水稻小粒基因SG101的鉴定和精细定位

doi:10.16819/j.1001-7216.2017.6152

摘要/Abstract

摘要：

【目的】籽粒大小是决定水稻产量的重要农艺性状之一,开展水稻籽粒大小相关基因的克隆和功能研究对于阐述水稻产量形成的遗传调控机制具有重要意义。【方法】利用甲基磺酸乙酯诱变粳稻品种中花11,筛选获得一小粒突变体,命名为sg101(small grain 101)。通过形态学、细胞学手段调查了SG101的突变对籽粒大小、穗部主要性状及颖壳细胞数目和大小的影响,通过测定叶夹角和胚芽鞘长度分析其对外施油菜素内酯的差异响应,结合定量PCR技术分析了油菜素内酯合成途径和信号途径相关基因表达情况,并利用图位克隆的手段精细定位了水稻小粒基因SG101。【结果】与野生型相比,突变体sg101粒长和粒宽均极显著减小,从而导致千粒重极显著降低。此外,sg101还表现出结实率降低、穗长变短、二次枝梗数减少、植株变矮等。细胞学观察发现sg101的颖壳细胞大小没有改变,但细胞数目明显减少。定量PCR检测表明sg101中的细胞周期相关基因表达显著下降。另外,突变体sg101对外施油菜素内酯响应迟钝,其油菜素内酯合成途径和信号途径相关基因表达亦显著降低。【结论】遗传分析表明sg101突变体由隐性单基因控制,通过图位克隆的方法将SG101精细定位于第1染色体上,物理距离为265 kb的区间内。这为该基因的克隆及深入的功能研究奠定了基础。

关键词: 水稻, 小粒, sg101, 油菜素内酯

Abstract:

【Objective】Grain size is an important agronomic trait in determining grain yield. The characterization and identification of grain size related genes will be beneficial to expound the genetic regulatory mechanisms behind yield formation in rice. 【Method】Here, we report the characterization of a rice (Oryza sativa L.) mutant, small grain 101 (sg101), gained by EMS mutagenesis for Zhonghua 11. The effects of sg101 on grain size, panicle-related traits, cell number and size of lemma were assessed by morphological and cytological methods. The responses to brassinolide(BR) were detected based on the variation in leaf angle and coleoptile length. The expression level of BR signal and synthesis related genes were tested by RT-PCR. Map-based cloning was executed for fine mapping of SG101.【Result】The grain length, grain width and grain weight were significantly reduced in sg101. Moreover, sg101 showed decreased panicle length, secondary rachis branch number, plant height and seed setting rate. The paraffin section observation under a scanning electron microscope indicated that the cell number in sg101 was significantly decreased, while the cell size was similar to wild type, suggesting that SG101 affected the grain size by regulating the cell division. Quantitative RT-PCR analysis displayed that the expression of cell cycle related genes was reduced in sg101. After treatment with BR, sg101 showed smaller leaf angle and shorter coleoptile than those of the wild type and the expression of BR biosynthetic and signal pathway related genes was down-regulated in sg101. Genetic analysis revealed that sg101 phenotype was controlled by a single recessive nuclear gene.【Conclusion】SG101 was mapped on the long arm of chromosome 1 between two STS markers S5 and S6 with a 265 kb physical distance. It laid a base for the further cloning and functional analysis of SG101.

Key words: rice, small grain, sg101, brassinolide

中图分类号:

汪玉琼, 杨窑龙, 冷语佳, 黄李超, 陈龙, 代丽萍, 涂政军, 高易宏, 胡江, 朱丽, 张光恒, 任德勇, 高振宇, 董国军, 陈光, 郭龙彪, 叶国友, 钱前, 曾大力. 水稻小粒基因SG101的鉴定和精细定位[J]. 中国水稻科学, 2017, 31(6): 580-589.

Yuqiong WANG, Yaolong YANG, Yujia LENG, Lichao HUANG, Long CHEN, Liping DAI, Zhengjun TU, Yihong GAO, Jiang HU, Li ZHU, Guangheng ZHANG, Deyong REN, Zhenyu GAO, Guojun DONG, Guang CHEN, Longbiao GUO, Guoyou YE, Qian QIAN, Dali ZENG. Identification and Fine Mapping of Small Grain Gene SG101 in Rice (Oryza sativa L.)[J]. Chinese Journal OF Rice Science, 2017, 31(6): 580-589.

图/表 10

表1 本研究用于RT-PCR分析的引物

Table 1 Primers used for real-time RT-PCR in the study.

引物 Marker	前引物序列 Forward primer (5′-3′)	后引物序列 Reverse primer (5′-3′)
UBQ5	CTCGCCGACTACAACATCCA	TCTTGGGCTTCCTCTACGTCTT
E2F2	TGTTGGTGGCTGCCGATAT	CGCCAGGTGCACCCTTT
CYCT1;2	GCATTTGTTGCAGCTCAAG	TCACCACTTCGCTGACTTATTG
CYCD4;1	GCCATGGAGTTGATACATCCAA	CCAGTAGGGCTCCGTGGAAT
CYCD7;1	CCTTCCACACTGACGGTACAGTT	TGCCGCTGCCAAATAGACA
CYCB2;2	CTCAAGGCTGCACAATCTGACA	GCATTGACGGCTGGAATTTG
D2	TTCAACCCATGGAGGTGGAA	GCACGGTGGGGAAGTTGACGA
OsCPOD	TTCTTCTCCATCCCCTTTCCTCTCGCCA	CACCCTCCGCCTCAAGAAGCTCCTCAA
DWARF4Q	GAGATGGTTTTCACGCAATGTG	ACCCTTGTAGTGCACGTCCTTG
BU1	GTAGCCAGCTTGATCTCATCTC	GGGACGACTCTACTGCATCA
BZRF	CTCGGCAGCGTCGAGGTGC	AGGAATTGTTGCTGAGCTTC
D61	CTCGGCAGCGTCGAGGTGC-3'	AGGAATTGTTGCTGAGCTTC
PAVL1	GCACTCCTCGTTGGATCTCGAT	GCAGCAGACGGAAGATGGATT
OsLIC	GGAGTTTCGAGCGTATCTGGAA	TGGACAGAGGAAGCAGGAGACT
OsMDP1	TTATTGACCGGTACAACTCGCA	TCCAGTCCATCGATCTCATCC

