Phenotyping and Gene Cloning of a Small-grain Dwarf Mutant sgd1(t) in Rice

doi:10.16819/j.1001-7216. 2016.5025

Abstract

Abstract:

A small-grain dwarf mutant, designated as sgd1(t) , was identified from the T-DNA insertion mutant lines of Nipponbare.The mutant,genetically stable,was characterized by dwarf plant, small grain, dark green leaves and thick husk. Scanning electron microscope analysis on cell morphology of stem and hull revealed that stem cells in sgd1(t) failed to form normal cell column and vascular bundles, while epidermal cells with irregular shape in hulls were tightly packed, resulting in confusion in cell arrangement, and sgd1(t) was a GA-sensitive dwarf mutant. Genetic analysis showed that this trait of dwarfism was controlled by a pair of recessive nuclear gene. Through map-based cloning, the gene sgd1 (t) was mapped in an interval of 230 kb between the markers DF13 and DF26 on the short arm of chromosome 9. There was a dwarf gene BC12/GDD1 in this region. Sequence analysis showed that sgd1(t) had a single-base substitution (G to T) in the fourth exon of the gene, resulting in the replacement of glycine by valine in 186th conservative amino acid.

Key words: rice (Oryza sativa L.), small grain and dwarfism, gene cloning, BC12/GDD1, single-base substitution

摘要：

在日本晴T-DNA突变体库中筛选得到小粒矮秆突变体sgd1(t) ,经多代自交稳定遗传。sgd1(t) 出现植株矮化、粒型圆小、叶色深绿和颖壳厚实等表型。茎秆及颖壳细胞扫描电镜结果表明,sgd1(t) 茎秆细胞不能形成正常细胞列、维管束发育异常;颖壳表皮细胞排列紧密但不规则;GA信号转导途径响应正常。遗传分析表明突变体sgd1(t) 的矮秆性状受一对隐性核基因控制。采用图位克隆方法,将sgd1(t) 定位于第9染色体短臂Indel标记DF13和DF26之间,物理距离约为230 kb。该区间存在已克隆的矮秆基因BC12/GDD1。测序结果显示,sgd1(t) 在该基因第4个外显子发生由G到T的单碱基突变,导致第186位保守氨基酸由甘氨酸突变为缬氨酸。

关键词: 水稻, 小粒矮秆, 基因克隆, BC12/GDD1, 单碱基突变

CLC Number:

Q343.5
Q785

Peng WANG, Yue CAI, Wei-wei CHEN, Jing MA, Xin-gang CHEN, Xiao-jie TANG, Xiao-man YOU, Fei KONG, Jie ZHANG, Hai-gang YAN, Guo-xiang WANG, Ling JIANG, Wen-wei ZHANG, Jian-min WAN. Phenotyping and Gene Cloning of a Small-grain Dwarf Mutant sgd1(t) in Rice[J]. Chinese Journal OF Rice Science, 2016, 30(1): 1-9.

汪鹏, 蔡跃, 陈韦韦, 马婧, 陈新刚, 唐小洁, 尤小满, 孔飞, 张杰, 燕海刚, 汪国湘, 江玲, 张文伟, 万建民. 水稻小粒矮秆突变体sgd1(t)的表型分析及基因克隆[J]. 中国水稻科学, 2016, 30(1): 1-9.

Figures/Tables 9

Table 2 Indel markers designed for fine-mapping.

标记 Marker	正向引物序列(5'-3') Sequence of forward primer(5'-3')	反向引物序列(5'-3') Sequence of reverse primer(5'-3')
D28	CATATCAACTAGCCCTACCG	GTCCATTATTGGCGTCCC
DF16	CAGATGGAGGTTACTCTGCTTCG	TAGTCAATGTGCCACCAGTAGGG
DF26	ATGGAATTAACCGTGGCT	GCTTTGGCCTCCATCAG
DF13	TATTGCACCTGCCTATTCG	TTGATGCCACCATCCTCTT
DF12	TCTCATAAGCCCAAATCGT	TAGTAGTCGTCGGCGTCAT
DF15	ATCAGGGCATTCACCTCCG	TAGCACCCCACAGCTCAAA
BC3	TGATAGTGCAACGGCAAGG	GTGGAGTTGTCAGCAGTGG

Fig. 1. Morphological characterization of Nipponbare and sgd1(t). A, Gross morphology of Nipponbare and sgd1(t) at the mature stage, bar=30 cm; B, Morphology of seed and brown rice of Nipponbare and sgd1(t) (n=5),bar=5 mm; C and D, Diagram of the length of the panicles and internodes (n=10). P,Ⅰ,Ⅱ, Ⅲ, Ⅳ, Ⅴ indicate the panicle and the five upper internodes, respectively.

