CN107937435A - 一种增强作物耐储藏性的方法 - Google Patents

一种增强作物耐储藏性的方法 Download PDF

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CN107937435A
CN107937435A CN201810014648.2A CN201810014648A CN107937435A CN 107937435 A CN107937435 A CN 107937435A CN 201810014648 A CN201810014648 A CN 201810014648A CN 107937435 A CN107937435 A CN 107937435A
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gene22715
seed
resistant storage
storage properties
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张瑛
滕斌
张雨萱
宣红
吴敬德
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Rice Research Institute of Anhui Academy of Agricultural Sciences
Anhui Academy of Agricultural Sciences
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Abstract

本发明公开了一种增强作物耐储藏性的方法，利用种子特异性过表达启动子或基因编辑或分子标记辅助或Q‑PCR筛选高表达α‑淀粉酶基因gene22715（Os08g0473600）（KO1176），使种子α‑淀粉酶基因gene22715（Os08g0473600）（KO1176）特异性过表达，以达到增强实现生物体由贮能向供能转化关键过程的作用，从而大量产生葡萄糖，以供给种子储藏过程物质和能量代谢的需要，从而增强作物的耐储藏性。

Description

一种增强作物耐储藏性的方法
技术领域
本发明涉及作物种子储藏领域，具体涉及一种增强作物耐储藏性的方法。
背景技术
种子寿命是指种子在一定环境条件下能保持生活力的期限，其长短受着遗传因素的控制。研究表明，大麦生命力最强，燕麦、小麦次之，玉米较差，黑麦最差。农作物种子的衰老导致种子的生活力下降，寿命缩短，商品性下降，既给国家财政带来负担，又直接造成种子企业及农业种植大户的巨大经济损失。
淀粉存在于作物种子的储藏组织中，其含量约占种子总量的70%-80%，是种子主要的营养和贮能物质。葡萄糖在生物学领域具有重要地位，是活细胞的能量来源和新陈代谢中间产物，即生物的主要供能物质。因此，由淀粉水解产生葡萄糖的代谢途径是实现生物体由贮能向供能转化的关键过程。种子是具有生命力的活体，其在储藏过程中，易受外界环境条件的影响，不可避免地要进行缓慢的物质和能量代谢。因而，能量即供能物质的多少可以决定其储藏寿命。综上所述，可以看出，实现生物体由贮能向供能转化的由淀粉水解产生葡萄糖的代谢途径是决定种子耐储藏性的关键代谢过程。
发明内容
本发明的目的在于提供一种增强作物耐储藏性的方法。
本发明是通过以下技术方案实现的：
本发明提供的增强作物耐储藏性的方法为使种子α-淀粉酶基因gene22715（Os08g0473600）（KO1176）特异性过表达。
其中使α-淀粉酶基因gene22715（Os08g0473600）（KO1176）特异性过表达的方法为利用种子特异性过表达启动子或利用基因编辑或利用分子标记辅助或Q-PCR筛选高表达α-淀粉酶基因gene22715（Os08g0473600）（KO1176）。
此外，本发明提供的增强作物耐储藏性的方法应用的作物为水稻、玉米、花生、大豆、小麦、棉花中的任意一种。
本发明的优点是：
本发明基于α-淀粉酶是淀粉水解产生葡萄糖代谢途径的关键酶，α-淀粉酶可以水解淀粉内部的α-1，4-糖苷键，水解产物为糊精、低聚糖和单糖，酶作用后可使糊化淀粉的黏度迅速降低，变成液化淀粉，最后进一步转化为α-D-葡萄糖，从而供给种子物质和能量代谢的需要，本发明使种子α-淀粉酶基因gene22715（Os08g0473600）（KO1176）特异性过表达，以达到增强实现生物体由贮能向供能转化关键过程的作用，从而大量产生葡萄糖，以供给种子储藏过程物质和能量代谢的需要，从而增强作物的耐储藏性。
