CN117305352B - 玉米ZmNAC78基因在调控玉米籽粒铁含量中的应用 - Google Patents
玉米ZmNAC78基因在调控玉米籽粒铁含量中的应用 Download PDFInfo
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Abstract
本发明公开了玉米ZmNAC78基因在调控玉米籽粒铁含量中的应用。所述玉米ZmNAC78基因的核苷酸序列如序列表SEQ ID NO:1所示,所述玉米ZmNAC78基因表达的蛋白的氨基酸序列如序列表SEQ ID NO:2所示。本发明的玉米ZmNAC78基因在玉米植株中过表达,可以提高玉米籽粒铁含量,在玉米体内敲除该基因,则降低了玉米籽粒铁含量,证明玉米ZmNAC78基因在玉米体内调控铁的含量,该研究为培育高铁含量玉米品种奠定了基础。
Description
技术领域
本发明属于生物技术领域,具体涉及玉米ZmNAC78基因在调控玉米籽粒铁含量中的应用。
背景技术
补充剂、饮食多样化和商业食品强化已被用于增加人类饮食中的微量营养元素含量,通过基因工程进行生物强化有很好的前景。基因工程利用与铁吸收和代谢相关的基因已经成功地用于提高水稻和其他作物可食用部分的铁含量。例如,AtNAS1、PvFERRITIN和AfPHYTASE的协同表达增加了水稻胚乳中的铁含量(Wirth et al., 2009);过表达TaFer1- A可使小麦铁含量提高50-85%(Borg et al., 2012);同时过表达突变的AtIRT1和AtFER1可以提高木薯的铁含量(Narayanan et al., 2019)。
培育籽粒铁含量高的生物强化玉米,是缓解撒哈拉以南非洲(Sub-SaharanAfrica, SSA)地区(多以玉米为主食)缺铁性贫血的有效途径,但生物强化玉米品种的培育一直受到限制,面临的一个挑战是籽粒中铁含量与玉米产量呈负相关,此外已经发现的调控玉米籽粒铁含量的关键基因也十分有限。因此,在不降低玉米产量的情况下,寻找能够提高玉米籽粒铁含量的新基因资源势在必行。
发明内容
本发明的目的在于提供玉米ZmNAC78基因在调控玉米籽粒铁含量中的应用。
玉米ZmNAC78基因在调控玉米籽粒铁含量中的应用。
所述玉米ZmNAC78基因的核苷酸序列如序列表SEQ ID NO:1所示,所述玉米ZmNAC78基因表达的蛋白的氨基酸序列如序列表SEQ ID NO:2所示。
所述调控为过表达玉米体内的ZmNAC78基因。
一种提高玉米籽粒铁含量的方法,为在玉米体内过表达ZmNAC78基因。
本发明的有益效果:本发明的玉米ZmNAC78基因在玉米植株中过表达,可以提高玉米籽粒铁含量,在玉米体内敲除该基因,则降低了玉米籽粒铁含量,证明玉米ZmNAC78基因在玉米体内调控铁的含量,该研究为培育高铁含量玉米品种奠定了基础。
附图说明
图1为zmnac78 ems 突变体植株及其铁含量。
图2 为利用CRISPR-Cas9的方法定向靶向ZmNAC78进行基因敲除示意图。
图3 为zmnac78敲除突变体籽粒中铁含量。
图4 为过表达ZmNAC78的转基因玉米籽粒中铁含量。
具体实施方式
为了便于理解本发明,下面将对本发明进行更全面的描述。但是,本发明可以以许多不同的形式来实现,并不限于本文所描述的实施例。相反地,提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
以下实施例中所用的实验材料:玉米自交系材料:B73、Zong31、KN5585;菌株:大肠杆菌菌株DH5α、农杆菌菌株GV3101;过表达载体为CUB。
实施例1 ZmNAC78的EMS突变体中的籽粒铁含量显著降低
从玉米EMS诱导突变数据库(http://elabcaas.cn/memd/public/index.html#/)中获得了一个B73背景下的EMS突变体。EMS突变体在ZmNAC78的第4外显子中含有C/T取代,导致CDS的提前终止。将此突变体命名为zmnac78 ems ,zmnac78 ems 突变体的提前终止不影响玉米的生长发育。而zmnac78 ems 突变体籽粒铁含量为22.6 mg/kg,比对照B73籽粒铁含量低40%(如图1所示)。
实施例2 ZmNAC78的敲除突变体中的籽粒铁含量显著降低
以铁含量较高的玉米自交系综31(Zong31)为受体,利用CRISPR-Cas9的方法定向靶向ZmNAC78。首先在ZmNAC78的第3个和第4个外显子设计两个特异的靶向位点,然后连接到单子叶植物的CRISPR-Cas9***的pBUE411载体上,分别靶向ATG密码子后的677-695 bp和1185-1203 bp核苷酸序列。在zmnac78敲除突变体收获的T1代植株中,通过PCR扩增靶标序列测序发现,第一个敲除事件缺失了35 bp,记为KO#1;第二个敲除事件缺失了71 bp,记为KO#2,这两个敲除事件碱基的缺失导致CDS提前终止(图2)。与zmnac78ems的观察结果一致,zmnac78敲除突变体在生长发育过程中与受体综31相似。虽然zmnac78敲除突变体的籽粒长度、籽粒宽度和百粒重均有所降低(表1),但zmnac78敲除突变体籽粒中铁含量仍远低于综31(图3)。
表1转 ZmNAC78基因玉米的农艺性状
数据以平均值±SD (n≥10)表示,对于多组比较,数据采用单因素方差分析(ANOVA)分析,然后采用Tukey多重比较检验,同一字母的平均值在P < 0.05时没有显著差异。
实施例3过表达ZmNAC78能显著增加玉米籽粒中的铁元素
为进一步研究ZmNAC78在调控玉米籽粒铁含量中产生的影响,构建了Ubiquitin启动子的过表达载体pCUB-NAC78,将该载体转化到铁含量较低的玉米自交系KN5585中。根据ZmNAC78的表达丰度,挑选其中的两个转基因株系作为过表达材料用于进一步研究,分别为OE#1和OE#2(图4),采用5’- CGAATGGGTCAAGGACAAAC-3’和5’- GAACACTCGGCATAAAGCGT-3’组成的引物对进行实时荧光定量PCR检测。对过表达ZmNAC78株系进行田间考种观察,发现ZmNAC78过表达株系并不影响玉米的营养生长和成熟籽粒的百粒重(图4, 表1),但可以显著提高籽粒中的铁含量(图4)。过表达ZmNAC78的转基因玉米籽粒中铁含量范围为49.4 ~70.5 mg/kg,是野生型KN5585的2.5 ~ 3.6倍(图4)。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (3)
1.玉米ZmNAC78基因在调控玉米籽粒铁含量中的应用,其特征在于,所述玉米ZmNAC78基因的核苷酸序列如序列表SEQ ID NO:1所示,所述玉米ZmNAC78基因表达的蛋白的氨基酸序列如序列表SEQ ID NO:2所示。
2.根据权利要求1所述玉米ZmNAC78基因在调控玉米籽粒铁含量中的应用,其特征在于,所述调控为过表达玉米体内的ZmNAC78基因。
3.一种提高玉米籽粒铁含量的方法,其特征在于,所述方法为在玉米体内过表达ZmNAC78基因,所述ZmNAC78基因的核苷酸序列如序列表SEQ ID NO:1所示,所述ZmNAC78基因的表达的蛋白的氨基酸序列如序列表SEQ ID NO:2所示。
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