Table 1 Comparison of agronomic traits between Nipponbare and sgd1(t) .

农艺性状 Agronomic traits	日本晴 Nipponbare	sgd1(t)
株高Plant height/cm	106.79±9.90	36.64±3.01^**
单株分蘖数 Tiller number per plant	19.6±3.44	22.0±4.96^**
粒长 Grain length/mm	7.81±0.38	5.42±0.30^**
粒宽 Grain width/mm	3.21±0.15	2.37±0.45^**
粒厚 Grain thickness/mm	2.17±0.11	1.13±0.25^**
千粒重 1000-grain weight/g	25.58±1.34	18.21±1.51^**
每穗粒数 No. of grains per panicle	114.50±20.17	30.62±6.10^**

Fig. 2. Leaves of Nipponbare and sgd1(t). A, Leave color; B, Pigment contents (n=3). Chl a, Chlorophyl a; Chl b, Chlorophyl b; Chl a/b, Ratio of chlorophyl a to chlorophyl b; Chl a+b, Sum of chlorophyl a and chlorophyl b.

Fig. 3. Electronic microscopy analysis of culm cells of Nipponbare and sgd1(t). A and B, Longitudinal sections, bar=100 μm; C and D, Transverse sections, bar=1 mm; E and F, Magnifications of indicated regions in C and D, bar=100 μm. SC, Sclerenchyma cells; VB, Vascular bundle; PC, Parenchyma cells.

Fig. 4. Electronic microscopy analysis of the glume cells of Nipponbare and sgd1(t). A and B, Epidermis cells, bar=200 μm; C and D,Transverse sections, bar=50 μm.

Fig. 5. Seeding phenotype(A) and length of second leaf sheath(B) with GA3 treatment between wild type and sgd1(t) (n=3).

Fig. 6. Distribution of plant height in F2 population derived from sgd1(t) /Dular.

Fig. 7. Map-based cloning of sgd1(t). A, Physical mapping of sgd1(t); B, Mutation site of sgd1(t); C, Comparison of amino acid sequences of BC12/GDD1.

