要的作用。
myb155突变体的玉米胚乳中的淀粉粒进行了淀粉粒度分布测定,总共对淀粉粒的数量分布、淀粉粒的表面积分布、淀粉粒体积分布和淀粉链长分布进行了测定,结果如图7。将淀粉粒分为三类,直径小于3μm的淀粉粒称小型淀粉粒、3-16μm称中型淀粉粒,大于16μm的称大型淀粉粒。
从图7A中能发现myb155突变体中的淀粉粒的中型淀粉粒的数量要略小于野生型,同时它的大型和小型淀粉粒的数量要略多于野生型,这也就说明了突变体中的淀粉粒结构发生了改变。图7B中能看出myb155突变体小型淀粉粒的表面积分布略高于野生型,而中型的淀粉粒的表面积分布却明显小于野生型,大型淀粉粒虽然在胚乳中的数量较少,但是在突变体中的大直径淀粉粒的表面积还是要比野生型多一些的。从淀粉粒的体积分布上来观察也存在着相同结果,如图7C。从图7D中发现,经过异淀粉酶水解α-1,6糖苷键后,myb155突变体中的长链支链淀粉淀粉和支链淀粉的含量变少了。同时对其聚合性质进行了测定,如图7E。发现,相对于野生型中的长链支链淀粉的数量发生了明显的降低,同时直链淀粉含量也发生了降低,这也会使之链淀粉的结构发生改变,从而影响到淀粉粒的结构发生了变化。上述结果中能够发现一个很明显的现象,就是在相同质量下,突变体中的小型淀粉粒和大型径淀粉粒数量相较于野生型数量增多,并且myb155突变体中大型淀粉粒约占总量的12.5%,而野生型玉米中的大型淀粉粒仅占了11%。因为小型淀粉粒是由大型淀粉粒分化而来,也就对该实验结果提供了更明确的证据。
2.6.3 myb155突变体玉米籽粒淀粉粒扫描电镜观察
从成熟的籽粒中挑选了四个位置进行电镜扫描,结果如图8。从图片中能观察出在野生型A位置的淀粉粒直径明显大于野生型中A位置的淀粉粒并且淀粉粒之间的位置也是比较松散的。在B位置图片中发现野生型的淀粉粒排列紧凑且淀粉粒直径相对于突变体偏大,但是myb155突变体的淀粉粒排列就比较稀疏,存在着一定的空腔结构。从C位置的扫描电镜图片中,发现在同等比例的情况下,突变体中的淀粉粒要少于野生型,但是其直径要略大于野生型的淀粉粒,而且突变体中淀粉粒之间的缝隙明显变大,空腔结构也明显变多。在D位置的淀粉粒也存在着很大的差异,首先突变体中的淀粉粒直径要很明显的小于野生型,淀粉粒之间的缝隙也变得很大,空腔结构也变得很多很大,但是相对于野生型中的淀粉粒,突变体中的淀粉粒显得更加圆润,不像野生型中的淀粉粒显得那么棱角分明。
相对于野生型中的淀粉粒,myb155突变体的淀粉粒要显得排列更稀疏,数量相对变少,但也正因为这种稀疏的排列结构导致了myb155突变体中淀粉粒的大小发生了变化,为大型淀粉粒的生物合成提供了空间结构,也许正是因为大型淀粉粒的数量过多,造成了淀粉颗粒形成时空间上的浪费,因而导致了胚乳中的空腔结构,当淀粉中的直链淀粉含量较多时,淀粉粒则越接近球型。并且也与淀粉测定结果所对应上,myb155突变体的直链淀粉要相对于野生型高出4.9%。
2.7 myb155突变体籽粒组织切片和透射电镜观察
2.7.1 myb155突变体籽粒组织切片
对授粉后的10d和15d的籽粒进行切片观察,如图9。在授粉后的第10d时,可以明显的看到myb155突变体的BETL细胞形态发生了明显的褶皱,这种性状会严重影响到营养物质的运输。同时,糊粉层细胞的形态变得相对较大,排列松散,糊粉层作为多种蛋白的载体细胞,这种形态会影响到蛋白的含量以及相应的功能。并且突变体中胚乳细胞排列较为松散,淀粉合成相对缓慢。在授粉后的第15d,发现突变体BETL细胞与野生型相比没有太多差异,说明ZmMYB155基因的缺失影响了BETL细胞的发育,同时观察到糊粉层细胞较野生型的密致排列发生了明显的改变,并且胚乳细胞内淀粉粒的数量也明显少于野生型。结果表明ZmMYB155基因的缺失影响了BETL细胞的发育以及糊粉层细胞的形态,同时因为营养物质运输受到了阻碍,也使得胚乳细胞的发育变得迟缓,淀粉合成受到了一定的影响。
2.7.2 myb155突变体籽粒透射电镜观察
