Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (5): 777-785.doi: 10.3864/j.issn.0578-1752.2019.05.001

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Cloning and Functional Analysis of a Root Development Related Gene OsKSR7 in Rice (Oryza sativa L.)

ZHOU JiaQin1,ZHU JunZhao2,YANG SiXue2,ZHU ZhouJie2,YAO Jie2,ZHENG WenJuan2,ZHU ShiHua2,DING WoNa2()   

  1. 1 School of Marine Science, Ningbo University, Ningbo 315211, Zhejiang
    2 College of Science and Technology, Ningbo University, Ningbo 315212, Zhejiang
  • Received:2018-07-18 Accepted:2018-08-26 Online:2019-03-01 Published:2019-03-12
  • Contact: WoNa DING E-mail:dwn@zju.edu.cn

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Abstract:

【Objective】The root system of rice is an important agronomic trait closely related to shoot growth and yield. Identifying new root development-related genes in rice will help further clarification of the underlying molecular mechanisms.【Method】In the present study, a mutant with significantly shorter roots was isolated from an EMS (ethyl methane sulfonate)-generated mutant library of rice and designated as Osksr7 (Oryza sativa kasalath short root 7 ). By using solution culture and field planting, analysis of young seedling phenotype and main agronomic traits of mature plants was conducted. The F2 populations from crossing of Osksr7 with indica Kasalath and japonica Nipponbare were used for genetic analysis and map-based cloning, respectively. Candidate genes were examined by DNA sequencing. Complementation analysis of the Osksr7 mutant with the protein-coding region of OsKSR7 driven by the 35S promoter was performed using Agrobacterium tumefaciens -mediated transformation. 【Result】 At the seedling stage, the elongation of primary roots, adventitious roots, lateral roots and root hairs in Osksr7 was severely impaired. The length of primary roots, adventitious roots and lateral roots of Osksr7 was only 33%, 38.9% and 35.3% of those of the wild type, respectively. Nevertheless, the number of adventitious roots of Osksr7 was significantly increased when compared with the wild type. At the maturation stage, the agronomic traits of Osksr7 were also significantly compromized, including the shoot height, panicle number, clum thickness, seed setting rate, 1000-grain weight and length and width of flag leaves. Among them, the panicle number and seed setting rate of Osksr7 dramatically decreased to only 56.3% and 37.3% of those of the wild type, respectively. Genetic analysis showed that the growth of F1 plants from the crossing of Osksr7 with indica Kasalath was similar to the wild type and the segregation ratio of wild type and mutant phenotype plants in the corresponding F2 population fitted a ratio of 3:1, indicating that the mutant trait of Osksr7 was controlled by a single recessive nuclear gene. The OsKSR7 locus was further mapped between InDel markers IND1 and IND2 on chromosome 11 with a physical distance of 143 kb, where there were 25 predicted genes with annotation. Sequencing analysis found a point mutation (T 73 to A) in the first exon of the gene LOC_Os11g24560 within this region in Osksr7 , resulting in an amino acid substitution (Trp 25 to Arg). The gene encodes a putative rice homolog of the SEC23 subunit of the coat protein complex II (COPII) involved in ER-to-Golgi transport. RT-PCR analysis revealed no significant difference in the expression level of LOC_Os11g24560 between the wild type and Osksr7 . Transformation of Osksr7 with the coding sequence of LOC_Os11g24560 driven by the 35S promoter could successfully restore its growth defects, confirming that the mutation in LOC_Os11g24560 was responsible for the mutant phenotype of Osksr7 .【Conclusion】 Osksr7 is a rice short root mutant, and yield-related agronomic traits are significantly suppressed in Osksr7 . OsKSR7 is confirmed to be within the locus LOC_Os11g24560 , which encodes the SEC23 subunit of the coat protein complex II (COPII). OsKSR7 is not allelic to any previously reported rice root gene and is a newly identified regulator of root development in rice.

