水稻脆秆突变体bc1-wu3的鉴定与基因克隆

doi:10.16819/j.1001-7216.2017.6130

摘要/Abstract

摘要：

目的茎秆机械强度与植株抗倒伏性直接相关。发掘脆秆突变体,克隆其相关基因有助于了解茎秆机械强度遗传机制,为抗倒伏育种提供理论依据。方法在经⁶⁰Co-γ诱变的粳稻品种武育粳3号后代群体中获得一个脆秆突变体,命名为bc1 -wu3（brittle culm 1 from Wuyujing3）,以突变体bc1 -wu3为母本,Kasalath为父本构建相应的F₂分离群体,采用图位克隆的方法定位相应脆秆基因。结果与野生型相比,突变体的叶片、叶鞘、茎秆等组织在全生育期始终表现为脆性,易折断;穗长和粒长显著降低,粒宽显著增加。茎秆细胞细胞壁糖成分测定表明,细胞壁中纤维素含量极显著下降,而木糖、葡萄糖及阿拉伯糖含量则显著增加。茎秆切片观察发现突变体茎秆的厚壁细胞层数减少,厚壁细胞细胞壁极显著变薄。遗传分析表明,bc1- wu3脆性性状受1对隐性核基因控制。采用图位克隆的技术将该基因定位于第3染色体标记MK12与MK18之间,物理距离为57 kb,在此定位区段内包含1个已克隆的脆性基因BC1（LOC_Os03g30250）。测序结果表明,突变体bc1-wu3中BC1基因第2外显子内（CDS 659 处）有1个碱基的替换(G-T),导致编码氨基酸由半胱氨酸变异为苯丙氨酸。实时荧光定量PCR结果表明,BC1基因在脆秆突变体bc1-wu3茎秆中的表达量显著降低。结论据此推断本研究中定位的脆秆基因bc1-wu3为BC1新等位基因。相关结果加深了对BC1基因功能的认识,有助于阐明水稻茎秆强度遗传机制。

关键词: 水稻, 脆秆突变体, BC1基因, 遗传分析, 基因定位

Abstract:

【Objective】Culm strength, an important agronomic trait, is related to lodging resistance of rice. The research of brittle culm mutants and genes has great significance to demonstrate the regulation of genetic mechanism of culm mechanical strength and breed lodging resistance varieties.【Method】In this study, a brittle culm mutant, named bc1-wu3(brittle culm 1 from Wuyujing 3), was obtained from the japonica variety Wuyujing 3 after ⁶⁰Co-γ induced mutagenesis. F₂ population derived from bc1-wu3/Kasalath was used to mapping this corresponding gene by position cloning approach.【Result】The mutant was characterized by the brittle leaf, leaf sheath, and culm during the whole growing stage. Compared with the wild type, the panicle length and grain length of bc1-wu3 decreased significantly, and the grain width presented a significant increase. There was a significant decrease in the cellulose content in culm, the number of sclerenchyma cell layers and the thickness of sclerenchyma cell wall, but the contents of xylose, glucose and arabinose increased significantly in bc1-wu3. Genetic analysis showed that the brittle trait in bc1-wu3 was controlled by one single recessive nuclear gene, and the locus was mapped to a 57 kb genomic region on chromosome 3 between molecular markers M12 and Mk18. In the mapped region the cloned brittle culm gene BC1(LOC_Os03g30250) was included, which might be the candidate gene of bc1-wu3. Sequence analysis revealed that there was a single-base substitution(G-T) in the second exon of BC1 gene in bc1-wu3 mutant, leading to a residue substitution from cysteine to phenylalanine. The data from real-time RT-PCR indicated that there was significant decrease in the expression of BC1 gene in the culm of the mutant. 【Conclusion】Based on these results,we speculate that the bc1-wu3 gene is allelic to BC1 gene. These results would deepen our understanding for the function of BC1and help us to clarify the genetic mechanisms of culm strength.

Key words: Oryza sativa L., brittle culm mutant, BC1 gene, genetic analysis, gene mapping

许作鹏, 仲崇元, 张丽佳, 刘巧泉. 水稻脆秆突变体bc1-wu3的鉴定与基因克隆[J]. 中国水稻科学, 2017, 31(2): 157-165.

Zuopeng XU, Chongyuan ZHONG, Lijia ZHANG, Qiaoquan LIU. Identification and Gene Cloning of the Brittle Culm Mutant bc1-wu3 in Rice[J]. Chinese Journal OF Rice Science, 2017, 31(2): 157-165.

图/表 9

表1 本研究中使用的PCR引物

Table 1 PCR primers used in this study.

