中国水稻科学 ›› 2017, Vol. 31 ›› Issue (4): 355-363.DOI: 10.16819/j.1001-7216.2017.6165 355
孙立亭1,3, 林添资1,3, 王云龙1, 牛梅1, 胡婷婷1, 刘世家1, 王益华1, 万建民1,2,*()
收稿日期:
2016-12-20
修回日期:
2017-02-07
出版日期:
2017-07-25
发布日期:
2017-07-10
通讯作者:
万建民
基金资助:
Liting SUN1,3, Tianzi LIN1,3, Yunlong WANG1, Mei NIU1, Tingting HU1, Shijia LIU1, Yihua WANG1, Jianmin WAN1,2,*()
Received:
2016-12-20
Revised:
2017-02-07
Online:
2017-07-25
Published:
2017-07-10
Contact:
Jianmin WAN
摘要:
目的叶色突变相关基因鉴定和克隆有助于研究光合作用,补充并完善叶绿体发育机理和色素合成代谢途径,为开展水稻的高光效育种提供理论依据。方法从粳稻品种Dongjin的组培后代中分离出一个白条纹突变体st13,成熟期测定野生型和st13的主要农艺性状,苗期测定色素含量并观察叶绿体的超微结构;将st13和Dongjin进行正反交,观察F1植株表型,并对F2表型分离进行卡方检验,对st13进行遗传分析;利用st13×南京11(籼稻品种)的F2和F2:3群体,对st13突变基因定位;采用qPCR分析叶绿体发育和叶绿素合成相关基因在st13与野生型相对表达量。结果与野生型Dongjin相比,该突变体的株高、单株有效穗数、穗长、结实率和千粒重等主要农艺性状显著下降。苗期的色素含量降低,分蘖期无差异。突变体的叶绿体中既有含丰富的类囊体膜结构的正常叶绿体,也存在无类囊体结构的叶绿体。遗传分析和基因定位结果表明,st13的突变表型受1对隐性核基因控制,突变基因位于第3条染色体长臂InDel(Insertion-Deletion)标记I3-21和I3-22之间。进一步在这两个标记之间设计了6对InDel标记,最终将基因定位在94 kb区间内,此区间共有8个候选基因。结论这8个候选基因中,有5个假定的蛋白,其他三个都是有功能注释的蛋白,而这三个蛋白在水稻中均未见报道,因此,st13突变是由一个新的叶色基因突变引起的;同时st13中叶绿体发育、叶绿素合成和光合系统相关基因的表达也发生了显著改变,推测ST13可能是调控叶绿体发育的关键基因。
孙立亭, 林添资, 王云龙, 牛梅, 胡婷婷, 刘世家, 王益华, 万建民. 水稻白条纹突变体st13的表型分析及基因定位[J]. 中国水稻科学, 2017, 31(4): 355-363.
Liting SUN, Tianzi LIN, Yunlong WANG, Mei NIU, Tingting HU, Shijia LIU, Yihua WANG, Jianmin WAN. Phenotypic Analysis and Gene Mapping of a White Stripe Mutant st13 in Rice[J]. Chinese Journal OF Rice Science, 2017, 31(4): 355-363.
材料 Material | 株高 Plant height/cm | 有效穗数 No. of effective panicles | 剑叶长 Flag leaf length /cm | 穗长 Panicle length /cm | 结实率 Seed setting rate/% | 千粒重 1000-grain weight /g | |
---|---|---|---|---|---|---|---|
WT | 112.2±2.9 | 9.6±0.6 | 31.5±2.4 | 23.8±0.2 | 95.5±1.5 | 27.9±1.6 | |
st13 | 70.8±4.7** | 5.8±0.5** | 31.9±1.5 | 17.9±0.5** | 74.0±1.8** | 20.3±0.9** |
表1 野生型Dongjin与突变体st13的主要农艺性状比较
Table 1 Comparison of major agronomic traits between wild-type Dongjin and the st13 mutant.
材料 Material | 株高 Plant height/cm | 有效穗数 No. of effective panicles | 剑叶长 Flag leaf length /cm | 穗长 Panicle length /cm | 结实率 Seed setting rate/% | 千粒重 1000-grain weight /g | |
---|---|---|---|---|---|---|---|
WT | 112.2±2.9 | 9.6±0.6 | 31.5±2.4 | 23.8±0.2 | 95.5±1.5 | 27.9±1.6 | |
st13 | 70.8±4.7** | 5.8±0.5** | 31.9±1.5 | 17.9±0.5** | 74.0±1.8** | 20.3±0.9** |
图4 苗期野生型Dongjin和st13突变体中叶绿体发育及光合系统相关基因的荧光定量PCR分析
Fig. 4. Quantitative RT-PCR analyses of genes associated with chloroplast development and photosynthetic system in wild-type Dongjin and st13 mutant at the seedling stage.
图5 苗期野生型Dongjin和st13突变体中叶绿体生物合成相关基因的荧光定量PCR分析
Fig. 5. Quantitative RT-PCR analyses of genes associated with chlorophyll biogenesis in wild-type Dongjin and st13 mutant at the seedling stage.
