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作物学报 ›› 2020, Vol. 46 ›› Issue (5): 690-699.doi: 10.3724/SP.J.1006.2020.93051

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

玉米tasselseed突变体ts12的遗传分析与分子鉴定

秦利萍,董二飞,白洋,周练,任岚扬,张任凤,刘朝显(),蔡一林()   

  1. 西南大学玉米研究所 / 南方山地农业教育部工程研究中心, 重庆400715
  • 收稿日期:2019-09-16 接受日期:2020-01-15 出版日期:2020-05-12 网络出版日期:2020-01-23
  • 通讯作者: 刘朝显,蔡一林
  • 作者简介:秦利萍, E-mail:919494709@qq.com

Genetic analysis and molecular characterization of tasselseed mutant ts12 in maize

Li-Ping QIN,Er-Fei DONG,Yang BAI,Lian ZHOU,Lan-Yang REN,Ren-Feng ZHANG,Chao-Xian LIU(),Yi-Lin CAI()   

  1. Maize Research Institute, Southwest University / Engineering Research Center of South Upland Agriculture, Ministry of Education, Chongqing 400715, China
  • Received:2019-09-16 Accepted:2020-01-15 Published:2020-05-12 Published online:2020-01-23
  • Contact: Chao-Xian LIU,Yi-Lin CAI

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摘要：

性别决定与玉米雄穗和雌穗发育密切相关, 性别决定基因功能研究对性别决定分子机制的解析具有重要意义。利用甲基磺酸乙酯(EMS)处理B73花粉, 获得了一个玉米雄穗结实突变体tasselseed12 (ts12)。用扫描电镜对ts12突变体雄穗的形态学观察, 发现未成熟雄穗长13 mm时, 小穗呈现出明显的雌性化特征。利用图位克隆的方法, 把ts12定位于分子标记LM4和RM5之间, 物理距离约为290 kb, 该区间共有9个注释基因, 其中包括已报道的性别决定基因Tasselseed2 (Ts2)。通过克隆ts12突变体中Ts2基因编码序列, 发现Ts2基因编码区第196个碱基鸟嘌呤被替换为腺嘌呤, 导致该位点编码的甘氨酸被替换为精氨酸, 由此推测该保守位点突变可能是tasselseed表型产生的原因。ts12和ts2等位性测验结果表明所有F1、F2代植株雄穗均可产生花丝, 推测ts12是ts2基因一个新的等位突变体。以加外源茉莉酸(JA, 1 mmol L -1)处理ts12突变体, 发现处理后的小穗大部分可恢复正常。Ts2基因表达分析揭示在正常植株未成熟雄穗中的表达量最高, 其次是未成熟雌穗及叶片中; 在ts12未成熟雄穗和雌穗中, 该基因的表达量极显著降低。Ts2保守位点的突变及其引起的表达量的降低可能是tasselseed表型产生的原因。

关键词: ts12, ts2, 雄穗结实, 精细定位, 等位测验

Abstract:

Sex determination is closely associated with development of maize tassel and ear. Function study of the genes underlying maize sex determination is critical for elucidating its regulation network. One sex determination mutant with feminized tassel, named tasselseed12 (ts12), was created through B73 pollen treatment with chemical agent ethyl methyl sulfonate (EMS). The feminized tassel spikelets were observed under scanning electron microscope when the immature tassels were 13 mm long. Using map-based cloning strategy, the ts12 was mapped to an interval about 290 kb, flanked by markers LM4 and RM5, which harbored nine annotated genes including the reported sex determination gene Tasselseed2 (Ts2). The Ts2 coding sequence from ts12 mutant revealed the 196th base guanine was substituted by adenine, leading to the substitution of amino acids from glycine to arginine, which probably resulted in the tasselseed phenotype. An allelic test crossing ts2 with ts12 showed that all the F1, F2 plants could produce feminized tassels with lots of silks, which indicated ts12 was a new allelic mutant of ts2. The exogenous jasmonic acid (JA, 1 mmol L -1) could recover the normal phenotype of most of spikelets. The expression analysis of Ts12 in wild-type plants showed a high expression level in immature tassels, and moderate one in immature ears and leaves. However, its expression was dramatically decreased in immature tassels and ears of ts12. The mutation in conserved region and the reduced expression of Ts2 probably are the causes of tasselseed phenotype.