为研究胚乳细胞中淀粉粒形态上所发生的变化,采集授粉后15d的籽粒,进行了透射电镜观察,结果如图10。在透射电镜照片中,可以清晰的观察到细胞的完整形态,并且能够清楚的分辨出多种细胞器和完整的淀粉颗粒形态,在相同位置同一比例尺下的细胞中,可以很明显的看出突变体中的淀粉粒数量相对变少,并且体积上也发生了缩小的改变,同时造粉体的体积变大,线粒体的数量变少,这也会对淀粉合成造成一定的影响。在照片中可以发现野生型中淀粉颗粒体积较大,褶皱明显,而突变体材料中胚乳细胞中淀粉粒表面相对光滑,褶皱明显减少。而且淀粉粒的表面相对光滑,细胞内的造粉体体积变大,但是线粒体数量明显减少。突变体中糊粉层细胞的排列也发生了改变,相较于野生型的紧密排列,显得非常松散。
2.8 myb155转录组测序分析
为研究myb155突变体中淀粉含量下降以及可溶性还原糖含量的异常上升的原因,采集了授粉后的第9d的纯合myb155突变体和野生型的籽粒,进行转录组测序。每个样品总共产生了约6.4GB的数据,经过统计,约测出33700个基因。经过统计学分析,发现myb155突变体中存在1644个基因的表达量在授粉后的第9d籽粒中发生了显著变化,并且其中有445个基因发生了上调,1199个基因发生了下调,如图11A。对这1644个基因进行了KEGGPathway分类,总共分为CellμLar Processes、Environmental Information Processing、Genetic Information Processing、Metabolism、Organismal Systems五大类,发现Metabolism有912个基因的表达量发生了改变,占据了总变化的一半。同时对淀粉和蔗糖代谢途径的基因进行了筛选并做了KEGG Pathway分析,其中89个基因表达量发生显著变化,如图12B。据统计,在淀粉合成途径中发现有关淀粉相关合成的α-淀粉酶、淀粉合成酶、淀粉分支酶、糖转运蛋白等16个基因表达量发生了显著下降,但是β-淀粉酶的含量却发生了显著上升。在糖代谢过程中,发现蔗糖合酶、葡萄糖甘酶、β-葡萄糖甘酶、双糖转移酶、海藻糖6-磷酸合酶、己糖激酶等35个基因表达量发生了显著下调,内切葡聚糖酶、β-呋喃果糖苷酶等36个基因表达量发生了明显的升高。同时统计了ZmDof36、ZmNAC128、ZmbZIP91等15个调节淀粉合成相关的转录因子的表达量变化,发现ZmNAC128、ZmNAC130、ZmNAkd1等7个转录因子的表达量发生了下降,其中O11基因下降了4倍,O2下降了20倍。并且,BETL细胞特异表达基因中有4个基因表达量降低,ESR细胞中有一个特异基因表达量发生了明显下降,如图12C。根据转录组数据的结果,发现淀粉合成相关酶基因的表达量下降与糖代谢相关酶基因表达量的上升就是导致myb155突变体中淀粉含量下降以及可溶性还原糖含量的异常上升的原因。
2.9 myb155突变体淀粉合成相关基因表达量分析
为了验证转录组测序结果,挑选了在淀粉合成通路以及糖代谢过程中表达量发生了显著改变的一些功能基因进行RT-PCR,结果如图12。发现了ZmMYB155、ZmGBSSⅠa、ZmUPG2.3、ZmBT2a、ZmAGPS1A、ZmAGPL2、ZmPTST1等淀粉合成通路上的相关酶的基因表达量相对于野生型发生了明显下降。在玉米胚乳发育的整个时期,突变体中ZmMYB155基因的表达量明显低于野生型,ZmPTST1的表达量在胚乳的整个发育过程中都显著低于野生型,并且在授粉后的第5、10、15d都相差了近五倍。ZmGBSSⅠa基因在myb155突变体的胚乳中表达量很低,在授粉的第20d时,表达量与野生型相差了10倍。ZmUPG2.3基因在myb155突变体中的表达量也明显下降,在授粉后的第5、10d下降了约10倍左右,在第15到20d的时候几乎不表达。ZmAGPL2基因的表达量在授粉后的第5d低于野生型12倍,在第15、20d的时候表达量基本与野生型持平。ZmBT2a基因的表达量也明显低于野生型的表达量,整体的表达量都低了将近三倍。同时,ZmSS1a、ZmSS2a、ZmAGPS1a、ZmAGPL1也都低于野生型的表达量。在淀粉合成的途径中,酶的调控发挥着不可或缺的作用,在myb155突变体中,淀粉合成通路上的相关酶基因表达量发生了变化。切片时发现BETL细胞形态发生了褶皱,所以对BETL基因进行了一个定量分析,发现ZmBETL3、ZmBETL4、ZmBETL9等基因的表达量发生了降低,这也是导致BETL细胞形态发育不良的原因。同时对一些转录因子的表达量进行了测定,发现O2、O11这两个基因发生了下降,ZmDOF36的表达量却发生了上升。说明ZmMYB155基因的缺失不仅影响着淀粉合成通路上的基因表达,同时还影响着糖代谢和BETL细胞基因的表达。
