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作物学报 ›› 2022, Vol. 48 ›› Issue (7): 1832-1842.doi: 10.3724/SP.J.1006.2022.12028

• 研究简报 • 上一篇    下一篇

水稻叶片早衰突变体ospls7的生理特性及其基因定位

黄福灯1(), 黄妍2, 金泽艳2, 贺焕焕2, 李春寿1,*(), 程方民2, 潘刚2   

  1. 1浙江省农业科学院作物与核技术利用研究所, 浙江杭州 310021
    2浙江大学农业与生物技术学院, 浙江杭州 310058
  • 收稿日期:2021-04-20 接受日期:2021-10-20 出版日期:2022-07-12 网络出版日期:2021-11-15
  • 通讯作者: 李春寿
  • 作者简介:黄福灯, E-mail: pahfd@126.com第一联系人:

    ** 同等贡献

  • 基金资助:
    国家自然科学基金项目(31771688);国家自然科学基金项目(31971819);浙江省十四五水稻育种重大专项(2021C02063-1)

Physiological characters and gene mapping of a precocious leaf senescence mutant ospls7 in rice (Orzo sativa L.)

HUANG Fu-Deng1(), HUANG Yan2, JIN Ze-Yan2, HE Huan-Huan2, LI Chun-Shou1,*(), CHENG Fang-Min2, PAN Gang2   

  1. 1Institute of Crop and Nuclear Technology Utilization, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, Zhejiang, China
    2College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, Zhejiang, China
  • Received:2021-04-20 Accepted:2021-10-20 Published:2022-07-12 Published online:2021-11-15
  • Contact: LI Chun-Shou
  • About author:First author contact:

    ** Contributed equally to this work

  • Supported by:
    National Natural Science Foundation of China(31771688);National Natural Science Foundation of China(31971819);Major Projects of Rice Breeding in the 14th Five Year Plan of Zhejiang Province(2021C02063-1)

摘要:

衰老是叶片发育的最后阶段, 但叶片, 尤其是功能叶, 早衰将影响作物的产量与品质, 因此, 研究叶片早衰的分子生理机制对培育耐早衰优良品种具有重要意义。通过60Co-γ辐射诱变旱稻Monolaya获得一个稳定遗传的叶片早衰突变体ospls7, 本文对其形态、叶片衰老生理特征、茎节细胞特性以及衰老性状遗传与基因定位等方面进行了研究。大田条件下, 突变体ospls7叶片早衰性状始于三至四叶期幼苗, 主要表现为: 叶尖及中上部叶边缘黄色褐化并最终枯萎, 成熟期穗长和各茎节长度均极显著短于野生型对照, 最终导致植株矮化。究其原因, 可能是由于突变体茎节细胞变短。叶片衰老生理结果表明, 与野生型对照相比, 孕穗期突变体ospls7倒二叶和倒三叶的叶绿素总含量、净光合速率、可溶性蛋白、过氧化氢酶活性均极显著降低, 致使其叶片中H2O2大量累积并引起丙二醛含量急剧增加。同时, 孕穗期突变体ospls7剑叶、倒二叶和倒三叶的内源ABA含量均极显著高于野生型对照, qRT-PCR结果证实ABA大量累积的原因在于ABA合成基因OsNCED3OsAAO3显著上调, 而其代谢基因OsABA8ox2OsABA8ox3则显著下调。遗传分析结果表明, 突变体ospls7的叶片早衰性状受单隐性核基因调控, 进一步利用图位克隆技术将该基因定位于10号染色体的InDel标记ID74-33/34和SSR标记RM25040之间, 物理距离为207 kb, 这些结果为最终克隆OsPLS7基因并研究其功能奠定了基础。

关键词: 水稻, ospls7, 叶片早衰, 生理分析, 基因定位

Abstract:

Leaf senescence is the final stage of leaf development, however, premature aging of leaves, especially functional leaves, leads to reduction of yield and quality. Thus, it is very important for developing novel crop germplasms with delayed leaf-senescence characteristics through investigating the molecular and physiological mechanism of leaf senescence. In this study, a stable precocious leaf senescence mutant ospls7 was obtained from 60Co γ-radiated upland rice cultivar Monolaya, and its morphology, physiological characteristics of leaf senescence, cytological observation of internodes, genetic analysis and gene mapping were investigated. Under field conditions, leaf senescence was noticed as early as the 3-4-leaf seedling stage, featuring yellowing and browning at the edge of tip and the upper middle parts of old leaves and finally wilting. Due to shorter length of the parenchyma cells, panicle length and all internodes length of ospls7 were significantly shorter compared with wild type plants at the mature stage, resulting in dwarf phenotype in ospls7. Physiological analysis of leaf senescence indicated that compared to the wild type plants, the total chlorophyll contents, net photosynthetic rate, soluble protein content, and catalase (CAT) activity of the second and third leaves from top in ospls7 were significantly declined at the booting stage, which in turn resulting in the accumulation of H2O2 and a steady increase of malondialdehyde (MDA) contents in the mutant leaves. Moreover, due to significant up-regulation of ABA biosynthetic genes (OsNCED3 and OsAAO3) and significant down-regulation of the ABA catabolism genes (OsABA8ox2 and OsABA8ox3), the endogenous ABA levels in the leaves of ospls7 were significantly higher compared with the wild type at the booting stage. Genetic analysis and gene mapping showed that ospls7 was controlled by a single recessive nuclear gene, located in a region of 207 kb between SSR marker RM25040 and the InDel marker ID74-33/34 of chromosome 10. These results would further facilitate the cloning and functional analysis of OsPLS7 gene.