Key words: Oryza sativa L., short root mutant, genetic analysis, map-based cloning, functional complementation

Table 1

The characteristics of 7-day-old seedlings of wild type (WT) and Osk sr 7 mutant (means±SD)"

性状Trait WT Osksr7
主根长Primary root length (cm) 9.4±0.9 3.1±0.3**
苗高Plant height (cm) 11.2±0.8 8.5±0.5*
不定根长Adventitious root length (cm)a 3.6±0.7 1.4±0.2**
不定根数Adventitious root number 3.5±0.6 5.2±0.8*
侧根长Lateral root length (cm)b 1.7±0.3 0.6±0.1**

Fig. 1

Phenotypic characterization of 7-day-old seedlings of wild type (WT) and Osksr7 mutant A: Seedlings of the WT and Osksr7 mutant, bar=2 cm; B: The root of WT and Osksr7 mutant, bar=2 cm; C: The primary root of WT and Osksr7 mutant under stereoscope, bar=1 mm"

Fig. 2

Growth curve of primary root (A) and adventitious root (B) of Oskrs7 and wide type (WT)"

Table 2

The agronomic traits comparison between the wild type (WT) and Osk sr 7 mutant (means±SD)"

农艺性状Agronomic trait WT Osksr7
株高Plant height (cm) 167.3±3.3 138.7±4.0*
穗数Panicle number 20.8±1.8 11.7±2.5**
剑叶长Flag leaf length (cm) 63.8±2.9 52.2.2±3.0*
剑叶宽Flag leaf width (cm) 1.9±0.1 1.5±0.1*
穗长Panicle length (cm) 33.1±0.5 32.4±0.6
茎杆周长Stem circumference (cm) 2.4±0.1 1.7±0.1*
千粒重1000-grain weight (g) 17.5±1.3 13.7±1.5*
结实率Seed setting rate (%) 95.1±1.2 35.5±2.1**

Table 3

Genetic analysis of short root mutant Osksr7 "

杂交组合
Cross		 F1表型
F1 phenotype		 F2群体 F2 population χ2(3﹕1)
正常型株数Normal plants 短根株数Short root plants 总株数Total plants
Osksr7 /Kasalath 正常型 Normal type 257 81 338 0.19

Table 4

Molecular markers and primers used to map Osksr7 "

标记
Marker		 引物序列
Primer sequence (5′-3′)		 产物大小
Product size (bp)
RM21 F：ACAGTATTCCGTAGGCACGG
R：GCTCCATGAGGGTGGTAGAG		 157
RM4862 F：CAACTTTCTGGCATAAACTA
R：TGGTGAAAGATATTTCAGAC		 159
InD1 F：AGAACATAAGAGTAAAAACCA
R：AGTAGGTTTCACCATTTTGGA		 99
InD2 F：AGTGGCTACATTTAGTTTGCT
R：ACTGGGGATTGTATGGAGCAG		 123

Fig. 3

Map-based cloning of OsKSR7 gene A: Fine mapping of OsKSR7 on rice chromosome 11; B: Gene structure of OsKSR7 , Black boxes represent exons, white boxes indicate the untranslated regions"

Fig. 4

Complementation of the Osksr7 mutant A: The primary root the WT, Osksr7 and two lines of over-expression transgenic plants (OV1 and OV2) under stereoscope, bar=1 mm; B: Seedlings of the WT, Osksr7 and two lines of over-expression transgenic plants (OV1 and OV2) in the Osksr7 mutant background, bar=2 cm; C: RT-PCR analysis of OsKSR7 expression"

