CN107988225B - 一种玉米籽粒发育相关基因miR169o及其应用 - Google Patents
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Abstract
本发明公开了属于生物基因技术领域的一种玉米籽粒发育相关基因miR169o及其应用。所述miR169o的前体核苷酸序列如序列表SEQ ID NO:1所示；其成熟编码序列如序列表SEQ ID NO:2所示。在相同种植和管理条件下，zma‑miR169o过表达玉米株系籽粒单粒体积比对照提高8％‑20％，单粒重增加10％‑20％。其他农艺性状没有明显变化，因此其在提高玉米产量中具有重要应用价值。此外，本发明中获得的zma‑miR169o过表达材料，可以通过回交转育至优异自交系，以创制优良品种，用于玉米育种。
Description
技术领域
本发明属于生物基因技术领域，具体涉及一种玉米籽粒发育相关基因miR169o及其应用。
背景技术
籽粒产量是作物最重要、最复杂的性状之一，由相互关联的多基因控制的一系列生理生化过程的最终体现。玉米籽粒产量由单位面积株数、单株粒数和百粒重决定。在种植密度一定的条件下,单株粒数和粒重直接决定籽粒产量。玉米籽粒中总重量的80％以上为胚乳，胚乳细胞的发育、增殖和充实情况决定了籽粒的重量和品质。
MiRNAs是一类长约22个碱基非编码的RNA分子，它以降解mRNA或抑制其翻译等方式特异地对靶基因进行转录后调控。miRNA的靶基因60％以上为各种调控因子。miRNA像心脏一样在植物的生长发育、逆境应答和种子形成等过程中发挥着关键的调控作用，甚至一个miRNA可以通过靶向不同下游基因而发挥多种调控功能。miR169家族是植物中最大的miRNA家族，有18个前体基因可以加工成为10个成熟的miRNA，分别为miR169a/b，miR169c/r，miR169d，miR169e，miR169f/g/h，miR169i/j/k，miR169l，miR169o，miR169p和miR169q/m/n。miR169的靶基因为NF-YA转录因子，在拟南芥等模式植物中的研究表明，部分miR169/NF-Y调控单元参与了植物逆境应答，关于miR169参与籽粒发育的研究尚不清楚，也没有玉米miR169成员的功能被报道。
专利201610373785.6公开了山核桃miR169在提前植物花期中的应用。应用山核桃miR169，有望调控山核桃的花期，使得山核桃童期缩短，提前开花，结实。但是，该miR169调控的山核桃重量和质量没有增加，无增产意义。专利201010276128.2公开了miR169或其靶基因NFYA5在植物适应氮胁迫中的应用。将miR169在拟南芥中过量表达，增加了拟南芥对于缺氮胁迫的敏感性，相应的过量表达miR169的靶基因NFYA5，降低了拟南芥对于缺氮胁迫的敏感性，增强了拟南芥耐低氮胁迫的能力。通过对这种复杂的调控网络的分析与深入了解，可进一步应用于氮高效农作物的培育。该专利的技术对于直接增加作物产量没有指导意义。
发明内容
本发明的目的在于提供一种玉米籽粒发育相关基因miR169o，用于培育高产农作物。
一种玉米籽粒发育相关基因miR169o，所述miR169o的前体核苷酸序列如序列表SEQ ID NO:1所示；其成熟编码序列如序列表SEQ ID NO:2所示。
包含上述miR169o前体核苷酸序列的表达载体。
包含上述表达载体的工程菌，细胞株。
扩增上述miR169o前体核苷酸序列的引物。
上述miR169o及miR169o的前体基因在培育高产农作物中的应用。
所述农作物为玉米，大豆，水稻，高粱，小麦，油菜中的一种或一种以上。
本发明的有益效果：在相同种植和管理条件下，zma-miR169o过表达玉米株系籽粒单粒体积比对照提高8％-20％，单粒重增加10％-20％。其他农艺性状没有明显变化，因此其在提高玉米产量中具有重要应用价值。此外，本发明中获得的zma-miR169o过表达材料，可以通过回交转育至优异自交系，以创制优良品种，用于玉米育种。