Key words: rice, ospls7, precocious leaf senescence, physiological analysis, gene mapping

附表1

用于ABA合成代谢基因qRT-PCR分析的引物序列"

基因
Gene
基因号
Locus ID
正向引物
Forward primer (5'-3')
反向引物
Reverse primer (5'-3')
OsZEP1 LOC_Os04g37619 GGTGCGATAACGTCGTTGATC GTATGGTCTATAAGTGGTAGC
OsNCED1 LOC_Os02g47510 ACCATGAAGTCCATGAGGCT TCTCGTAGTCTTGGTCTTGG
OsNCED2 LOC_Os12g24800 ATGGAAACGAGGATAGTGGT CTTATTGTTGTGCGAGAAGT
OsNCED3 LOC_Os03g44380 CTCCCAAACCATCCAAACCG TGAGCATATCCTGGCGTCGT
OsNCED4 LOC_Os07g05940 ATCTCCTTCTCCCTCCTCCCA TCGCACCCTGCTTGATCTTGC
OsNCED5 LOC_Os12g42280 TCCGAGCTCCTCGTCGTGAA AGGTGTTTTGGAATGAACCA
OsSDR LOC_Os03g59610 GACCTGACGAGACGATGTCC GCAACCTTGCTTTCCAACC
OsAAO1 LOC_Os07g18154 TTCGCCATTTGTTCGTAA CAGAGGAGGTTGCTCAAG
OsAAO2 LOC_Os07g18158 CCCTTGACGCCAACACTG CCGCTTTCGCCACTTATT
OsAAO3 LOC_Os07g07050 CGCCTGGTAAAGTGTCTA AATTGCTCCTTGAGTGGT
OsABA8ox1 LOC_Os02g47470 AAGCTGGCAAAACCAACATC CCGTGCTAATACGGAATCCA
OsABA8ox2 LOC_Os08g36860 CTACTGCTGATGGTGGCTGA CCCATGGCCTTTGCTTTAT
OsABA8ox3 LOC_Os09g28390 AGTACAGCCCATTCCCTGTG ACGCCTAATCAAACCATTGC

图1

突变体ospls7及其野生型的表型 A: 幼苗期; B: 孕穗期; C: 孕穗期叶片, 其中F代表剑叶, 2~4分别为倒二叶至倒四叶; D: 成熟期穗及茎节, P代表小穗, 1~6分别为倒一节至倒六节; E: 2020年成熟期各茎节长度; F~I: 野生型对照(F, H)和突变体(G, I)的倒二茎节(F, G)和叶片(H, I)的纵切面。A~E的标尺为20 cm, F~G的标尺为50 μm, H~I的标尺为20 μm。**表示在0.01水平上差异显著(t-test)。"

表1

突变体ospls7及其野生型的主要农艺性状"

性状
Trait
2019 2020
野生型WT 突变体ospls7 野生型WT 突变体ospls7
株高 Plant height (cm) 92.27±3.13 74.23±4.65** 89.63±1.67 74.08±2.92**
穗长 Panicle length (cm) 24.87±1.04 19.34±1.15* 23.81±0.95 18.97±1.13*
有效穗数 Effective panicle number 7.75±0.96 5.23±0.45* 9.67±0.58 8.52±0.58*
每穗粒数 Grain number per panicle 176.61±15.83 112.63±11.76** 176.68±9.07 100.24±10.51**
结实率 Seed-setting rate (%) 82.81±5.83 69.84±3.97** 81.76±5.76 72.14±6.93**
千粒重 1000-grain weight (g) 19.57±0.94 15.98±0.59** 18.94±0.53 15.88±0.71**
单株产量 Yield per plant (g) 21.91±2.13 7.09±1.04** 27.38±3.78 8.84±1.37**