[1] GIEHL R F, GRUBER B D, VON W N .It's time to make changes: modulation of root system architecture by nutrient signals. Journal of Experimental Botany, 2014,65(3):769-778.
doi: 10.1093/jxb/ert421 pmid: 24353245
[2] MEISTER R, RAJANI M S, RUZICKA D, SCHACHTMAN D P .Challenges of modifying root traits in crops for agriculture. Trends in Plant Science, 2014,19(12):779-788.
doi: 10.1016/j.tplants.2014.08.005 pmid: 25239776
[3] VILLORDON A Q, GINZBERG I, FIRON N .Root architecture and root and tuber crop productivity. Trends in Plant Science, 2014,19(7):419-425.
doi: 10.1016/j.tplants.2014.02.002 pmid: 24630073
[4] 石庆华, 黄英金, 李木英, 徐益群, 谭雪明, 张佩莲 .水稻根系性状与地上部的相关及根系性状的遗传研究. 中国农业科学, 1997,30(4):62-68.
SHI Q H, HUANG Y J, LI M Y, XU Y Q, TAN X M, ZHAGN P L . Studies on the heredity of root characteristics and correlation between the characteristics of roots and upperground parts in rice. Scientia Agricultura Sinica , 1997,30(4):62-68. (in Chinese)
[5] 洛育 .黑龙江省水稻根系性状与地上部性状的关系. 中国农学通报, 2010,26(14):165-168.
LUO Y . Dependence relation of aerial part traits and root traits of rice in Heilongjiang province. Chinese Agricultural Science Bulletin , 2010,26(14):165-168. (in Chinese)
[6] ROGERS E D, BENFEY P N .Regulation of plant root system architecture: implications for crop advancement. Current Opinion in Biotechnology, 2015,32(32C):93-98.
doi: 10.1016/j.copbio.2014.11.015 pmid: 25448235
[7] REBOUILLAT J, DIEVART A, VERDEIL J L, ESCOUTE J, GIESE G, BREITLER J C, GANTET P, ESPEOUT S, GUIDERDONI E PÉRIN C,. Molecular genetics of rice root development. Rice, 2009,2(1):15-34.
doi: 10.1007/s12284-008-9016-5
[8] E Z-G GE L,WANG L,. Molecular mechanism of adventitious root formation in rice. Plant Growth Regulation, 2012,68(3):325-331.
doi: 10.1007/s10725-012-9721-3
[9] YU P, GUTJAHR C, LI C, HOCHHOLDINGER F .Genetic control of lateral root formation in cereals. Trends in Plant Science, 2016,21(1):951-961.
doi: 10.1016/j.tplants.2016.07.011 pmid: 27524642
[10] CHEN Z C, YAMAJI N, KASHINO-FUJII M, MA J F .A cation-chloride cotransporter gene is required for cell elongation and osmoregulation in rice. Plant Physiology, 2016,171(1):494-507.
doi: 10.1104/pp.16.00017 pmid: 26983995
[11] DING W, LIN L, ZHANG B, XIANG X, WU J, PAN Z, ZHU S .OsKASI, a β-ketoacyl-[acyl carrier protein] synthase I, is involved in root development in rice (Oryza sativa L.). Planta , 2015,242(1):203-213.
doi: 10.1007/s00425-015-2296-2 pmid: 25893869
[12] JIA L, WU Z, HAO X, CARRIE C, ZHENG L, WHELAN J, WU Y, WANG S, WU P, MAO C .Identification of a novel mitochondrial protein, short postembryonic roots 1 (SPR1), involved in root development and iron homeostasis in Oryza sativa . New Phytologist , 2011,189(3):843-855.
doi: 10.1111/j.1469-8137.2010.03513.x pmid: 21039568
[13] JIA L, ZHANG B, MAO C, LI J, WU Y, WU P, WU Z .OsCYT-INV1 for alkaline/neutral invertase is involved in root cell development and reproductivity in rice (Oryza sativa L.). Planta , 2008,228(1):51-59.
doi: 10.1007/s00425-008-0718-0 pmid: 18317796