References 25

[1]	Peng J,Richards D E,Hartley N M,et al.‘Green revolution’genes encode mutant gibberellin response modulators.Nature. 1999,400(6741):256-261.
[2]	Itoh H, Ueguchi-Tanaka M, Sentoku N,et al.Cloning and functional analysis of two gibberellin 3 beta -hydroxylase genes that are differently expressed during the growth of rice.Proc Natl Acad Sci USA, 2001,98(15):8909-8914.
[3]	Ueguchi-Tanaka M,Ashikari M,Nakajima M,et al.GIBBERELLIN INSENSITIVE DWARF1 encodes a soluble receptor for gibberellin.Nature, 2005,437(7059):693-698.
[4]	Sasaki A,Ashikari M,Ueguchi-Tanaka M,et al.Green revolution:A mutant gibberellin-synthesis gene in rice.Nature, 2002,416(6882):701-702.
[5]	Gomi K,Sasaki A,Itoh H,et al.GID2,an F-box subunit of the SCF E3 complex,specifically interacts with phosphorylated SLR1 protein and regulates the gibberellin-dependent degradation of SLR1 in rice.Plant J, 2004, 37(4):626-634.
[6]	Yamamuro C,Ihara Y,Wu X,et al.Loss of function of a rice brassinosteroid insensitive1 homolog prevents internode elongation and bending of the lamina joint.Plant Cell, 2000,12(9):1591-1606.
[7]	Hong Z,Ueguchi-Tanaka M,Umemura K,et al.A rice brassinosteroid-deficient mutant,ebisu dwarf (d2), is caused by a loss of function of a new member of cytochrome P450.Plant Cell, 2003,15(12):2900-2910.
[8]	Li H,Jiang L,Youn J H,et al.A comprehensive genetic study reveals a crucial role of CYP90D2/D2 in regulating plant architecture in rice (Oryza sativa).New Phytol, 2013,200(4):1076-1088.
[9]	Arite T,Iwata H,Ohshima K,et al.DWARF10,an RMS1/MAX4/DAD1 ortholog,controls lateral bud outgrowth in rice.Plant J, 2007,51(6):1019-1029.
[10]	Arite T,Umehara M,Ishikawa S,et al.d14, a strigolactone-insensitive mutant of rice,shows an accelerated outgrowth of tillers.Plant Cell Physiol, 2009,50(8):1416-1424.
[11]	Zhou F,Lin Q,Zhu L,et al.D14-SCFD3-dependent degradation of D53 regulates strigolactone signalling.Nature, 2013,504(7480):406-410.
[12]	Sasaki A,Itoh H,Gomi K,et al.Accumulation of phosphorylated repressor for gibberellin signaling in an F-box mutant.Science, 2003,299(5614):1896-1898.
[13]	Hirano K,Asano K,Tsuji H,et al.Characterization of the molecular mechanism underlying gibberellin perception complex formation in rice.Plant Cell, 2010,22(8):2680-2696.
[14]	黄耀祥. 半矮秆、早长根深、超高产、特优质中国超级稻生态育种工程. 广东农业科学, 2001,3(3): 2-6.
	Huang Y X.Ecological breeding engineering for maximum yield,super high quality of China super-rice with semi-dwarf and early growth and deep root.Guangdong Agric Sci, 2001, 3(3):2-6. (in Chinese with English abstract)
[15]	陈韦韦. 水稻小粒矮秆突变体sgd1(t)的表型分析及基因精细定位. 南京:南京农业大学,2011.
	Chen W W.Phenotype analysis and gene fine mapping of sgd1(t),a small-grain dwarfness mutant in rice (Oryza Sativa L.). Nanjing: Nanjing Agricultural University,2011. (in Chinese with English abstract)
[16]	何祖华,Etohkossi,石春海,等. 水稻株高基因对GA3敏感性及与酶的关系. 中国水稻科学. 1993,7(3): 143-147.
	He Z H,Etohkossi,Shi C H,et al. Sensitivity of plant height genes to GA3 and their relationship with enzymes in rice.Chin J Rice Sci,1993,7(3):189-196. (in Chinese with English abstract)
[17]	Lichtenthaler H K.Chlorophylls and carotenoids:Pigments of photosynthetic biomembranes. Methods Enzymol,Academic Press,1987,350-382.
[18]	Mitsunaga S, Tashiro T, Yamaguchi J.Identification and characterization of gibberellin-insensitive mutants selected from among dwarf mutants of rice.Theor Appl Genet, 1994,87(6):705-712.
[19]	Hedden P, Phillips A L.Gibberellin metabolism:New insights revealed by the genes.Trends Plant Sci, 2000,5(12):523-530.
[20]	Gomi K,Matsuoka M.Gibberellin signalling pathway.Curr Opin Plant Bio, 2003,6(5):489-493.
[21]	Li J,Jiang J,Qian Q,et al.Mutation of rice BC12/GDD1,which encodes a kinesin-like protein that binds to a GA biosynthesis gene promoter,leads to dwarfism with impaired cell elongation.Plant Cell, 2011,23(2): 628-640.
[22]	Zhang M,Zhang B,Qian Q,et al.Brittle Culm 12,a dual-targeting kinesin-4 protein,controls cell-cycle progression and wall properties in rice.Plant J, 2010,63(2):312-328.
[23]	马良勇,包劲松,李西明,等. 水稻矮生基因的克隆和功能研究进展. 中国水稻科学, 2009,23(1): 1-11.
	Ma L Y,Bao J S,Li X M,et al.Progress on cloning and functional analysis of dwarfism related genes in rice.Chin J Rice Sci, 2009,23(1):1-11. (in Chinese with English abstract)
[24]	Mazumdar M,Misteli T.Chromokinesins:Multitalented players in mitosis.Trends Cell Biol, 2005,15(7): 349-355.
[25]	Bannigan A,Scheible W,Lukowitz W,et al.A conserved role for kinesin-5 in plant mitosis.J Cell Sci, 2007, 120:2819-2827.