附图说明
图1所示为实施例1中通过分子标记辅助对农艺性状优异籼、粳稻材料父本的耐储藏性状进行改良的技术路线。
具体实施方式
实施例一
以水稻为例，通过分子标记辅助选择耐储藏水稻α-淀粉酶高表达基因gene22715（Os08g0473600）（KO1176）的分子标记对农艺性状优异籼、粳稻材料父本的耐储藏性状进行改良，可克服常规育种中表型选择难、周期长、效率低、结果可靠性差的缺点。培育成的水稻新品种与优异籼、粳稻材料父本相比，耐储藏性状可得到明显提高，其它性状诸如生育期、农艺性状、产量、抗性和适应性均一致，它们为一对近等基因系，可以在原水稻推广地区直接应用。
图1中如F1农艺性状存在明显缺陷，可通过携α-淀粉酶高表达基因gene22715（Os08g0473600）（KO1176）水稻品系与过渡材料形成F1，再用优异籼、粳稻材料父本作为轮回亲本进行改良，另外，MAS分子标记也可用Q-PCR代替筛选。
具体步骤如下：
1、选择携α-淀粉酶高表达基因gene22715（Os08g0473600）（KO1176）水稻品系为父本，与需改良水稻材料杂交获得F1杂交种子；
2、进行亲本的多态性检测，对紧邻α-淀粉酶高表达基因gene22715（Os08g0473600）（KO1176）座位的微卫星标记进行多态性检测，两侧各筛选出1-2个微卫星标记；
3、采用回交和分子标记辅助选择相结合的方法将α-淀粉酶高表达基因gene22715（Os08g0473600）（KO1176）转入受体亲本中，在α-淀粉酶高表达基因gene22715（Os08g0473600）（KO1176）座位两侧各选1个具多态性的SSR标记对耐储藏基因进行辅助选择，回交试验方案如图1所示，从BC1F1代起，选取20个单株进行代换片段检测，耐储藏基因座位两侧SSR标记按与供体亲本多态性一致的标准在BC1F1代进行单株选择，获选单株继续回交产生BC2F1代；BC2F1代选取20个单株用于继续进行标记检测，根据结果，选取单株继续回交产生BC3F1；BC3F1代选取20个单株继续进行标记检测，根据结果，选取单株继续回交产生BC4F1；BC4F1代自交，于BC4F2和BC4F3代选取单株进行标记检测，选择到携有供体纯合耐储藏基因的改良株系；
4、分别收获BC4F2或BC4F3代携带有耐储藏基因纯合染色体片段的种子，进行α-淀粉酶高表达基因gene22715（Os08g0473600）α-淀粉酶酶活性测定和耐储藏特性检测。
实施例二
利用α-淀粉酶高表达基因gene22715（Os08g0473600）序列、质粒EHA105转1301和高表达启动子对α-淀粉酶高表达基因gene22715（Os08g0473600）（KO1176）进行过表达，或通过CRISPR/CAS9基因编辑，获得α-淀粉酶高表达基因gene22715（Os08g0473600）（KO1176）高表达材料，并利用转录组RNA-Seq技术和人工加速老化对该基因进行耐储藏功能验证。
本发明中关于KO1176的说明：
在KEGG PATHWAY Database中利用KO，输入1176，点回车键后再点K01176，在Taxonomy中找出所需要延长种子寿命的作物简称，再在Genes中找到相关的耐储藏基因。以水稻为例，先找到水稻简称DOSA, 再找到Os08g0473600-1即α-淀粉酶基因序列。

Claims (3)

1.一种增强作物耐储藏性的方法，其特征在于，使种子α-淀粉酶基因gene22715（Os08g0473600）（KO1176）特异性过表达。
2.根据权利要求1所述的一种增强作物耐储藏性的方法，其特征在于，使α-淀粉酶基因gene22715（Os08g0473600）（KO1176）特异性过表达的方法为利用种子特异性过表达启动子或利用基因编辑或利用分子标记辅助或Q-PCR筛选高表达α-淀粉酶基因gene22715（Os08g0473600）（KO1176）。
3.根据权利要求1所述的一种增强作物耐储藏性的方法，其特征在于，所述方法应用的作物为水稻、玉米、花生、大豆、小麦、棉花中的任意一种。
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