[14] ZHAO Y, HU Y F, DAI M Q, HUANG L M, ZHOU D X .The WUSCHEL-related homeobox gene WOX11 is required to activate shoot-borne crown root development in rice. The Plant Cell, 2009,21(3):736-748.
doi: 10.1105/tpc.108.061655 pmid: 19258439
[15] DING W, TONG H, ZHENG W, YE J, PAN Z, ZHANG B, ZHU S .Isolation, characterization and transcriptome analysis of a cytokinin receptor mutant osckt1 in rice. Frontiers in Plant Science , 2017,8:88.
doi: 10.3389/fpls.2017.00088 pmid: 5281565
[16] 丁沃娜, 童艳丽, 吴晶, 朱世华 .一个水稻短根毛突变体的鉴定和基因定位. 中国农业科学, 2011,44(21):4333-4339.
doi: 10.3864/j.issn.0578-1752.2011.21.001
DING W N, TONG Y L, WU J, ZHU S H . Identification and gene mapping of a novel short root hair mutant in rice. Scientia Agricultura Sinica , 2011,44(21):4333-4339. (in Chinese)
doi: 10.3864/j.issn.0578-1752.2011.21.001
[17] CHEN S, JIN W, WANG M, ZHANG F, ZHOU J, JIA Q, WU Y, LIU F, WU P .Distribution and characterization of over 1000 T-DNA tags in rice genome. The Plant Journal, 2003,36(1):105-113.
doi: 10.1046/j.1365-313X.2003.01860.x pmid: 12974815
[18] MICHELMORE R W, PAPAN I, KESSELI R V .Identification of markers linked to disease-resistance genes by bulked segregant analysis: A rapid method to detect markers in specific genomic regions by using segregating populations. Proceedings of the National Academy of Sciences of the USA, 1991,88(21):9828-9832.
doi: 10.1073/pnas.88.21.9828
[19] QIN C, CHENG L, ZHANG H, HE M, SHEN J, ZHANG Y, WU P .OsGatB, the subunit of tRNA-dependent amidotransferase, is required for primary root development in rice. Frontiers in Plant Science, 2016,7:599.
doi: 10.3389/fpls.2016.00599
[20] VENDITTI R, WILSON C DE MATTEIS M A,. Exiting the ER: What we know and what we don’t. Trends in Cell Biology, 2014,24(1):9-18.
doi: 10.1016/j.tcb.2013.08.005 pmid: 24076263
[21] MILLER E A, BARLOWE C .Regulation of coat assembly-sorting things out at the ER. Current Opinion in Cell Biology, 2010,22(4):447-453.
doi: 10.1016/j.ceb.2010.04.003 pmid: 20439155
[22] BI X, CORPINA R A, GOLDBERG J .Structure of the Sec23/24-Sar1 pre-budding complex of the COPII vesicle coat. Nature, 2002,419(6904):271-277.
doi: 10.1038/nature01040 pmid: 12239560
[23] ROBINSON D G, HERRANZ M C, BUBECK J, PEPPERKOK R, RITZENTHALER C .Membrane dynamics in the early secretory pathway. Critical Reviews in Plant Sciences, 2007,26(4):199-225.
doi: 10.1080/07352680701495820
[24] SCHWARZ K, IOLASCON A, VERISSIMO F, TREDE N S, HORSLEY W, CHEN W, PAW B H, HOPFNER K P, HOLZMANN K, RUSSO R, ESPOSITO M R, SPANO D, DE FALCO L, HEINRICH K, JOGGERST B, ROJEWSKI M T, PERROTTA S, DENECKE J, PANNICKE U, DELAUNAY J, PEPPERKOK R, HEIMPEL H .Mutations affecting the secretory COPII coat component SEC23B cause congenital dyserythropoietic anemia type II. Nature Genetics, 2009,41(8):936-940.
doi: 10.1038/ng.405 pmid: 19561605
[25] ABOULELA M, NAKAGAWA T, OHSHIMA A, NISHIMURA K, TANAKA Y .The Arabidopsis COPII components, AtSEC23A and AtSEC23D, are essential for pollen wall development and exine patterning. Journal of Experimental Botany, 2018,69(7):1615-1633.
