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"

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