CN117487846A - SnRK1a1基因在调节玉米籽粒性状、平衡玉米产量和品质中的用途 - Google Patents
SnRK1a1基因在调节玉米籽粒性状、平衡玉米产量和品质中的用途 Download PDFInfo
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Abstract
本发明公开了SnRK1a1基因在调节玉米籽粒性状、平衡玉米产量和品质中的用途。本发明通过敲除SnRK1a1基因，发现不仅可平衡产量和品质，打破育种过程中产量和品质不兼容现象，同时还可以提高生物量和缩短生育期。
Description
技术领域
本发明属于基因工程技术领域，具体涉及SnRK1a1基因在调节玉米籽粒性状、平衡玉米产量和品质中的用途。
背景技术
玉米(Zea mays L.)是重要的粮食、饲料、能源及工业原料，自2010年以来，我国玉米种植面积和产量反超水稻，成为第一大粮食作物。随着人民生活水平的提高，一方面肉蛋奶需求推动了玉米生产的不断增加，另一方面，玉米的营养品质也越来越被重视。然而，在玉米育种改良过程中，追求不断高产的同时发现营养品质难以改善。另外，玉米籽粒主要的储藏蛋白—醇溶蛋白氨基酸比例极不协调，缺乏赖氨酸和色氨酸等必需氨基酸，导致玉米无论作为饲料还是食用，营养品质都很差。因此，玉米育种在提高产量的同时需不断改善营养品质。
玉米优质和高产相互拮抗，一直是育种面临的难题。玉米蛋白品质改良最初的突破源于60年发现opaque2(o2)突变体具有高蛋白含量。o2突变体醇溶蛋白含量显著下降，而对应的非醇溶蛋白含量显著上升，改善了赖氨酸等必需氨基酸比例，使玉米蛋白品质大幅度提高。但这个令人振奋的发现很快在育种过程中遇到了困难。o2突变也带来了很多不良性状，例如产量降低、籽粒粉质、不耐储藏、抗病性差等，使其难以达到育种推广的基本要求。国家小麦和玉米改良中心在70年代启动了QPM育种计划，试图克服o2突变体粉质以及产量降低等问题。虽然QPM育种计划取得了一定的成功，但是由于QPM修饰位点是由数量性状所控制的，使其在大规模推广应用上仍有一定的困难。
在玉米育种过程中，增加产量最普遍的方式为利用杂种优势。自上个世纪50年代我国玉米杂交种广泛推广，我国玉米产量显著提升。但是相对于双亲而言，杂交种蛋白含量显著下降。例如我国推广面积最大的品种之一郑单958是利用郑58和昌7-2杂交选育出的一代杂交玉米品种，因其高产、稳产、不秃尖，无空秆、耐干旱，耐高温、抗倒、抗病等众多优良特性，郑单958一直保持畅销，被种植户称为“不老的神话”。但就蛋白品质而言，其亲本郑58蛋白含量为9.18％，昌7-2蛋白含量为11.2％，而郑单958蛋白含量仅为8.47％。打破育种过程中品质与产量不兼容现象，创制新型的高产优质玉米为我国育种亟待解决的重要科学问题。
发明内容
针对现有技术中的上述不足，本发明提供一种SnRK1a1基因在调节玉米籽粒性状、平衡玉米产量和品质中的用途，SnRK1a1基因不仅可平衡产量和品质，打破育种过程中产量和品质不兼容现象，同时还可以提高生物量和缩短生育期。
为实现上述目的，本发明解决其技术问题所采用的技术方案是：
SnRK1a1基因在调节玉米籽粒性状中的用途。
进一步地，籽粒性状包括粒长、粒宽和籽粒横切面面积。
进一步地，通过抑制SnRK1a1基因的表达，或敲除SnRK1a1基因提升籽粒粒长、粒宽和籽粒横切面面积。
一种抑制SnRK1a1基因表达的试剂在制备提升玉米籽粒粒长的制剂中的用途。
一种抑制SnRK1a1基因表达的试剂在制备提升玉米籽粒粒宽的制剂中的用途。
一种抑制SnRK1a1基因表达的试剂在制备提升玉米籽粒横切面面积的制剂中的用途。
一种抑制SnRK1a1基因表达的试剂在制备平衡玉米产量和品质的制剂中的用途。
上述SnRK1a1基因或制剂在玉米种质资源改良中的用途。
本发明的有益效果：
本研究通过CRISPR-Cas9技术创制了玉米snrk1a1敲除株系，发现snrk1a1敲除突变体粒长、粒宽和籽粒横切面面积均显著增加，同时snrk1a1敲除突变体淀粉、醇溶蛋白和总蛋白含量均显著增加，并且还可提高生物量和缩短生育期，平衡玉米籽粒品质和产量。
附图说明