2.10 ZmMYB155基因下游靶基因的筛选
ZmMYB155的突变导致玉米籽粒形体上变得要小一些,并且胚乳中的淀粉含量也有所下降,同时还有变化的是参与到胚乳淀粉生物合成的相关酶的基因的表达量也出现了降低的变化。ZmMYB155作为转录因子,并且在胚乳中特异高表达,并且又具有转录自激活活性,可知ZmMYB155基因可以调控淀粉合成中某些基因的特异性表达,进而调控玉米胚乳中淀粉的合成。
把在玉米胚乳淀粉合成过程中发生调控功能的基因的转录起始位点前2000bp长度的碱基序列从NCBI网站上下载下来,设计引物克隆。结果如图13,能够发现ZmMYB155能与ZmGBSSⅠa、ZmUPG2.3、ZmAGPS1A、ZmBT2、ZmPTST1的启动子序列结合。
同时用烟草做了荧光素酶互补实验,将构建好的p0800-BT2、p0800-GBSSⅠa、p0800-PTST1、p62sk-MYB155、p0800、p62sk等载体分别转到农杆菌感受态中,将验证的阳性单克隆菌液注射入烟草中暗培养36h。根据活体荧光成像仪的成像结果发现注射了p0800-BT2+p62sk-MYB155、p0800-GBSSⅠa+p62sk-MYB155、p0800-PTST1+p62sk-MYB155的荧光区域强度要明显高于p0800-BT2+p62sk、p0800-GBSSⅠa+p62sk、p0800-PTST1+p62sk和p0800+p62sk-MYB155的萤光亮度,如图14。因此证明酵母单杂试验中ZmMYB155能与ZmGBSSⅠa、ZmBT2、ZmPTST1的启动子序列结合。
3、结论
本发明通过对胚乳内高表达基因的生物信息学研究,并经定量分析发现ZmMYB155属于胚乳高表达基因,亚细胞定位发现ZmMYB155基因定位在细胞核和细胞膜上,并且验证其拥有自激活活性,且自激活区段位于第一个MYB结构域上。
myb155突变体其籽粒形体和胚变小,籽粒以粉质胚乳为主,直支链淀粉、蛋白质、油脂含量明显降低,可溶性糖含量明显升高。在授粉后10d的籽粒切片终发现BETL细胞形态发生了褶皱现象,糊粉层细胞和胚乳细胞分布松散,且突变体材料淀粉合成较缓慢。扫描电镜和透射电镜照片显示突变体中淀粉粒数量较少、淀粉颗粒形体变小,表面光滑且排列更加松散。
转录组测序和q-PCR结果表明,淀粉合成通路、糖代谢等途径的相关基因表达量发生了明显的变化,BETL细胞特异基因表达量降低。酵母单杂和烟草LUC实验表明ZmMYB155基因可以与一些淀粉合成、糖代谢等基因的启动子序列结合。同时BETL层细胞形态的改变会影响营养物质的运输,糊粉层细胞结构发生改变,对种子的萌发也会有一定的影响。ZmMYB155基因作为转录因子其在蛋白在细胞膜和细胞核都有荧光信号,可以使其通过调控ZmBT2、ZmPTST1这两种转运蛋白的基因表达来正向调控淀粉合成前期糖转运的过程,并且ZmMYB155基因还可以单独调控ZmGBSSⅠa基因来调控直链淀粉的合成。
以上显示和描述了本发明的基本原理和主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
Claims (1)
1.一种玉米ZmMYB155基因在调控玉米籽粒形态和淀粉合成过程中的应用,其特征在于,所述玉米ZmMYB155基因的核苷酸序列如SEQ ID NO.1所示,所述玉米ZmMYB155基因的缺失使得玉米籽粒形体和胚的形态缩小,直支链淀粉、蛋白质、油脂含量降低,淀粉合成速度降低,淀粉粒数量减少,淀粉粒形体变小;所述玉米ZmMYB155基因的缺失使得玉米可溶性糖含量上升。
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Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2944190A1 (en) * | 2014-05-15 | 2015-11-18 | ETH Zurich | Means and methods for modifying the amylose content in plant starch |