引物名称	正向引物	反向引物	实验目的
Primer name	Forward primer (5′-3′)	Reverse primer (5′-3′)	Purpose
RM6676	TGCATAACACCCAAATGCCTTC	TATGTTACAATGTTCACGGTCC	定位Mapping
STS-3-86.6	AGGATTTGGGCTGTCAATGC	AGCCTACCACAAGACATATAATCG	定位Mapping
MK2	TGGTTCAATCATGTATAGGTCGTG	CATAAGTTATTAGTTAACACAAACC	定位Mapping
MK12	GAATTGTGAGCATACAGCAA	ATTCGATTTTGTGGATTGAC	定位Mapping
MK16	TGGCGTGCTAGACCTGATGTTTG	TGTCTGATAAGGCTCTCAATGCG	定位Mapping
MK18	TCTTCTCTCATCGTCATCCT	ACAAGCCAACAAAGCTAGAG	定位Mapping
MK21	AGCCTTTGCTCTCTCTCTCT	CTTTGTATCTGTGCGAGAAA	定位Mapping
bc1-1	AAATGTTCCGCCGTCGCTAT	GTCGCCCATGATGCACTCG	测序Sequence
bc1-2	CGCAGCAGACGCAACAGC	TTGAACCCCTATGGAGTA	测序Sequence
bc1-3	CGGGACGAACCCAAGATT	GACGGTGGAAGGGACGAT	测序Sequence
bc1-4	ACCCACCAACTTCACCCT	AGTCCTTTCGCATCAGCA	测序Sequence
bc1-5	GCTCTCTACCACAACCTCTTA	TGTTTCTTTTTTCCTCTCAAT	测序Sequence
bc1-6	CGTCCCCTACAACCAGC	GTCCTTTCGCATCAGCA	测序Sequence
bc1-RT	CTTTGAAATTGCCTGATAGA	AAAGTTTGTGGTGTGATTT	RT-PCR
OsActin	GGAAGTACAGTGTCTGGATTGGAG	TCTTGGCTTAGCATTCTTGGGT	RT-PCR

图1 野生型(WT)与突变体bc1-wu3表型的比较

Fig. 1. Comparison of the phenotypes between wild type(WT) and bc1-wu3.

图2 野生型(WT)与突变体bc1-wu3种子颖壳细胞形态的比较

Fig. 2. Comparison of the size of grain glume cell between wild type(WT) and bc1-wu3.

表2 野生型(WT)和突变体bc1-wu3的农艺与产量性状

Table 2 Agronomic and yield traits of wild type(WT) and bc1-wu3.

材料	抽穗期	株高	分蘖数	穗长	结实率	千粒重	粒长	粒宽
Material	Heading date /d	Plant height/cm	No. of tillers	Panicle length/cm	Seed-setting rate/%	Thousand-grain weight/g	Grain length/mm	Grain width/mm
WT	103.2±0.3	84.1±3.4	10.0±2.1	16.5±1.7	96.5±1.0	26.69±0.19	7.11±0.91	3.15±0.85
bc1-wu3	103.1±0.3	82.7±2.1	9.0±2.2	14.7±1.1^*	95.2±0.9	26.77±0.17	6.97±0.89^*	3.35±0.54^*

图3 野生型(WT)和突变体bc1-wu3的茎秆机械强度

Fig. 3. Culm strength of wild type(WT) and bc1-wu3.

表3 野生型(WT)与突变体bc1-wu3茎秆细胞细胞壁中糖组分含量比较

Table 3 Comparison of the sugar composition contents of culms between wild type(WT) and bc1-wu3. mg/g

材料	鼠李糖	岩藻糖	阿拉伯糖	木糖	甘露糖	半乳糖	葡萄糖	纤维素
Material	Rhamnose	Fucose	Arabinose	Xylose	Mannose	Galactose	Glucose	Cellulos
WT	1.86±0.02	1.05±0.01	23.46±0.51	217.64±2.60	1.20±0.41	6.95±0.26	45.76±1.89	426.52±7.13
bc1-wu3	2.02±0.04	1.08±0.01	29.61±0.85^*	310.93±9.65^**	1.21±0.40	8.74±0.22	55.42±0.72^*	261.17±2.53^**

图4 野生型(WT)和突变体bc1-wu3茎秆细胞结构

Fig. 4. Scanning electron micrographs of the culm phenotype of wild type(WT) and bc1-wu3. A和B–茎秆横截面;C和D–茎秆厚壁细胞细胞壁厚度;E–厚壁细胞细胞壁厚度定量比较,数据为平均值±标准差(n=100)。**表示在0.01水平差异显著(t检验)。A and B, Comparison of the cross sections of internodes. Bars=20 μm. C and D, The sclerenchyma cells of the internodes under a scanning electron microscope. Bars =2 μm. E, Quantification of the thickness of the sclerenchyma cell wall. Error bars, mean ±SD(n=100). **P<0.01(t test).

图5 bc1-wu3基因精细定位及候选基因序列分析

Fig. 5. Fine mapping of bc1-wu3 gene and sequence analysis of the candidate gene. A–bc1-wu3的初步定位; B–bc1-wu3定位于MK12与MK18之间,物理距离为57 kb; C–MK12与MK18之间的57 kb范围内含10个ORF;D–LOC_Os03g30250为候选基因,序列比对分析结果表明,bc1-wu3候选基因的CDS序列中第659个碱基G突变为T,导致bc1-wu3编码的氨基酸由半胱氨酸(Cysteine)变为苯丙氨酸(Phenylalanine)。A, Primary mapping of bc1-wu3 gene; B, Fine mapping of bc1-wu3 gene in the 57 kb region between molecular markers MK12 and MK18; C, The region between marker MK12 and MK18 contains 10 ORF; D, The locus named LOC_Os03g30250 is identified as candidate gene. Sequence analysis result demonstrates that the CDS of bc1-wu3 carries one point mutation (G/T), resulting in the substitution from Cysteine to Phenylalanine.

图6 BC1基因在水稻茎秆中的表达量

Fig. 6. Expression analysis of the BC1 gene in culm of rice.

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