图1为snrk1a1敲除突变体载体图谱；
图2为snrk1a1敲除突变体缺失位点及免疫印迹分析蛋白含量；其中，A，snrk1a1敲除突变体缺失位点。KO1和KO2突变体分别是11个碱基和17个碱基的缺失；B，野生型和snrk1a1敲除突变体中SnRK1a1和O2蛋白量分析；
图3为snrk1a1敲除突变体表型分析；其中，(A)snrk1a1敲除突变体和对应野生型成熟棒子、粒长和粒宽分析；(B)snrk1a1敲除突变体和对应野生型籽粒横切面面积分析；(C)统计学分析snrk1a1敲除突变体和对应野生型籽粒粒长；(D)统计学分析snrk1a1敲除突变体和对应野生型籽粒粒宽；(E)统计学分析snrk1a1敲除突变体和对应野生型籽粒横切面面积；(F)统计学分析snrk1a1敲除突变体和对应野生型籽粒百粒重；
图4为snrk1a1敲除突变体淀粉和蛋白分析；其中，(A)Western blot检测淀粉相关蛋白含量；(B)野生型与snrk1a1敲除突变体淀粉含量分析；(C)野生型与snrk1a1敲除突变体总蛋白含量分析；(D)野生型与snrk1a1敲除突变体醇溶蛋白和非醇溶蛋白分析；(E)统计学分析野生型与snrk1a1敲除突变体醇溶蛋白含量；(F)统计学分析野生型与snrk1a1敲除突变体非醇溶蛋白含量；
图5为snrk1a1敲除突变体生物量增加和生育期提前；其中，(A)野生型与snrk1a1敲除突变体植株形态；(B)野生型与snrk1a1敲除突变体植株根形态；(C)野生型与snrk1a1敲除突变体株高统计分析；(D)统计分析野生型与snrk1a1敲除突变体生育期；(E)统计分析野生型与snrk1a1敲除突变体根重；
图6为SnRK1a1过表达籽粒表型考察；其中，(A)野生型与SnRK1a1过表达棒子表型；(B)野生型与SnRK1a1过表达籽粒长度比较；(C)统计学分析野生型与SnRK1a1过表达籽粒长度；(D)统计学分析野生型与SnRK1a1过表达籽粒面积；(E)统计学分析野生型与SnRK1a1过表达籽粒百粒重；
图7为SnRK1a1过表达籽粒品质考察；其中，(A)统计学分析野生型与SnRK1a1过表达籽粒淀粉含量；(B)WB检测淀粉合成相关蛋白含量；(C)SDS-PAGE分析野生型与SnRK1a1过表达籽粒醇溶蛋白和非醇溶蛋白含量；(D)统计学分析野生型与SnRK1a1过表达籽粒醇溶蛋白和非醇溶蛋白含量；(E)统计学分析野生型与SnRK1a1过表达籽粒总蛋白含量；
图8为snrk1a1位点导入郑58和B73性状表型；其中，(A)snrk1a1位点导入郑58棒子表型考察；(B)snrk1a1位点导入B73棒子表型考察；(C)snrk1a1位点导入郑58和B73百粒重分析；(D)snrk1a1位点导入郑58和B73籽粒面积分析；
图9为snrk1a1位点导入郑58和B73理化性状考察表型；其中，(A)snrk1a1位点导入郑58和B73籽粒淀粉含量分析；(B)snrk1a1位点导入郑58和B73籽粒醇溶蛋白分析；(C)snrk1a1位点导入郑58和B73籽粒非醇溶蛋白分析；(D)统计学分析snrk1a1位点导入郑58和B73籽粒醇溶蛋白含量；(E)统计学分析snrk1a1位点导入郑58和B73籽粒非醇溶蛋白含量。
具体实施方式
下面对本发明的具体实施方式进行描述，以便于本技术领域的技术人员理解本发明，但应该清楚，本发明不限于具体实施方式的范围，对本技术领域的普通技术人员来讲，只要各种变化在所附的权利要求限定和确定的本发明的精神和范围内，这些变化是显而易见的，一切利用本发明构思的发明创造均在保护之列。
实施例1构建snrk1a1敲除遗传材料
在SnRK1a1基因编码区的第一个外显子设计gRNA，并构建至敲除载体pCAMBIA3301-Ubi-Cas9中(图1)。将构建好的敲除载体转至B104自交系幼胚中，经三轮抗生素筛选，成功获得T0代snrk1a1敲除突变体。经两代回交并自交一代，获得没有转基因载体的snrk1a1纯合敲除株系。经测序分析，共获得两种敲除材料，一种是11个碱基的缺失(缺失位点为从启动子ATG开始的第331位碱基到341位碱基)，另外一个是17个碱基的缺失(缺失位点为从启动子ATG开始的第325位碱基到341位碱基)，将获得两种敲除材料分别命名为KO1和KO2(图2A)。