CN105349559A (zh) * | 2015-12-23 | 2016-02-24 | 山东大学 | 玉米ZmWx基因在提高玉米产量和改良籽粒性状中的应用 |
CN107177601A (zh) * | 2017-07-03 | 2017-09-19 | 安徽省农业科学院烟草研究所 | 一种玉米淀粉合成调控基因ZmMIKC2a及其应用 |
WO2019062895A1 (zh) * | 2017-09-30 | 2019-04-04 | 海南波莲水稻基因科技有限公司 | 玉米基因ZmABCG20在调控作物雄性育性中的应用以及与玉米雄性生育力相关的DNA分子标记及其应用 |
CN109929851A (zh) * | 2019-01-29 | 2019-06-25 | 安徽农业大学 | 一种玉米籽粒淀粉合成调控基因ZmDof36及其应用 |
CN110117596A (zh) * | 2019-04-30 | 2019-08-13 | 山东农业大学 | 一种玉米种子灌浆调控基因ZmDef1及应用 |
CN114149999A (zh) * | 2021-12-16 | 2022-03-08 | 安徽农业大学 | 一种玉米淀粉合成调控基因ZmSSP1及其应用 |
CN114410663A (zh) * | 2020-10-28 | 2022-04-29 | 中国农业科学院作物科学研究所 | 玉米ZmDek701基因在调控植物籽粒品质中的应用及其突变体 |
-
2022
- 2022-12-21 CN CN202211646082.8A patent/CN116042653B/zh active Active
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2944190A1 (en) * | 2014-05-15 | 2015-11-18 | ETH Zurich | Means and methods for modifying the amylose content in plant starch |
CN105349559A (zh) * | 2015-12-23 | 2016-02-24 | 山东大学 | 玉米ZmWx基因在提高玉米产量和改良籽粒性状中的应用 |
CN107177601A (zh) * | 2017-07-03 | 2017-09-19 | 安徽省农业科学院烟草研究所 | 一种玉米淀粉合成调控基因ZmMIKC2a及其应用 |
WO2019062895A1 (zh) * | 2017-09-30 | 2019-04-04 | 海南波莲水稻基因科技有限公司 | 玉米基因ZmABCG20在调控作物雄性育性中的应用以及与玉米雄性生育力相关的DNA分子标记及其应用 |
CN109929851A (zh) * | 2019-01-29 | 2019-06-25 | 安徽农业大学 | 一种玉米籽粒淀粉合成调控基因ZmDof36及其应用 |
CN110117596A (zh) * | 2019-04-30 | 2019-08-13 | 山东农业大学 | 一种玉米种子灌浆调控基因ZmDef1及应用 |
CN114410663A (zh) * | 2020-10-28 | 2022-04-29 | 中国农业科学院作物科学研究所 | 玉米ZmDek701基因在调控植物籽粒品质中的应用及其突变体 |
CN114149999A (zh) * | 2021-12-16 | 2022-03-08 | 安徽农业大学 | 一种玉米淀粉合成调控基因ZmSSP1及其应用 |
Non-Patent Citations (1)
Title |
---|
玉米胚乳转录调控网络构建及重要节点转录因子的鉴定;张红;中国知网数据库;第1-92页 * |
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