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作物学报 ›› 2021, Vol. 47 ›› Issue (10): 1903-1912.doi: 10.3724/SP.J.1006.2021.03060

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

玉米籽粒缺陷突变基因dek54的精细定位及候选基因分析

周练(), 刘朝显, 陈秋栏, 王文琴, 姚顺, 赵子堃, 朱思颖, 洪祥德, 熊雨涵, 蔡一林*()   

  1. 西南大学玉米研究所 / 农业科学研究院 / 南方山地作物逆境生物学国家级培育基地, 重庆400715
  • 收稿日期:2020-10-15 接受日期:2021-01-13 出版日期:2021-10-12 网络出版日期:2021-03-02
  • 通讯作者: 蔡一林
  • 作者简介:E-mail: zhoulianjojo@swu.edu.cn
  • 基金资助:
    国家自然科学基金项目(31601312)

Fine mapping and candidate gene analysis of maize defective kernel mutant dek54

ZHOU Lian(), LIU Chao-Xian, CHEN Qiu-Lan, WANG Wen-Qin, YAO Shun, ZHAO Zi-Kun, ZHU Si-Ying, HONG Xiang-De, XIONG Yu-Han, CAI Yi-Lin*()   

  1. Maize Research Institute / Academy of Agricultural Sciences / State Cultivation Base of Crop Stress Biology for Southern Mountainous Land, Southwest University, Chongqing 400715, China
  • Received:2020-10-15 Accepted:2021-01-13 Published:2021-10-12 Published online:2021-03-02
  • Contact: CAI Yi-Lin
  • Supported by:
    National Natural Science Foundation of China(31601312)

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摘要：

玉米籽粒与产量和营养品质密切相关, 控制籽粒发育基因的功能研究对解析籽粒发育分子机制, 提高玉米产量, 改善籽粒营养品质提供重要依据。利用甲基磺酸乙酯(ethyl methanesulfonate, EMS)处理B73花粉, 筛选到一个玉米籽粒缺陷突变体defective kernel 54 (dek54)。dek54表现出成熟籽粒变小、皱缩、颜色发白等特征; 遗传分析表明dek54是一个单基因控制的隐性突变体。石蜡切片显示dek54淀粉胚乳细胞形状不规则且排列致密, 扫描电镜观察成熟籽粒胚乳中心区域发现dek54淀粉粒周围蛋白体比野生型少且排列疏松。dek54成熟籽粒的总蛋白、醇溶蛋白、各氨基酸组分和全氮含量相比野生型都显著降低。利用F2分离群体中的1566个dek54单株, 把dek54定位在7号染色体标记SSR6和SSR7之间, 物理位置约为290 kb。该区间有3个基因, 基因测序发现Zm00001d019294基因第2个外显子上第351个碱基由G突变为A, 从而导致蛋白翻译的提前终止。该基因在玉米籽粒中特异性表达, 且在12 DAP (days after pollination)籽粒中表达量最高。通过CRISPR/Cas9系统进行靶向突变确定候选基因Zm00001d019294导致该突变表型。Dek54编码一个与ZmNRT1.5 (nitrate transporter)具有较高同源性的MFS (major facilitator superfamily)家族蛋白并定位在玉米原生质体的细胞质膜。该研究为揭示dek54在玉米籽粒发育的分子机制奠定了重要基础。

关键词: 玉米, defective kernel 54, 籽粒发育, 精细定位

Abstract:

Maize kernel is closely related to yield and nutritive quality. Study on the function of maize kernel development relative genes provides important basis for the molecular mechanism analysis, yield increasing and nutritive quality improving. B73 pollen was treated with ethyl methylmethanesulfonate (EMS) and a defective maize kernel defective kernel 54 (dek54) was screened. dek54 had small mature kernel, wrinkled and whitened seed coat phenotype. Genetic analysis indicated that dek54 is a recessive mutant controlled by a single gene. Paraffin sections showed starchy endosperm cells of dek54 had irregular shape and dense arrangement at developmental stage. Scanning electron microscopy observation indicated that protein bodies around starch granules in the central region of the dek54 mature kernel endosperm were fewer and arranged more loosely compare to wild type. Total protein, zein, amino acids components contents and total nitrogen content of dek54 mature kernel were significantly lowered compared with the wild type. dek54 was located on chromosome 7 within the interval of the physical distance of about 290 kb between markers SSR6 and SSR7. Sequencing revealed that the 351th base G on the 2nd exon of Zm00001d019294 gene changed into A, which led to the premature termination of the protein translation. Zm00001d019294 gene was specifically expressed in immature maize kernel, and has the highest expression in 12 DAP (days after pollination) immature kernel. Targeted mutation was performed using CRISPR/Cas9 system to identify that mutant phenotype was caused by candidate gene Zm00001d019294. Dek54 encoded an MFS (major facilitator superfamily) protein and had high homology with ZmNRT1.5 (nitrate transporter). Besides, Dek54 protein was localized in the plasma membrane of maize protoplasts. The study of dek54 laid the foundation for the molecular mechanism analysis of maize kernel development.

