中國農業科學 ?? 2019, Vol. 52 ?? Issue (5): 777-785.doi: 10.3864/j.issn.0578-1752.2019.05.001

? 作物遺傳育種·種質資源·分子遺傳學 ? 上一篇    下一篇

水稻根系發育基因OsKSR7 的克隆與功能分析

周佳琴1,朱俊兆2,楊思學2,諸周潔2,姚婕2,鄭文娟2,朱世華2,丁沃娜2()   

  1. 1 寧波大學海洋學院,浙江寧波 315211
    2 寧波大學科學技術學院,浙江寧波 315212
  • 收稿日期:2018-07-18 接受日期:2018-08-26 出版日期:2019-03-01 發布日期:2019-03-12
  • 通訊作者: 丁沃娜 E-mail:[email protected]
  • 作者簡介:周佳琴,E-mail: [email protected]。|朱俊兆,E-mail: [email protected]。周佳琴和朱俊兆為同等貢獻作者。
  • 基金資助:
    國家自然科學基金(31300246);浙江省自然科學基金(LY17C020002);寧波市自然科學基金(2017A610291);寧波市自然科學基金(2017A610292)

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:[email protected]

摘要:

【目的】 水稻根系是與地上部性狀和產量密切相關的重要農藝性狀。通過鑒定新的水稻根系發育相關基因,為深入解析水稻根系發育遺傳機理奠定基礎。【方法】 從甲基磺酸乙酯(ethyl methane sulfonate,EMS)誘變的水稻Kasalath突變體庫中篩選到1個根系發育缺陷的突變體Osksr7Oryza sativa kasalath short root 7 )。通過溶液培養和田間種植,對該突變體進行苗期表型鑒定及成熟期主要農藝性狀考察。將Osksr7 分別與野生型秈稻Kasalath和粳稻Nipponbare雜交,F2群體進行遺傳分析和突變基因的圖位克隆,對預測的候選基因進行測序驗證。構建由35S啟動子驅動OsKSR7 的回復載體,通過農桿菌介導轉入突變體成熟胚誘導的愈傷組織進行轉基因互補驗證。【結果】 與野生型相比,Osksr7 幼苗期的主根、不定根、側根和根毛的伸長都受到抑制,主根、不定根和側根的長度分別只有野生型的33%、38.9%和35.3%,但不定根數有顯著增加。農藝性狀調查發現,Osksr7 的株高、穗數、莖稈粗細、結實率、千粒重和劍葉長寬等性狀都受到顯著影響,其中,穗數和結實率的差異極顯著,分別只有野生型的56.3%和37.3%。遺傳分析表明,突變體Osksr7 和秈稻Kasalath雜交的F1表型正常,F2群體中正常植株與短根突變植株的分離比符合3﹕1,表明突變體Osksr7 的突變性狀受1對隱性核基因控制;利用SSR和InDel分子標記將突變基因定位在水稻第11染色體上IND1與IND2之間,物理距離約為143 kb的區間。在該區間有25個預測注釋基因,候選基因測序比對發現,突變體Osksr7 中的一個編碼轉運蛋白的基因LOC_Os11g24560 第一個外顯子上ATG后73 bp處的T突變為A,導致編碼的第25位氨基酸由色氨酸突變為精氨酸。生物信息學分析表明LOC_Os11g24560 是介導蛋白質從內質網(ER)運向高爾基體(Golgi)的COPII有被小泡的SEC23亞基在水稻中的同源基因。RT-PCR表明LOC_Os11g24560 的表達水平在野生型和Osksr7 突變體中無顯著差異,35S啟動子驅動的LOC_Os11g24560 的回復載體能夠使Osksr7 突變體的表型回復成野生型,證實Osksr7 的表型是由LOC_Os11g24560 突變引起。【結論】 Osksr7 是一個水稻短根突變體,其產量相關的幾個重要農藝性狀顯著受抑制,突變基因為LOC_Os11g24560 ,編碼COPII有被小泡的SEC23亞基,與已報道的水稻根系基因都不等位,是一個新的水稻根系發育調控基因。

關鍵詞: 水稻, 短根突變體, 遺傳分析, 圖位克隆, 功能互補

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

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