doi: 10.1093/jxb/ery015 pmid: 29390074
[26] FROMME J C, ORCI L, SCHEKMAN R .Coordination of COPII vesicle trafficking by Sec23. Trends in Cell Biology, 2008,18(7):330-336.
doi: 10.1016/j.tcb.2008.04.006 pmid: 18534853
[27] KUEHN M J, HERRMANN J M, SCHEKMAN R .COPII-cargo interactions direct protein sorting into ER-derived transport vesicles. Nature, 1998,391(6663):187-190.
doi: 10.1038/34438 pmid: 9428766
[28] MANCIAS J D, GOLDBERG J .The transport signal on Sec22 for packaging into COPII-coated vesicles is a conformational epitope. Molecular Cell, 2007,26(3):403-414.
doi: 10.1016/j.molcel.2007.03.017 pmid: 17499046
[29] BOYADJIEV S A, FROMME J C, BEN J, CHONG S S, NAUTA C, HUR D J, ZHANG G, HAMAMOTO S, SCHEKMAN R, RAVAZZOLA M, ORCI L, EYAID W .Cranio-lenticulosutural dysplasia is caused by a SEC23A mutation leading to abnormal endoplasmic-reticulum-to-Golgi trafficking. Nature Genetics, 2006,38(10):1192-1197.
doi: 10.1088/0031-9155/53/6/010 pmid: 16980979
[30] FROMME J C, RAVAZZOLA M, HAMAMOTO S, AL-BALWI M, EYAID W, BOYADJIEV S A, COSSON P, SCHEKMAN R, ORCI L .The genetic basis of a craniofacial disease provides insight into COPII coat assembly. Developmental Cell, 2007,13(5):623-634.
doi: 10.1016/j.devcel.2007.10.005 pmid: 2262049
[31] LANG M R, LAPIERRE L A, FROTSCHER M, GOLDENRING J R, KNAPIK E W .Secretory COPII coat component Sec23a is essential for craniofacial chondrocyte maturation. Nature Genetics, 2006,38(10):1198-1203
doi: 10.1038/ng1880 pmid: 16980978
[32] ZENG Y, CHUNG K P, LI B, LAI C M, LAM S K, WANG X, CUI Y, GAO C, LUO M, WONG K B, SCHEKMAN R, JIANG L .Unique COPII component AtSar1a/AtSec23a pair is required for the distinct function of protein ER export in Arabidopsis thaliana . Proceedings of the National Academy of Sciences of the USA , 2015,112(46):14360-14365.
doi: 10.1073/pnas.1519333112 pmid: 26578783
[33] TANAKA Y, NISHIMURA K, KAWAMUKAI M, OSHIMA A, NAKAGAWA T .Redundant function of two Arabidopsis COPII components, AtSec24B and AtSec24C, is essential for male and female gametogenesis. Planta , 2013,238(3):561-575.
doi: 10.1007/s00425-013-1913-1 pmid: 23779001
[34] CONGER R, CHEN Y, FORNACIARI S, FASO C, HELD MA, RENNA L, BRANDIZZI F .Evidence for the involvement of theArabidopsis SEC24A in male transmission. Journal of Experimental Botany , 2011,62(14):4917-4926.
doi: 10.1093/jxb/err174 pmid: 21705385
[35] NAKANO R T, MATSUSHIMA R, UEDA H, TAMURA K, SHIMADA T, LI L, HAYASHI Y, KONDO M, NISHIMURA M, HARA-NISHIMURA I .GNOMLIKE1/ERMO1 and SEC24a/ ERMO2 are required for maintenance of endoplasmic reticulum morphology in Arabidopsis thaliana . The Plant Cell , 2009,21(11):3672-3685.
doi: 10.1105/tpc.109.068270 pmid: 19933201
[36] QU X, CHATTY P R, ROEDER A H .Endomembrane trafficking protein SEC24A regulates cell size patterning in Arabidopsis . Plant Physiology , 2014,166(4):1877-1890.
doi: 10.1104/pp.114.246033 pmid: 25315606
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