CN118256517A - 控制水稻抽穗期基因yth07及其编码蛋白质和应用 - Google Patents
控制水稻抽穗期基因yth07及其编码蛋白质和应用 Download PDFInfo
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Abstract
本发明公开了一个水稻抽穗期基因YTH07及其编码蛋白质和应用。该基因编码一个含有YT521‑B‑like homology(YTH)结构域的蛋白，基因序列如SEQ ID NO.1所示，氨基酸序列如SEQ ID NO.2所示。遗传转化实验证明YTH07在长、短日照条件下均促进水稻抽穗。由于yth07敲除突变体较野生型晚抽穗天数在长、短日照下较稳定，因此通过杂交或者基因编辑将其导入常规品种对于躲避极端天气的危害和提高区域适应性有重要意义。
Description
技术领域
本发明属于基因工程领域，具体涉及一个控制水稻抽穗期微效基因YTH07及其编码蛋白质和应用。
背景技术
随着全球疫情流行，极端天气频发，粮食安全的保障已经迫在眉睫。水稻作为全球重要的粮食作物之一，养育着全球一半以上的人口。抽穗期是水稻的重要农艺性状之一，既决定了水稻品种的季节适应性和区域适应性，也通过调节光合产物的积累影响着水稻的产量和品质。因此，挖掘调控水稻抽穗期的关键基因，深入理解其分子作用机制，完善水稻抽穗期调控网络，有利于育种家选育适应不同地区的优良品种。
通过正向遗传学、反向遗传学和群体遗传学，科学家已经克隆了很多的抽穗期基因，但是大部分基因的效应都较大，往往伴随着水稻产量和株高的显著改变。而抽穗期略微延迟(3-7天)可以使水稻充分利用营养生长阶段的光温条件，提高水稻品质和质量。而抽穗期略微提前则可以使水稻避免在开花时遭遇高温胁迫。近年来水稻品种的产量增长势头不足，表明在种质资源开发遭遇了瓶颈期，微效基因的聚合可能是提高产量的另一有效方法。目前关于微效抽穗期基因的挖掘和利用还比较少。
挖掘抽穗期微效基因可以为提高水稻品种的区适应性提供基因资源储备和理论基础，对抽穗期品种的改良具有重要意义。
发明内容
本发明的目的在于公开控制水稻抽穗期微效基因YTH07及其编码蛋白质和应用。
本发明的目的可通过如下技术方案实现：
本发明提供一种控制水稻抽穗期微效基因YTH07，基因序列如SEQ ID NO.1所示，编码一个含有YT521-B-like homology(YTH)结构域的蛋白，氨基酸序列如SEQ ID NO.2所示。
本发明还提供一种敲除或沉默YTH07的引物，序列如SEQ ID NO.3和SEQ ID NO.4所示。
本发明还提供一种敲除或沉默YTH07的sgRNA，序列如SEQ ID NO.5所示。
本发明还提供一种突变基因yth07，序列如SEQ ID NO.6或SEQ ID NO.7所示。
本发明还提供所述的控制抽穗期微效基因YTH07或突变基因yth07在水稻品种改良中的应用。
本发明还提供所述的控制抽穗期微效基因YTH07或突变基因yth07在水稻抽穗期调控育种中的应用。
本发明还提供所述的控制抽穗期微效基因YTH07或突变基因yth07在培育抽穗期延迟的水稻品种中的应用，具体为将野生型品种敲除或沉默基因YTH07，或将突变基因yth07转入野生型品种。
本发明还提供一种培育抽穗期延迟的水稻品种的方法，具体为将野生型品种敲除或沉默基因YTH07或者将突变基因yth07转入野生型品种，获得抽穗期较野生型延迟的水稻品种。
本发明的有益效果：
用基因编辑的方法将野生型材料中的YTH07进行敲除，可获得yth07的功能缺陷型基因型(抽穗期较野生型略微延迟)的水稻品种。通过本发明的方法可以提高水稻品种的区域适应性，为增加抽穗期改良的遗传多样性提供了技术支持。相对于现有技术，具有如下效果：
(1)本发明通过反向遗传学鉴定了一个调控水稻抽穗期的微效促进子YTH07，转基因敲除可以获得YTH07功能丧失后出现略微延迟抽穗表型的水稻品种。
(2)用基因编辑的方法将yth07基因转移到野生型材料中，可获得yth07的功能缺陷型基因型、抽穗期较野生型稍晚的的水稻品种。
(3)yth07较野生型略微延迟抽穗的表型在长、短日照下较稳定，因此突变基因yth07可以通过杂交或者基因编辑方法导入常规品种。
(4)通过本发明的基因可以调整水稻抽穗期时间，从而调整水稻品种的地域适应性，获得生育期适应不同地区的水稻品种，为抽穗期改良的遗传多样性提供技术支持。
附图说明
图1是对YTH07进行基因编辑的两种基因型。
图2为野生型Dongjin与敲除突变体yth07的表型及其在长、短日照下的抽穗期统计图。
具体实施方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。
实施例1
一、基因敲除载体构建
a)利用http://CRISPR.hzau.edu.cn/CRISPR/网站设计YTH07的敲除引物，引物序列为：
YTH07-CRISPR-F：
AGATGATCCGTGGCAGCCTCTAGAGTCACGCCTTGGTTTTAGAGCTATGC(SEQ ID NO.3)
