水稻矮秆多分蘖突变体mz3的遗传分析和基因定位

doi:10.16819/j.1001-7216.2016.6037

摘要/Abstract

摘要：

通过EMS诱变粳稻品种中花11获得一个稳定遗传的矮秆多分蘖突变体mz3。遗传分析表明该突变性状受一对隐性基因控制,并利用mz3与籼稻品种南京11杂交建立的F₂群体,将该基因定位在水稻第6染色体长臂上的SSR标记RM19353与RM510之间约747 kb范围内。由于该区间包含控制水稻株高和分蘖的D3基因,结合表型分析,推测突变基因与D3可能为一对等位基因。设计7对引物分别对中花11与突变体mz3的基因进行测序,结果显示,与中花11相比,D3基因在mz3中第636位核苷酸由G突变为A,使得编码色氨酸的密码子TGG突变为终止密码子TGA,导致翻译提前终止。进一步对定位群体中10个隐性极端个体测序,结果显示所有极端个体都带有该突变位点。亚细胞定位结果表明,突变体编码的D3蛋白与野生型一样定位在细胞核中,荧光双分子互补试验结果表明,突变体D3蛋白不能与D14蛋白发生互作,推测突变体编码的D3截短蛋白缺少了与D14互作的氨基酸序列,从而阻碍了独脚金内酯信号传递。因此,mz3表型很可能由D3基因突变引起。

关键词: 水稻, 矮秆多分蘖, D3, 基因定位

Abstract:

A dwarf and high-tillering mutant, temporarily termed as mz3 was obtained from Zhonghua 11, a japonica rice variety by EMS mutagenesis. Genetic analysis indicated that the mutant phenotype was controlled by a recessive gene. An F₂ population was developed by crossing mz3 with Nanjing 11, an indica rice variety for mapping of the mutant gene, which was ultimately restricted within a physical distance of about 747 kb between two SSR markers RM19353 and RM510 on the long arm of rice chromosome 6. Gene prediction revealed that D3, a gene conferring rice plant height and tiller number was located in this region. Sequence analysis identified a single G to A nuclear acid substitution at the position 636 from the ATG start codon, forming a premature translational termination codon. Sequencing of 10 randomly selected recessive plants from mz3/Nanjing 11 mapping population confirmed the same mutation site carried by the 10 individuals. Subcelluar location indicated that D3 protein of the mutant was located in the nuclei as wild type. BiFC analysis showed that D3 protein of the mutant could not interact with D14 protein for lacking of essential amino acids that bind with D14, blocking signal transduction of strigolactones. So we speculated that the mutant phenotype of mz3 was probably caused by mutation in D3 gene.

Key words: rice, dwarf and high-tillering, D3, gene mapping

中图分类号:

苏晓妹, 方宇星, 刘钰龙, 刘峰, 张所兵, 张云辉, 鲍依群. 水稻矮秆多分蘖突变体mz3的遗传分析和基因定位[J]. 中国水稻科学, 2016, 30(5): 479-486.

Xiao-mei SU, Yu-xing FANG, Yu-long LIU, Feng LIU, Suo-bing ZHANG, Yun-hui ZHANG, Yi-qun BAO. Genetic Analysis and Gene Mapping of a Dwarf and High-tillering Mutant mz3 in Rice (Oryza sativa L.)[J]. Chinese Journal OF Rice Science, 2016, 30(5): 479-486.

图/表 6

图1 野生型中花11与突变体mz3的表型

Fig.1. Phenotype of wild type Zhonghua 11 and mz3.

图2 野生型与突变体的农艺性状对比 **表示0.01水平上差异显著; ns表示差异不显著。

Fig. 2. Comparison of the agronomic traits of wild type Zhonghua 11 and mz3. **P<0.01; ns, Not significantly different by Student’s t-test.

图3 突变基因的精细定位

Fig. 3. Fine mapping of the mutant gene.

图4 候选基因的序列分析与后代验证 A-突变体mz3的基因突变位点; B-10株隐性极端个体e1~e10测序结果。 ZH11,中花11(野生型)。

Fig. 4. Sequence analysis of candidate gene and offspring validation. A, Mutation site of mz3; B, Sequencing results of 10 extreme recessive individuals (e1-e10). ZH11, Zhonghua 11(wild type).

图5 D3蛋白的亚细胞定位 A-野生型的D3蛋白定位在细胞核中; B-突变型的D3蛋白定位在细胞核中 (Bar=10 μm)。

Fig. 5. Subcellular localization of D3 protein. A, D3 protein of wild type located in the nuclei; B, D3 protein of the mutant located in the nuclei (Bar=10 μm).

图6 D3与D14的双分子荧光互补分析 A-野生型的D3与D14能够在核中结合; B-突变体的D3蛋白不能与D14蛋白在细胞核中发生互作; C-野生型的D3与p2YN空载体共转化; D-突变体的D3与p2YN空载体共转化; E-p2YC空载体与D14共转化。(Bar=10 μm)

Fig. 6. Bimolecular fluorescence complementation (BiFC) analysis of D3 and D14. A, Wild type D3 can interact with D14 in the nuclei; B, Mutant D3 can not interact with D14 in the nucleus; C, Co-transformation of wild type D3 with empty p2YN vector; D, Co-transformation of mutant D3 with empty p2YN vector; E, Co-transformation of empty p2YC vector with D14. (Bar=10 μm).

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