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作物学报 ›› 2023, Vol. 49 ›› Issue (3): 662-671.doi: 10.3724/SP.J.1006.2023.22012

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

水稻早衰基因ESL8的遗传与定位

朱晓彤1,2(), 叶亚峰2, 郭均瑶2, 杨惠杰2, 王紫瑶1, 詹玥2, 吴跃进2, 陶亮之2, 马伯军1, 陈析丰1,*(), 刘斌美2,*()   

  1. 1浙江师范大学化学与生命科学学院, 浙江金华 321004
    2中国科学院合肥物质科学研究院, 安徽合肥 230031
  • 收稿日期:2022-03-03 接受日期:2022-07-21 出版日期:2023-03-12 网络出版日期:2022-08-19
  • 通讯作者: 陈析丰,刘斌美
  • 作者简介:E-mail: 1634453295@qq.com
  • 基金资助:
    安徽省自然科学基金项目(2108085MC99);合肥市“借转补”科技专项(J2020G45);安徽省科技重大专项(202003c08020006);浙江省自然科学基金重大项目(LD19C130001)

Heredity and fine mapping of an early-senescence leaf gene ESL8 in rice

ZHU Xiao-Tong1,2(), YE Ya-Feng2, GUO Jun-Yao2, YANG Hui-Jie2, WANG Zi-Yao1, ZHAN Yue2, WU Yue-Jin2, TAO Liang-Zhi2, MA Bo-Jun1, CHEN Xi-Feng1,*(), LIU Bin-Mei2,*()   

  1. 1College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321004, Zhejiang, China
    2Hefei Institutes of Physical Science, Chinese Academy of Science, Hefei 230031, Anhui, China
  • Received:2022-03-03 Accepted:2022-07-21 Published:2023-03-12 Published online:2022-08-19
  • Contact: CHEN Xi-Feng,LIU Bin-Mei
  • Supported by:
    Natural Science Foundation of Anhui Province(2108085MC99);Hefei Science and Technology Project(J2020G45);Anhui Science Technology Major Project(202003c08020006);Natural Science Foundation of Zhejiang Province(LD19C130001)

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摘要：

早衰突变体是研究植物细胞凋亡分子机制的重要遗传材料。从水稻“科辐粳7号”诱变库中, 筛选到一个早衰突变体esl8 (early senescence leaf 8)。与野生型相比, 该突变体叶片在抽穗期表现出严重的早衰, 其株高、分蘖数、穂长、每穗粒数以及结实率, 在成熟期均极显著降低, 但千粒重无显著变化; 该突变体叶片的叶绿素含量显著下降, 组织化学染色进一步检测到细胞死亡、活性氧与丙二醛过量积累。遗传分析发现, 该突变体的早衰表型受单隐性核基因控制。采用图位克隆技术将esl8基因精细定位在12号染色体的FM12-14和FM12-15分子标记之间, 物理距离为359 kb。候选基因预测与PCR测序结果表明, esl8基因是OsSL/ELL1基因的一个新等位变异, 突变位点发生在该基因的保守区域。以上结果为进一步研究该基因编码蛋白的功能及其早衰分子机制提供理论依据。

关键词: 早衰, 水稻, esl8, 细胞凋亡, 定位克隆

Abstract:

Early-senescence mutants are genetic materials important for the researches on the molecular mechanism of cell apoptosis in plants. An early-senescence mutant named esl8 (early senescence leaf 8) has been screened from the mutagenesis library of rice variety Kefujing 7. Compared with the wild-type control, the leaves of the esl8 mutant displays a severe of early-senescence phenotype at heading stage in rice, and its agronomic traits, including plant height, tiller number, grain length, grains per panicle, and seed setting rate, were obviously impaired except for the 1000-grain weight. In leaves of the esl8 mutant, the chlorophyll content was abnormally decreased, and the programmed cell death accompanied by excessive accumulation of the reactive oxygen species and the malondialdehyde were detected by histochemical staining. Genetic analysis indicated that the early-senescence phenotype of the esl8 mutant was controlled by a recessive nuclear gene. Based on the strategy of map-based cloning, the esl8 gene was finely mapped into a 359-kb region flanking by two molecular markers (FM12-14 and FM12-15) on chromosome 12. The prediction and verification of candidate genes by PCR sequencing confirmed that esl8 was a new variational allele of the OsSL/ELL1 gene. The sequence of mutation esl8 occurred in the conserved region of the corresponding wild-type gene. Our results provide a theoretical basis for further study on the biofunction and molecular mechanism of the protein encoding by ESL8 in early-senescence process.