YTH07-CRISPR-R：
TTCTAGCTCTAAAACGGCTGAGGGGTATACACATTCTGAGCCTCAGCGCAGCAGCTTA(SEQ IDNO.4)
b)以CRISPR-JIEYAO-1/2质粒(Wang et al.,2023)为模板，利用上述YTH07-CRISPR-F和YTH07-CRISPR-R引物扩增U6c启动子和sgRNA scaffold(SEQ ID NO.5)。扩增程序为：94℃2min；98℃10s，58℃30s，68℃1min/1kb，共33个循环；68℃10min；4℃10min。扩增完成后使用琼脂糖凝胶电泳检测，并回收相应条带。将回收的片段和经BsaⅠ线性化的pYLCRISPR/Cas9Pubi-H骨架(Ma et al.,2015)进行重组，反应体系如下：
以上混合体系在50℃下反应20min。
c)重组产物冰上静置2min，转入大肠杆菌感受态细胞中，冰上静置30min；
e)42℃水浴热激90s，冰上静置2min；
f)加700μL液体LB培养基，37℃，220rpm摇床复苏1h；
g)在含有卡那霉素的固体LB培养基平板上均匀涂布，37℃培养箱倒置培养16h；
h)挑取单克隆活化后进行测序，对测序正确的单克隆提取质粒；
i)利用冻融法将重组正确的敲除载体转入农杆菌中，获得YTH07敲除载体的农杆菌菌株。
二、转基因植株的获取
1、转基因愈伤侵染
a)将水稻(Dongjin，公知公用品种，粳稻品种)成熟胚诱导2周后长出的愈伤转移至继代培养基再诱导培养半个月；
b)诱导成功的愈伤与包含步骤一制备的YTH07敲除载体的农杆菌菌液(OD600在0.6-1之间)均匀混合孵育30min，用灭菌滤纸吸干菌液转入共培养基，黑暗培养3天；
c)0.2％的羧苄杀死未侵染成功的农杆菌；愈伤转入筛选培养基筛选15天；
d)挑取生长健康的愈伤再次转移至筛选培养基，再次筛选15天；
e)将筛选后成活的愈伤转移至分化培养基上分化21天；
f)分化结束后转移至生根培养基15天，然后移栽至温室或者大田生长。
2、转基因植株鉴定
在敲除靶点上下游250bp附近设计扩增引物，提取T0代转基因植株叶片DNA进行PCR扩增，测序分析编辑方式，测序鉴定引物如下所示：
yth07-JD-F：ACTAGGAAGTGCCTCCACAT
yth07-JD-R：ATGTGCAACGTAGAGCAGCA
经过序列测序分析，鉴定到了两种编辑方式纯合的yth07敲除突变体，其中yth07-1在第二个外显子出现一个碱基T的插入(突变序列如SEQ ID NO.6所示)，yth07-4则是第二个外显子存在2个碱基(CG)的缺失(突变序列如SEQ ID NO.7所示)，这两种编辑方式均导致移码突变，使其所编码的蛋白提前终止，如图1所示。
种植上述纯合的T1转基因植株，在南京自然长日照条件下(NLD)和海南自然短日照(NSD)条件下调查敲除突变体和野生型Dongjin的抽穗期，并进行拍照。
结果如图2所显示，两个独立的yth07敲除家系在南京自然长日照和海南自然短日照下均表现出较野生型Dongjin晚3-5天抽穗，说明YTH07是一个正调控水稻抽穗期的因子，本发明方法可用于增强水稻的区域适应性。
用传统杂交或者基因编辑的方法将突变基因转入野生型材料，获得yth07纯合基因型，具有较野生型晚抽穗的材料，可以定向调节目的品种的抽穗期。
Claims (10)
1.控制水稻抽穗期微效基因YTH07，其特征在于，基因序列如SEQ ID NO.1所示，氨基酸序列如SEQ ID NO.2所示。
2.敲除权利要求1所述基因的引物。
3.根据权利2所述的引物序列，其特征在于，序列如SEQ ID NO.3和SEQ ID NO.4所示。
4.敲除权利要求1所述基因YTH07的sgRNA。
5.根据权利要求4所述的sgRNA，其特征在于，序列如SEQ ID NO.5所示。
6.敲除权利要求1所述基因YTH07的敲除载体、转基因细胞系或重组菌。
7.一种突变基因yth07，序列如SEQ ID NO.6或SEQ ID NO.7所示。
8.权利要求1所述的控制抽穗期微效基因YTH07、权利要求2或3所述的引物、权利要求4或5所述的sgRNA、权利要求6所述的敲除载体、转基因细胞系或重组菌或权利要求7所述的突变基因yth07在水稻品种改良或在水稻抽穗期调控育种中的应用。
9.权利要求1所述的控制抽穗期微效基因YTH07、权利要求2或3所述的引物、权利要求4或5所述的sgRNA、权利要求6所述的敲除载体、转基因细胞系或重组菌或权利要求7所述的突变基因yth07在培育抽穗期延迟的水稻品种中的应用，具体为将野生型品种敲除或沉默基因YTH07，或将突变基因yth07转入野生型品种。
10.一种培育抽穗期延迟的水稻品种的方法，具体为将野生型品种敲除或沉默权利要求1所述的基因YTH07或者将权利要求7所述的突变基因yth07转入野生型品种，获得抽穗期较野生型延迟的水稻品种。
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