图1 野生型中花11和突变体sg101的表型比较**代表突变体与野生型之间差异达0.01显著水平。图2和图3同。

Fig. 1. Phenotypes of Zhonghua 11(WT) and sg101. **Significant difference at 0.01 level between wild type and mutant sg101. The same as that in Fig. 2 and Fig. 3.

表2 野生型中花11和突变体sg101的穗部性状比较

Table 2 Comparison of panicle-related traits between Zhonghua 11 and sg101.

性状 Trait	野生型 Wild type	突变体 sg101
株高Plant height/cm	88.8±2.4	67.6±6.1
穗长Panicle length/cm	22.7±1.1	17.1±1.2
着粒密度Spikelet density	11.8±1.3	9.9±1.4
一次枝梗数Primary rachis branch number per panicle	15.0±1.0	13.0±3.0
二次枝梗数Secondary rachis branch number per panicle	51.3±11.1	33.6±7.1
每穗颖花数Spikelet number per panicle	270.0±40.0	170.0±29.3
结实率Seed setting rate/%	70.0±4.3	5.0±3.2

图2 野生型中花11和突变体sg101籽粒的比较 A–野生型和sg101发育成熟的颖;B,C分别为野生型和sg101颖壳石蜡切片;D–野生型和sg101横切面细胞数目的比较;E,F分别为野生型和sg101颖壳内表皮的扫描电镜图;G–野生型和sg101颖壳细胞长度的比较。

Fig. 2. The comparison of grain between Zhonghua 11(WT) and sg101. A, Developed lemma; B and C, Paraffin section of the lemmas of wild-type and sg101, respectively. D, Cell number of the lemma; E and F, SEM analysis of the lemma outer surface of wild-type and sg101, respectively. G, Cell length of the outer epidermal lemma.

图3 细胞周期相关基因在野生型中花11和突变体sg101中的表达差异

Fig. 3. Expression levels of cell cycle-related genes in the wild type(WT) and sg101.

图4 中花11和突变体sg101对2,4-epiBL处理的响应 A–不同2,4-epiBL浓度处理,上面为中花11,下面为sg101,bar=2 cm;B, C分别为不同2,4-epiBL浓度处理叶夹角和胚芽鞘长度的变化。*,**分别代表突变体与野生型之间差异达0.05,0.01显著水平。

Fig. 4. Lamina joint test to 2,4-epiBL in Zhonghua 11(WT) and sg101. A, Performance of lamina joint to 2,4-epiBL in wild-type(up) and sg101(down). Scale bars, 2.0 cm; B and C, Changes of lamina inclination and coleoptile length to 2,4-epiBL, respectively. *, **Significant difference at 0.05 and 0.01 level between wild type and mutant sg101, respectively.

表3 SG101的遗传分析

Table 3 Genetic analysis of SG101.

分离群体 Segregation population	F₁		F₂		χ²_(3:1)	P值 P-value
分离群体 Segregation population	正常表型 Wild-type phenotype	突变表型 sg101 phenotype	正常表型 Wild-type phenotype	突变表型 sg101 phenotype	χ²_(3:1)	P值 P-value
sg101/TN1	4	0	116	34	0.436	0.509
sg101/南京6号 sg101/Nanjing 6号	8	0	258	74	1.301	0.254
sg101/9311	5	0	154	43	1.058	0.304
TN1/sg101	4	0	128	38	0.394	0.530
南京6号/sg101 Nanjing 6/sg101	3	0	102	29	0.573	0.449
9311/sg101	6	0	178	56	0.143	0.076

图5 BR合成和信号途径相关基因在野生型中花11和突变体sg101中的表达

Fig. 5. Expression levels of BR synthesis and signal transduction pathway genes in Zhonghua 11(WT) and sg101.

图6 SG101的遗传定位

Fig. 6. Molecular mapping of SG101.

表4 本研究中SG101定位所用引物

Table 4 Primers used for fine mapping of SG101 in this study.

引物 Marker	前引物序列 Forward primer(5′-3′)
M1	GCTAGGCTTGATCCGAGAGA	GTCCCGCTTCTTCCCCTA
M2	GACCACGTCAAGCATAAGTTCA	AGCCTTTCAGGGAGGAAGAA
M3	CTCCCTACTTCTCCTCTTCG	CATACGCAACACCGCATCTT
M4	GGTGTTTGATCCCATTTGCT	TGGATGAAGAACTGCGCATA
S1	GGAACGGGGTGACAATTCTA	GCCTGTTTGAGGGAATGGTA
S2	ATTCTTACTTAATAATCAATAC	TACCTAGCTCGACTCAGACCCA
S3	GAGCAGGCAGACCTGCATAA	AAAACACTGCCTGTGATTCTGT
S4	CGACGGTGGGAGCAGAAGAAA	TCGGACTATCTCTGCTGGAATT
S5	ACTCCTATGTTTCAACCTG	GTGACCATCAATGAACCTT
S6	AAATCTAGTCAACACGCCGT	ACATGAGTTTAATTCCAAAATT

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