Key words: maize (Zea mays L.), defective kernel 54, kernel development, fine mapping

图1

dek54突变体表型鉴定 A: 成熟果穗F2分离群体; B: 籽粒表型; C: 籽粒纵切; D: 籽粒横切; E: F2分离群体上野生型和dek54突变体籽粒的百粒重、十粒长和十粒宽。标尺为0.5 cm。Em: 胚; En: 胚乳; SE: 粉质胚乳; VE: 玻璃质胚乳。"

附图1

野生型和dek54 成熟籽粒的发芽测试(发芽后5 d) 标尺为1 cm。"

图2

dek54突变体的组织学观察 A: 16 DAP野生型和dek54突变体籽粒的石蜡切片纵切观察。标尺为100 µm。AL: 糊粉层; SE: 淀粉胚乳。B: 野生型和dek54突变体成熟籽粒胚乳中心区域扫描电镜观察。标尺为50 µm。SG: 淀粉粒; PB: 蛋白体。"

图3

dek54突变体的生化成分分析 野生型和dek54突变体成熟籽粒的蛋白(A)、淀粉(B)、各氨基酸组分(C)和全氮含量(D)。"

表1

dek54定位SSR标记引物序列"

标记类型
Type		 标记名称
Name		 正向引物序列
Forward sequence (5′-3′)		 反向引物序列
Reverse sequence (5′-3′)
SSR SSR23403 ACTAGTATGTGGATTGCTCGTCG GCTGCTGAGCTGTATGTACCA
SSR SSR1 CCCTGACGAAAGCATGAATGAG ACAGATGACTCTGCACCTCAAG
SSR SSR2 TGCCCATAAGAGCTTCGAGGATA AGCCTTAGTTGTCAGTCCATCG
SSR SSR3 TGCCCATAAGAGCTTCGAGGATA AGCCTTAGTTGTCAGTCCATCG
SSR SSR4 CCATCTGTACTAATGGCACCTGA GCCAGCAAAGCTTTTCAAGAGT
SSR SSR5 GGCTACATAAGATGCAAAGCGG TACCCTTTGACAGAGCCTACCT
SSR SSR6 GGTGCCAAAAACATCTCCCAAC TAGCGTGGGGTCATAGCAACA
SSR SSR7 CATGGCCAAAATATCGCACGAG TGACGTACATGAACACCTCGG
SSR SSR8 GCTAGGTGCAGTGTCTCTGCTT CCTTGAACGTGGGGTAGGCT
SSR SSR10308 GCAAATGTTCTGTGCAAGGCTA GCCCCACAAGAACTCCATCTAT

表2

dek54定位区间基因注释"

编号
No.		 基因ID
Gene ID		 功能注释
Function annotation
1 Zm00001d019292 无注释 No annotation
2 Zm00001d019293 异柠檬酸/异丙基苹果酸脱氢酶 Isocitric acid/isopropyl malate dehydrogenase
3 Zm00001d019294 MFS家族 Major facilitator superfamily

图4

dek54的连锁SSR标记筛选和精细定位 A: umc2160的PCR扩增产物在WP和MP之间具有多态性; B: 通过33个dek54单株的基因分型确定SSR连锁标记umc2160; C: 通过F2群体1566个单株对dek54进行精细定位。dek54定位标记SSR6和SSR7之间物理距离约为290 kb的区间内。黑色垂直实线上方为分子标记名称, 下方数字为该标记鉴定的交换单株数量。"

图5

Zm00001d019294基因突变位点及时空表达分析 A: Zm00001d019294基因结构图, 红色箭头所示为突变位点; B: Zm00001d019294基因在不同组织及发育中的未成熟籽粒中的表达分析。"

图6

Zm00001d019294 CRISPR/Cas9靶向突变表型鉴定 A: Zm00001d019294的CRISPR/Cas9靶位点序列。sgRNA靶序列为绿色, PAM (protospacer-adjacent motif)序列为橙色, 红色字母和短线分别代表插入和缺失; B: dek54-cas9-1xMo17的F2成熟果穗; C: 成熟的WT和dek54-cas9-1籽粒; D: 成熟的WT和dek54-cas9-1籽粒的纵切。Em: 胚; En: 胚乳。标尺为0.5 cm。"

图7

Dek54蛋白保守结构域(A)及进化(B)分析"

图8

玉米原生质体中Dek54的亚细胞定位观察 Dek54-GFP载体与mCherry标记的质膜标记(mCherry-PM; CD3-1007)共定位。标尺为20 μm。"

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