Key words: early senescence, rice, esl8, cell apoptosis, mapping and cloning

表1

水稻ESl8基因精细定位分子标记的引物序列"

分子标记
Molecular marker		 正向序列
Forward sequence (5'-3')		 反向序列
Reverse sequence (5'-3')
LSR6 TCTTGCCTCGCTAGGGTTAG CCCACGTTTCTCTTGTCCTC
LSR14 TCCACCACTCTGACGTCTACTAACC CTGCGGGAAGTGTAGGAGAAGC
FM12-1 CCCAGTTTTGCTTAGCCTCTT TTTTGGCATACATCGATTGG
FM12-3 GCCATTTTTAGACGTGGCAAT TTTGCCACTTTTAGTTTTATCGTTT
FM12-7 GGCTGTTGACCATGCTGTAA TGAAGTCCTAGATGCACTTTTTCA
FM12-9 ACATCAGCAGCCATGGTTTT TTTCAACGGTAAAGTCATGTGG
FM12-11 ACATTATGGTTCACGCATGG CGGGACCGTAGGTAAAAAGG
FM12-14 GCCGATGAAACTCCGATAGC TTATCGTGTGAGGGGGAAAG
FM12-15 ACATCCGCGCTTTTCTTTT TAACAAGGGGCAAAGTGGTC

表2

水稻ESL8基因PCR扩增的引物序列"

引物名称
Primer name		 正向序列
Forward sequence (5'-3')		 反向序列
Reverse sequence (5'-3')
ESL8-1 TCTTGCCTCGCTAGGGTTAG CCCACGTTTCTCTTGTCCTC
ESL8-2 TCCACCACTCTGACGTCTACTAACC CTGCGGGAAGTGTAGGAGAAGC
ESL8-3 CCCAGTTTTGCTTAGCCTCTT TTTTGGCATACATCGATTGG
ESL8-4 GCCATTTTTAGACGTGGCAAT TTTGCCACTTTTAGTTTTATCGTTT

图1

水稻esl8突变体的早衰表型及理化特征 A: 苗期的植株表型; B: 抽穗期的植株表型; C: 抽穗期的单个分蘖表型; D: 台盼蓝染色; E: DAB染色; F: NBT染色; G: SOD酶活检测; H: MDA含量测定; I: 叶绿素含量测定。WT: 野生型对照科辐粳7号; esl8: 早衰突变体; 标尺为10 cm; **表示突变体与野生型对照之间存在显著性差异(P < 0.01)。"

表3

水稻突变体esl8与野生型对照的农艺性状统计"

材料名称
Material name		 株高
Plant height
(cm)		 穂长
Panicle length (cm)		 有效分蘖数
Effective
panicle number		 每穗粒数
Grain number
per panicle		 结实率
Seed-setting
rate (%)		 千粒重
1000-grain
weight (g)
WT 101.18±2.93 14.87±0.86 9.00±1.15 206.00±18.00 81.27±2.18 26.74±0.41
esl8 89.88±1.03** 13.69±0.56** 7.00±0.58** 129.00±10.00** 58.45±2.33** 25.98±0.33

图2

水稻ESL8基因定位与测序分析 A: ESL8基因初定位的物理图谱; B: ESl8基因精细定位的遗传图谱; n为定位分析所用到F2突变单株数, 标记下的数字为该标记检测到的交换事件数; C: ESL8基因突变位点的测序结果, WT为野生型对照科辐粳7号, ESl8为早衰突变体, 红框标出了TGCCCC碱基缺失与GCG碱基插入的位置。"

图3

ESL8的蛋白序列比对与系统进化树分析 A: esl8及其野生型基因的编码蛋白序列比对; B: ESL8及其拟南芥与水稻同源蛋白的系统发育树; C: ESL8及其同源蛋白的保守序列比对。"

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