组合 Combination | 正常株数 No. of normal plants | 白条纹株数 No. of plants with white stripes | 实际分离比 Segration ratio | χ2(3:1) | χ20.05 |
---|---|---|---|---|---|
Dongjin×st13 | 286 | 98 | 2.92:1 | 0.03 | 3.84 |
st13×Dongjin | 281 | 89 | 3.16:1 | 0.13 |
表2 野生型Dongjin与st13突变体正反交组合的F2分离比
Table 2 Segregation of F2 populations from wild-type Dongjin and st13 mutant.
组合 Combination | 正常株数 No. of normal plants | 白条纹株数 No. of plants with white stripes | 实际分离比 Segration ratio | χ2(3:1) | χ20.05 |
---|---|---|---|---|---|
Dongjin×st13 | 286 | 98 | 2.92:1 | 0.03 | 3.84 |
st13×Dongjin | 281 | 89 | 3.16:1 | 0.13 |
引物名称 Primer name | 前引物 Forward primer | 后引物 Reverse primer | BAC克隆 BAC clone | |
---|---|---|---|---|
I3-21 | GCGAGATGGGCAGCTACTAC | ACACAATGTCCAGCTTGCAG | OSJNBa0010D22 | |
P5 | CCTCCTCATAGCCCCAATCC | TCACTCTCCCATAGGTGGTC | OSJNBb0042N11 | |
P3-13 | CCTCCGCACGAACCCT | GTGGGAAACTGGAGACGAG | OSJNBa0087M10 | |
P3-14 | GGTTACATCTCCTTTTCGTTT | TCTTTGTTTGCTGCCATCT | OJ1519_A12 | |
S-57 | AAAACCAAAACTAGAAGC | ATGTCCCTGGAAATGTA | OSJNBa0004G03 | |
S-24 | CTCCGAGCGGCTGATTA | GGATAGTACTTCGTTTCGTA | OSJNBa0004G03 | |
P3-29 | CAAACGCAATCTCACATCACA | AAGCGAGTGGGAGGGTG | OSJNBa0091E13 | |
I3-22 | AGGTCTCGTGTCGTTCATCC | TGGAGGGAGCATGTCTATCA | OSJNBa0063J18 |
表3 目标区域内有多态的InDel标记
Table 3 Polymorphic InDel markers used in this study.
引物名称 Primer name | 前引物 Forward primer | 后引物 Reverse primer | BAC克隆 BAC clone | |
---|---|---|---|---|
I3-21 | GCGAGATGGGCAGCTACTAC | ACACAATGTCCAGCTTGCAG | OSJNBa0010D22 | |
P5 | CCTCCTCATAGCCCCAATCC | TCACTCTCCCATAGGTGGTC | OSJNBb0042N11 | |
P3-13 | CCTCCGCACGAACCCT | GTGGGAAACTGGAGACGAG | OSJNBa0087M10 | |
P3-14 | GGTTACATCTCCTTTTCGTTT | TCTTTGTTTGCTGCCATCT | OJ1519_A12 | |
S-57 | AAAACCAAAACTAGAAGC | ATGTCCCTGGAAATGTA | OSJNBa0004G03 | |
S-24 | CTCCGAGCGGCTGATTA | GGATAGTACTTCGTTTCGTA | OSJNBa0004G03 | |
P3-29 | CAAACGCAATCTCACATCACA | AAGCGAGTGGGAGGGTG | OSJNBa0091E13 | |
I3-22 | AGGTCTCGTGTCGTTCATCC | TGGAGGGAGCATGTCTATCA | OSJNBa0063J18 |
基因 | 功能注释 |
---|---|
Gene | Annotation |
Os03g0603300 | 保守假定蛋白 Conserved hypothetical protein |
Os03g0603500 | 保守假定蛋白 Conserved hypothetical protein |
Os03g0603600 | 甘油磷酸二酯酶 Glycerophosphoryl diester phosphodiesterase family protein |
Os03g0603700 | A22A肽酶 Peptidase A22A, presenilin family protein |
Os03g0604200 | 尿苷二磷酸葡萄糖脱氢酶 Similar to UDP-glucose dehydrogenase |
Os03g0604500 | 保守假定蛋白 Conserved hypothetical protein |
Os03g0604600 | 保守假定蛋白 Conserved hypothetical protein |
Os03g0604700 | 假定蛋白 Hypothetical protein |
表4 94 kb区间内的预测基因及其可能的功能
Table 4 Predicted genes and their putative functions in the 94 kb region.
基因 | 功能注释 |
---|---|
Gene | Annotation |
Os03g0603300 | 保守假定蛋白 Conserved hypothetical protein |
Os03g0603500 | 保守假定蛋白 Conserved hypothetical protein |
Os03g0603600 | 甘油磷酸二酯酶 Glycerophosphoryl diester phosphodiesterase family protein |
Os03g0603700 | A22A肽酶 Peptidase A22A, presenilin family protein |
Os03g0604200 | 尿苷二磷酸葡萄糖脱氢酶 Similar to UDP-glucose dehydrogenase |
Os03g0604500 | 保守假定蛋白 Conserved hypothetical protein |
Os03g0604600 | 保守假定蛋白 Conserved hypothetical protein |
Os03g0604700 | 假定蛋白 Hypothetical protein |
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