Key words: ts12, ts2, tasselseed, fine mapping, allelism test

附表1

ts12基因定位、候选基因克隆及表达分析引物序列"

引物名称
Primer name		 正向引物序列
Forward primer sequence (5′-3′)		 反向引物序列
Reverse primer sequence (5′-3′)		 标记类型
Genetic marker types
LM1 CACAGCAAGAGTACAGCATCAG TGATCCCAGTTCTCGATGTAGC SSR
LM2 GGAAATGTTTGACGTGACCTGC CACTTGGCAAACCAATCCAACG SSR
LM3 AATAGCGTGCTCCCGTGTTATC CTGTCGTTGCTTGCTTCTTCCA SSR
LM4 AATGGGATAAAGACCGGATTCT ATAAGCGTTGTAGGACAGGAGC InDel
RM1 TCGGAGAGGACACGGTTTAGTA CATGAACATTGGCGAAGCTACC SSR
RM2 GCTCCTGTGTGCAACTTGTTAG GCATATGAATCGGTGTTCGGTG SSR
RM3 CTGTTCCTTCGCTTTCACAGAA AGTAGCTCTCTAGTTGTGTCCC SSR
RM4 GCTGATTTAAGATATAAAGTGCCTCCC CCTGCTGGACCGGAGTAAAG InDel
RM5 AGAACGGTGATTTTTGTCTGGT GTTTTGCATGCTCATGTAGACG InDel
Ts2-JD ACAGCAGAGTAGAGTAGCACAC GCCATGGGAATGGGATATTTGG
Ts2-qPCR GTGGAGAAGATGGAGGAGGTGGT ATTGATTCACAAGCCGATGAGGTT
Actin TCACCCTGTGCTGCTGACCG GAACCGTGTGGCTCACACCA

图1

ts12表型观察 A和B: 野生型和ts12成熟雄穗; C: ts12雄穗上能正常发育产生花粉的小穗; D和E: 未成熟野生型雄穗(12 mm)和ts12雄穗(13 mm)扫描电镜观察。WT: 野生型; G: 雌蕊; ST: 雄蕊; C: 心皮。"

表1

ts12显隐性的分离比检验"

群体组配
Population construction		 F1植株雄穗表型
Phenotype of F1 plants		 F2分离群体 F2 segregation population F2植株总数
Total number of F2 plants		 χ2
野生型 WT ts12
ts12 × Mo17 正常 Normal 115 35 150 0.22#

图2

ts12连锁标记umc1169的筛选及验证 MT: ts12 DNA混合池; WT: 野生型DNA混合池。"

图3

ts12精细定位 黑色实线上方相邻标记之间的数字代表物理距离, 括号内数字代表标记在染色体上的确切位置, 标记下方的红色数字代表交换单株数目。"

表2

ts12定位区间内基因功能注释"

基因 ID Gene ID 基因注释 Gene annotation
Zm00001d028796 Meristematic receptor-like kinase
Zm00001d028797 Laccase-13
Zm00001d028798 Hypothetical protein
Zm00001d028799 CASP-like protein 4B1
Zm00001d028801 26S proteasome regulatory subunit 4 homolog A
Zm00001d028802 Hypothetical protein
Zm00001d028803 Protein SCAR2
Zm00001d028804 U2 small nuclear ribonucleoprotein B
Zm00001d028806 Sex determination protein Tasselseed2

图4

B73和ts12中Ts2基因CDS序列比对 红色下画线部分代表Ts2保守结构域A, 绿色方框代表Ts2中氨基酸发生替换的位点"

图5

ts12突变体和6个自交系中的Ts2基因突变位点序列比对。。 蓝色方框代表Ts2基因的单核苷酸突变位点"

图6

ts12和ts2突变体等位测验 A: ts2植株雌性化雄穗; B: ts12和ts2等位测验后代表型。"

图7

茉莉酸处理后ts12的表型及雄穗结实数 A: 未处理的ts12雄穗表型; B: 0.05%乙醇处理的ts12雄穗表型; C: JA处理的ts12雄穗表型; D: 雄穗结实数统计; 柱形图上方数字代表标准差; **代表JA处理植株雄穗结实数量与未处理植株和0.05%乙醇处理植株雄穗结实数在统计学上有极显著差异(P < 0.01)。"

图8

Ts2基因表达分析 Root: 根; Stem: 茎; Leaf: 叶; Silk: 花丝; Immature ear: 雌穗; Immature tassel: 雄穗; Embryo: 授粉后20 d胚; Endosperm: 授粉后20 d胚乳。**代表在特定组织中Ts2基因表达与野生型相比在统计学上有极显著差异(P < 0.01)。"

附图1

短链乙醇脱氢酶同源蛋白保守结构域A序列分析 SbTs2: 高粱Ts2; SiTs2: 小米Ts2; OsTs2: 水稻Ts2; BdTs2: 二穗短柄草Ts2; ATSDR2: 拟南芥Ts2直系同源蛋白; BHD: 来自小鼠的短链乙醇脱氢酶; 17βHSD1: 来自人的短链乙醇脱氢酶; NACMAN: 来自黄杆菌的短链乙醇脱氢酶; 3βHSD: 来自假单胞菌的短链乙醇脱氢酶; FABG: 来自大肠杆菌的短链乙醇脱氢酶。"

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