水稻脆秆突变体bc-s1的表型分析和基因定位

doi:10.11983/CBB15048

摘要/Abstract

摘要： 茎秆机械强度影响植株抗倒伏能力, 是备受关注的重要农艺性状之一。与野生型相比, 水稻(Oryza sativa)脆秆隐性突变体bc-s1茎秆抗折力和抗张力分别降低31.1%和67.2%, 茎秆纤维素和木质素含量分别降低24.97%和增高38.82%。细胞学分析显示, bc-s1茎秆厚壁细胞发生不规则变化, 次生壁增厚受阻。通过图位克隆和测序分析, 初步确定bc-s1突变体中纤维素合成酶催化亚基Os09g25490/OsCesA9基因第1外显子的第28个碱基G突变为A。该等位突变体的获得为进一步揭示OsCesA9调控细胞壁建成的生物学功能提供了新的研究材料。

关键词: bc-s1, 脆秆, 次生细胞壁, 纤维素合成酶, 水稻

Abstract: The mechanical strength of rice stem is an important agronomic trait that affects crop lodging. In this study, a brittle culm mutant, bc-s1, was identified from the rice mutant library. The mutant showed inherited traits of brittleness from bolting to mature stage. Meanwhile, the leaf, leaf sheath, culm, node and seed shell of the mutant showed brittleness during the mature stage. Compared with breaking and elongation force in the wild type, that in bc-s1 culms decreased 31.1% and 67.2%, respectively. Besides showing irregular shape of sclerenchyma cells, in bc-s1, cellulose and lignin content in culm decreased 24.97% and increased 38.82%, respectively. The BC-S1 gene was mapped on chromosome 9 between InDel markers Ha6 and Ha9 within 101 kb physical distance. According to sequencing results for bc-s1, a candidate open reading frame (Os09g25490/OsCesA9), which encodes a cellulose synthase catalytic subunit (CesA), showed a point mutation at the 28^th base of first extron, changing GGG to AGG and causing the substitution at the 10th amino acid residue from glycine to arginine. Therefore, bc-s1 is a new allele of OsCesA9 and provides a new material for uncovering the biological function of OsCesA9 in rice cell-wall biosynthesis.

Key words: bc-s1, brittle culm, secondary wall, cellulose synthase, rice

靳振明, 平宝哲, 沈浩珺, 杜淮清, 李瑞乾, 朱璐, 张大兵, 袁政. 水稻脆秆突变体bc-s1的表型分析和基因定位. 植物学报, 2016, 51(2): 167-174.

Zhenming Jin, Baozhe Ping, Haojun Shen, Huaiqing Du, Ruiqian Li, Lu Zhu, Dabing Zhang, Zheng Yuan. Characterisation and Gene Mapping of a Brittle Culm Mutant bc-s1 in Rice. Chinese Bulletin of Botany, 2016, 51(2): 167-174.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB15048

https://www.chinbullbotany.com/CN/Y2016/V51/I2/167

图/表 5

表1 图位克隆定位引物序列

Table 1 DNA markers used for positional cloning

Marker	Forward primer (5'-3')	Reverse primer (5'-3')	Type
Chr09_04	GGTAATGTCACAACTCAAAAAGC	CAAGATTGTAAACCCTGTCTATTG	InDel
Ha1	GCGAACCGATAAAACTGCTC	AGAGGTGTATCAAAGCAATCGAG	InDel
Ha4	TCACCTCTCAACTTAATCGA	AGTCCATCAAGCCATGATGC	InDel
Ha6	AGTTCGTCCGGTTTTGATCG	GTAGAATAAGCGAAACAGCA	InDel
Ha9	TCGACCATCAGCGATTTGAC	TTTTCCATGCGCGGTGTTTG	InDel
Chr09_05	GAATTTGAGTGGGTTTATACTAGC	GTTAACTTAGGCTATTTTGGCTTC	InDel

图1 水稻突变体bc-s1与野生型9522的表型与机械强度 (A) 抽穗期突变体bc-s1与野生型9522整株表型(Bar=20 cm); (B) 成熟期水稻茎秆第2节间手折脆性性状; (C) 成熟期水稻剑叶手折脆性性状; (D) 茎秆第2节间抗张力测定; (E) 茎秆第2节间抗折力测定。*和**分别表示t-检验的显著差异(P<0.05)和极显著差异(P<0.01)。

Figure 1 The phenotype and mechanical strength of bc-s1 mutant and wild-type 9522 in Oryza sativa (A) The whole plant phenotype of bc-s1 mutant and wild-type 9522 in the heading stage (Bar=20 cm); (B) An easily broken culm of bc-s1 compared with a wild-type 9522 culm in the mature stage; (C) An easily broken flag leaf of bc-s1 compared with a wild-type 9522 flag leaf in the mature stage; (D) The elongation force of the second upper internode; (E) The breaking force of the second upper internode. * and ** represent t-test at P<0.05 and P<0.01, respectively.

图2 水稻野生型9522与突变体bc-s1茎秆横切片扫描电镜观察 (A), (B) 野生型9522初生壁; (C), (D) 野生型9522次生壁; (E), (F) 突变体bc-s1初生壁; (G), (H) 突变体bc-s1次生壁。(A), (C), (E), (G) Bar=100 μm; (B), (D), (F), (H) Bar=5 μm

Figure 2 The cross-section of wild-type 9522 and bc-s1 mutant culms in Oryza sativa under a scanning electron microscope (A), (B) The primary wall of a wild-type 9522 culm; (C), (D) The secondary wall of a wild-type 9522 culm; (E), (F) The primary wall of a bc-s1 mutant culm; (G), (H) The secondary wall of a bc-s1 mutant culm; (A), (C), (E), (G) Bar=100 μm; (B), (D), (F), (H) Bar=5 μm

图3 水稻野生型9522与突变体bc-s1茎秆细胞壁组织切片观察与组分测定 (A), (B) 细胞壁木质素间苯三酚染色, 图(A)和(B)分别为野生型9522和脆秆突变体bc-s1第2节间茎秆组织横切片; (C), (D) 细胞壁纤维素荧光增白剂染色, 图(C)和(D)分别为野生型9522和脆秆突变体bc-s1第2节间茎秆组织横切片; (E) 野生型9522与突变体bc-s1茎秆细胞壁组分比较。*和**分别表示t-检验的显著差异(P<0.05)和极显著差异(P<0.01)。

Figure 3 Tissue section staining and components determination in the culm cell wall of wild-type 9522 and bc-s1 mutant in Oryza sativa (A), (B) The cross section of the second upper internodes of wild-type 9522 (A) and bc-s1 mutant (B) were stained with phloroglucinol; (C), (D) The cross section of the second upper internodes of wild-type 9522 (C) and bc-s1 mutant (D) were stained with Calcofluor White; (E) The comparison of cell-wall components between bc-s1 mutant and wild-type 9522 culm. * and ** represent t-test at P<0.05 and P<0.01, respectively.

图4 BC-S1基因的图位克隆 BC-S1基因定位于水稻第9号染色体的AP005579 BAC上, 基因编号为LOC_Os09g25490, 具有编码纤维素合成酶A催化亚基9 (OsCesA9)的功能。测序分析结果显示, BC-S1基因第1外显子的第28个碱基发生G变为A的点突变。

Figure 4 Map-based cloning of BC-S1 The BC-S1 gene (Os09g25490/OsCesA9), which encodes a cellulose synthase catalytic subunit (CesA), was mapped on BAC AP005579, chromosome 9. According to sequence comparison result, the BC-S1 showed a point mutation at the 28th base of first extron (changing GGG to AGG)

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