取授粉后18天的新鲜籽粒提取总蛋白，检测野生型和snrk1a1敲除突变体籽粒中SnRK1a1和O2蛋白质水平。Western blot结果显示snrk1a1敲除突变体不可检测到SnRK1a1蛋白，O2蛋白丰度显著上升(图2B)。
实施例2snrk1a1突变体对玉米性状和品质的影响
1、种植snrk1a1突变体及分离的野生型材料观察表型。相对于野生型，snrk1a1突变体成熟籽粒粒长、粒宽、籽粒横切面面积增加、百粒重均显著增加(图3)。
2、snrk1a1突变体蛋白和淀粉含量增加
取授粉后18天野生型和snrk1a1突变体籽粒提取总蛋白用于western blot分析。结果表明，与野生型相比，snrk1a1突变体中淀粉合成相关蛋白Sh1、SUS1、SS1、SS2、SBE1、SBE2a、SU2蛋白含量显著增加(图4A)。检测淀粉含量发现snrk1a1突变体中淀粉显著提升(图4B)。进一步考察snrk1a1突变体和对应野生型蛋白含量。相对野生型，snrk1a1敲突变体醇溶蛋白含量显著提高、非醇溶蛋白含量无显著差异、总蛋白含量显著提升(图4C～F)。
3、snrk1a1突变体植株生物量增加和生育期提前
考察snrk1a1突变体发现，相对于野生型，snrk1a1植株株高显著增加(图5A和5C)。观察植株根形态发现snrk1a1植株根生物量和根重均显著增加(图5B和5E)。考察生育期发现，snrk1a1植株生育期提前5到7天(图5D)，植株提前进入生殖生长阶段。
实施例3SnRK1a1过表达对玉米品质的影响
1、SnRK1a1过表达材料产量降低
用组成型表达Ubiquitin启动子驱动SnRK1a1，构建过表达载体并转至自交系B104幼胚中，经三次抗生素筛选成功获得3个独立的转化时间，分别命名为OE1、OE2和OE3(图6A)。经两代自交获得纯合过表达株系用于考察表型。相对于野生型，过表达籽粒粒长和粒面积均变小，百粒重降低(图6B-6E)。
2、SnRK1a1过表达材料品质降低
考察SnRK1a1过表达材料淀粉和蛋白含量。相对于野生型，SnRK1a1过表达成熟籽粒淀粉含量显著降低(图7A)。提取总蛋白检测淀粉相关蛋白的蛋白含量，WB检测发现淀粉合成相关蛋白Sh1、SUS1、SS1、SS2、SBE1、SBE2a和SU2含量显著降低(图7B)。SDS-PAGE胶检测以及定量分析发现SnRK1a1过表达成熟籽粒醇溶蛋白含量显著降低，非醇溶蛋白含量没有显著变化，总蛋白含量显著降低(图7C-E)。
3、snrk1a1突变体导入常见品种增加产量和品质
将snrk1a1突变体导入目前推广面积最大的郑单958的亲本郑58和应用面积最大的自交系B73杂交后自交考察表型。考察结果发现携带有snrk1a1缺失位点籽粒面积、百粒重均显著增加(图8)。测定淀粉和蛋白含量发现携带有snrk1a1缺失位点籽粒淀粉、醇溶蛋白和非醇溶蛋白含量均显著提高(图9)。
最后应说明的是，以上具体实施方式仅用以说明本发明的技术方案而非限制，尽管参照实例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围，其均应涵盖在本发明的权利要求范围当中。
Claims (8)
1.SnRK1a1基因在调节玉米籽粒性状中的用途。
2.根据权利要求1所述的用途，其特征在于，籽粒性状包括粒长、粒宽和籽粒横切面面积。
3.根据权利要求1或2所述的用途，其特征在于，通过抑制SnRK1a1基因的表达，或敲除SnRK1a1基因提升籽粒粒长、粒宽和籽粒横切面面积。
4.一种抑制SnRK1a1基因表达的试剂在制备提升玉米籽粒粒长的制剂中的用途。
5.一种抑制SnRK1a1基因表达的试剂在制备提升玉米籽粒粒宽的制剂中的用途。
6.一种抑制SnRK1a1基因表达的试剂在制备提升玉米籽粒横切面面积的制剂中的用途。
7.一种抑制SnRK1a1基因表达的试剂在制备平衡玉米产量和品质的制剂中的用途。
8.权利要求1中所述的SnRK1a1基因，或权利要求4～7任一项所述制剂在玉米种质资源改良中的用途。
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