图2

孕穗期突变体ospls7及其野生型叶片的光合特性 A~D: 突变体及其野生型叶片的叶绿素a (A)、叶绿素b (B)、总叶绿素含量(C)及叶绿素a/b比值(D); E~F: 突变体及其野生型叶片的净光合速率(E)及Fv/Fm值(F)。1: 剑叶; 2: 倒二叶; 3: 倒三叶。*表示在0.05水平上差异显著; **表示在0.01水平上差异显著(t-test)。"

图3

孕穗期突变体ospls7和野生型对照叶片中的O2?和H2O2含量及其CAT和SOD酶活分析 1: 剑叶; 2: 倒二叶; 3: 倒三叶。*表示在0.05水平上差异显著; **表示在0.01水平上差异显著(t-test)。"

图4

孕穗期突变体ospls7及其野生型叶片的MDA和可溶性蛋白含量 1: 剑叶; 2: 倒二叶; 3: 倒三叶。* 表示在0.05水平上差异显著; ** 表示在0.01水平上差异显著(t-test)。"

图5

外源ABA处理对突变体ospls7及其野生型(WT)幼苗的影响 A: 不同浓度ABA处理7 d后ospls7及野生型的表型; B~C: 处理7 d后ospls7及野生型的株高(B)和根长(C)。A的标尺为20 cm。* 表示在0.05水平上差异显著(t-test)。"

图6

孕穗期突变体ospls7及野生型对照叶片中的ABA含量及其合成代谢基因的qRT-PCR分析 1: 剑叶; 2: 倒二叶; 3: 倒三叶。* 表示在0.05水平上差异显著; ** 表示在0.01水平上差异显著(t-test)。"

附表2

用于OsPLS7基因定位的分子标记"

标记
Marker
正向引物
Forward primer (5'-3')
反向引物
Reverse primer (5'-3')
RM25000 CATTGAAGCAGGAGAAGGAGTTGC GATGCATCTGCTCCATCAATTCG
RM25011 AAGCTGCTGCTTCCACTTCACTTCG GTGGCCTCCTCGAGATCGAACG
RM25020 AATCCCTCTCGGCCCATCTCC CGAAGACGACGGCGATGACG
RM25030 GTGATGACGTGGACAAATCTCG GGGTAATCACTACTCACAGAGTTTGG
RM25034 TGTCATGTGGCAATATGAGAGC GACCTTTACCAAGCACATAGTCC
RM25038 CTTTAGAGGTTGCCGAACTGG GAGCGTTTGTAGGAAGTCTTATGG
RM25040 GGCTGGACTTCACTTGACTTTGG CCACACGACCATCTAAGTGAACAGG
ID74-31/32 TCATCTGGTGTTTTGTACCC GGAATGCCTTGGTTAGGTAT
ID74-33/34 GAAAATTGGAGGAGGAGGTA ACCCAAGGAGATAGCAAGTC
ID74-35/36 AGGTCTACCCAAAACAGGAG GTAAGGCCTGACTGGAAGTT

表2

定位区间内的基因及功能注释"

基因号
Locus ID
功能注释
Function annotation
LOC_Os10g07150 转座子蛋白 Transposon protein
LOC_Os10g07160 反转座子 Retrotransposon
LOC_Os10g07200 Hsp20/α晶体家族蛋白 Hsp20/alpha crystallin family protein
LOC_Os10g07210 Hsp20/α晶体家族蛋白 Hsp20/alpha crystallin family protein, putative, expressed
LOC_Os10g07229 脱氢酶 Dehydrogenase
LOC_Os10g07248 转座子蛋白 Transposon protein
LOC_Os10g07270 泛素羧基末端水解酶5 Ubiquitin carboxyl-terminal hydrolase 5
LOC_Os10g07280 表达蛋白 Expressed protein
LOC_Os10g07290 糖苷水解酶家族17 Glycosyl hydrolases family 17
LOC_Os10g07320 假定蛋白 Hypothetical protein
LOC_Os10g07340 表达蛋白 Expressed protein
LOC_Os10g07370 表达蛋白 Expressed protein
基因号
Locus ID
功能注释
Function annotation
LOC_Os10g07380 表达蛋白 Expressed protein
LOC_Os10g07390 假定蛋白 Hypothetical protein
LOC_Os10g07400 类RPP13抗病蛋白1 Disease resistance RPP13-like protein 1
LOC_Os10g07420 表达蛋白 Expressed protein
LOC_Os10g07430 表达蛋白 Expressed protein
LOC_Os10g07440 表达蛋白 Expressed protein
LOC_Os10g07450 表达蛋白 Expressed protein

图7

叶片早衰基因OsPLS7在10号染色体上的分子定位"

图8

定位区间内的叶中表达基因的qRT-PCR分析"

附图1

定位区间的基因表达热图"

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