附图说明
图1为表达载体pmiR169o::GUS结构示意图。
图2为表达载体pUBI::miR169o结构示意图。
图3为zma-miR169o在玉米籽粒中的表达模式；DAP为授粉后天数。
图4为zma-miR169o过表达玉米的籽粒表型，A为转基因玉米籽粒表型；B为单籽粒体积；C为单籽粒重量。
具体实施方式
下面结合附图和具体实施例对本发明做进一步说明。
实施例1玉米zma-miR169o前体序列cDNA的克隆
以玉米B73的cDNA文库为模板进行RT-PCR扩增，PCR引物如下：zma-miR169o F：5'-GTAGCCAAGAATGACTTGCC-3'(SEQ ID NO:3)；zma-miR169o R：5'-TGGCTAGCCAAGAAGACCTG-3'(SEQ ID NO:4)。
扩增条件如下：
扩增出DNA片段从琼脂糖胶中回收，克隆至pEASY-T1载体。酶切并测序鉴定，命名为pTmiR169o。测序结果表明：得到前体为序列1所示的miRNA，为zma-miR169o，全长138bp，具体序列如序列表SEQ ID NO:1所示。其加工成的成熟序列如序列2所示，共21个nt，其序列如序列表SEQ ID NO:2所示。
实施例2 pmiR169o:GUS表达载体的构建
1)设计扩增zma-miR169o前体序列上游2.0kb长的引物，在引物两端各添加BamHI和NcoI酶切位点序列，引物序列如下：
PmiR169oF：
5'-GGATCCGAGAGGGAGAGGGAAGTTGC-3'(SEQ ID NO:5)；
PmiR169oR：
5'-CCATGGCAAAAGGGGCTCTCCATCTC-3'(SEQ ID NO:6)。
以B73基因组DNA为模板，进行PCR扩增；
2)扩增产物进行回收，对回收后的产物和经BamHI和NcoI双酶切的pCAMBIA3301进行连接反应。
反应体系如下：
3)酶切鉴定阳性克隆，阳性克隆即为pmiR169o::GUS载体。图1示出了pmiR169o::GUS表达载体图谱，将阳性克隆进行测序验证，获得阳性质粒如图1所示，用于后续实验。
实施例3pUBI::miR169o表达载体的构建
1)设计扩增Zma-miR169o前体全长的引物，在引物两端各添加20nt载体同源序列，引物序列如下：
Zma-miR169oF：
5'-GCGTGGATCCGAGCTCACATGTAGCCAAGAATGACTTGCC-3'(SEQ ID NO:7)；
Zma-miR169oR：
5'-GTCACCTGTAATTCACACTCTGGCTAGCCAAGAAGACCTG-3'(SEQ ID NO:8)。
以pTmiR169o质粒为模板，进行PCR扩增；
2)扩增产物进行回收，对回收后的产物和改造过的pCAMBIA3301m进行重组反应，所用试剂为GBclonart Seamless Cloning Kit，
反应体系如下：
3)酶切鉴定阳性克隆，阳性克隆即为pUBI::miR169o载体。图2示出了pUBI::miR169o表达载体图谱，将阳性克隆进行测序验证，获得阳性质粒如图2所示，用于后续实验。
实施例4 pmiR169o::GUS和pUBI::miR169o转基因玉米的获得
1农杆菌转化及玉米转化：
将实例2和3中获得的植物表达载体pmiR169o::GUS和pUBI::miR169o转化到农杆菌EHA105中。玉米转化方法参照Frame et al.,进行。
2pmiR169o::GUS和pUBI::miR169o转基因玉米PCR检测：
检测pmiR169o::GUS转基因植物用引物
正向引物：pmiR169oF:5’-GGACGGTTGAACCAACCAAGTG-3’(SEQ ID NO:9)；
反向引物：GUSR:5’-GGGTCCTAACCAAGAAAATGAAG-3’(SEQ ID NO:10)；
检测pUBI::miR169o转基因植物用引物
正向引物：miR169oFW:5’-GAATGACTTGCCTATGCACGCC-3’(SEQ ID NO:11)；
反向引物：NOS70:5’-CCGGCAACAGGATTCAATCTTA-3’(SEQ ID NO:12)；
反应体系：
转基因植株DNA 1μl(20ng～50ng)；
反应条件：
72℃延伸5min，筛选出PCR阳性植株。
3转基因玉米Basta叶面喷洒检测及纯系的获得：
待PCR阳性植株长至5-6片叶时，用1000倍稀释的Basta喷施，每周一次，连续喷施3周后观察植株存活情况，存活的阳性苗连续自交筛选两代，获得T2代纯系，种植观察表型。
实施例5 zma-miR169o在玉米籽粒中表达模式的分析
利用GUS染色组化分析对pmiR169o::GUS转基因玉米籽粒中zma-miR169o的表达模式进行系统研究。从授粉当天(0DAP)开始至24DAP，每隔两天剥取整个籽粒，纵切进行GUS活性分析。结果表明，在籽粒发育的多核期和细胞化阶段(0DAP-4DAP)，zma-miR169o主要在枝梗(pedicel)、胎盘(placenta)和表皮(pericarp)中表达，这三个组织均来自母体，对籽粒发育过程中营养物质的传递和运输有重要作用；6-8DAP，其主要在胚乳基部转移层(BETL)表达，BETL参与母体组织营养物质向内部胚乳细胞的转运；10DAP开始，zma-miR169o在整个籽粒中大量表达，之后表达逐渐减弱，至24DAP几乎不表达(图3)。10-12DAP中央胚乳细胞快速增大和增多，大量积累淀粉和储藏蛋白，12DAP之后胚乳细胞从中央向外逐渐停止细胞增殖，至20-25DAP结束。基因表达模式的研究结果表明，zma-miR169o可能参与玉米籽粒发育过程中营养物质运输，胚乳细胞增殖和淀粉、蛋白的积累。
实施例6 pUBI::miR169o转基因玉米表型分析
克隆并构建了玉米zma-miR169o的植物过表达载体，转化玉米自交系，将转空载体对照及4个过表达miR169o基因的转基因玉米纯系种植和连续两代纯系的表型观察，图4示出了转基因玉米的籽粒表型和单粒体积与单粒重等统计结果，图4A为转基因玉米籽粒表型、图4B为单籽粒体积、图4C为单籽粒重量。发现zma-miR169o过表达转基因玉米籽粒体积增大，粒重增加，结果表明zma-miR169o可能参与了玉米籽粒的发育调控。
本发明把zma-miR169o前体序列构建到植物表达载体pCAMBIA3301上，启动子为Ubiqutin，得到pUBI:miR169o质粒(图2)，将其转化到农杆菌EHA105中，用农杆菌转化玉米并获得了转基因植株(图3)。把转基因植株与未转基因的野生型植株在相同条件下种植进行比较，结果表明，zma-miR169o能够增加玉米籽粒的大小和重量(图4)。
序列表
<110> 中国农业科学院生物技术研究所
<120> 一种玉米籽粒发育相关基因miR169o及其应用
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Claims (3)
1.一种miR169o基因用于制备提高玉米单籽粒体积和粒重的调控剂的用途，其特征在于，所述miR169o的前体核苷酸序列如序列表SEQ ID NO: 1所示；其成熟编码序列如序列表SEQ ID NO: 2所示。
2.包含权利要求1中所述miR169o的前体核苷酸序列的表达载体用于制备提高玉米单籽粒体积和粒重的调控剂的用途。
3.包含权利要求1中所述miR169o的前体核苷酸序列的表达载体的工程菌或细胞株用于制备提高玉米单籽粒体积和粒重的调控剂的用途。
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