水稻矮化小穗基因DSP2的鉴定与克隆

doi:10.16819/j.1001-7216.2022.210606

摘要/Abstract

摘要：

【目的】水稻株高和穗型在产量形成中发挥着重要作用。鉴定与克隆水稻株高和穗型发育相关基因,可以丰富水稻株高穗型发育调控的分子机理,为分子设计育种奠定理论基础和提供基因资源。【方法】在粳稻日本晴T-DNA插入群体中筛选到矮化小穗突变体dsp2-D(dwarf and small panicle 2-Dominant),对其主要农艺性状进行了分析;采用图位克隆法结合T-DNA标签分离法进行了基因定位和克隆;利用半定量PCR和qRT-PCR进一步确定dsp2-D的候选基因;遗传转化实验验证了DSP2的功能。【结果】与野生型日本晴相比,dsp2-D突变体表现为半矮化、穗轴和枝梗明显缩短、穗型直立、千粒重降低等特征;遗传分析表明该突变体受一对不完全显性单基因控制;利用图位克隆将DSP2定位于第2染色体标记RM208和RM7337之间,与RM3850共分离;随后遗传分析发现,T-DNA插入位点与dsp2-D表型共分离,利用TAIL-PCR分离T-DNA插入序列,显示T-DNA插入到上述RM208和RM7337之间的两个基因之间。RT-PCR检测发现,位于T-DNA插入位点下游的一个编码LOB家族转录因子基因的表达量明显增加,而其他5个基因的表达量变化不明显,表明该基因可能为DSP2的候选基因;在野生型日本晴中过量表达DSP2基因,转化植株出现半矮化、小穗的表型,与dsp2-D表型类似,从而验证了DSP2的功能。【结论】 DSP2基因的过量表达是产生dsp2-D突变表型的原因;DSP2基因对水稻株高和穗长发育具有负向调控作用;为进一步丰富株高和穗型的遗传调控网络打下了基础。

关键词: 水稻, 矮化, 穗长, 突变体, 基因克隆

Abstract:

【Objective】 Plant height and panicle length play important roles in rice production. Identifying and cloning genes related to rice plant height and panicle architecture will enrich the molecular mechanism of the regulation of rice plant height and panicle development, and will provide a theoretical foundation and genetic resources for molecular design breeding. 【Method】 A dwarf and small panicle mutant dsp2-D(dwarf and small panicle2-Dominant) was obtained from T-DNA insertion population of Nipponbare. The main agronomic traits of dsp2-D and the wild-type were characterized under conventional planting conditions in field. An F₂ mapping population was generated by crossing the dsp2-D mutant with Longtefu B (an indica rice variety) and DSP2 was identified by map-based cloning and T-DNA tag method. The candidate gene was determined by the relative expression analysis of three genes located upstream and downstream of the T-DNA insertion site, respectively, which was carried out by real-time RT-PCR. The function of DSP2 was confirmed by overexpressing DSP2 (DSP2-OE) in wild-type Nipponbare.【Result】 The plant height, panicle length, the length of primary and secondary inflorescence branches of dsp2-D was dramatically reduced compared with the wild type. Genetic analysis indicated that the phenotypes of dsp2-D were regulated by an incomplete dominant gene. The DSP2 gene was mapped to a region between RM208 and RM7337 on chromosome 2, and co-segregated with RM3850. Subsequently, T-DNA insertion site was found to be co-segregated with dsp2-D phenotype, and T-DNA insertion sequence was separated by TAIL-PCR, which showed that T-DNA was inserted into the two genes between RM208 and RM7337. Real-time PCR analysis indicated that the expression level of LOC_Os02g57490, which located down-stream of T-DNA site, was dramatically up-regulated. While the expression levels of the rest five detected genes were not significantly changed in dsp2-D. DSP2 (DSP2-OE) overexpressing transgenic plants showed semi-dwarf and small panicle,which were similar with the phenotypes in dsp2-D. The results confirmed that DSP2 gene was the target gene controlling dsp2-D.【Conclusion】 The overexpression of DSP2 caused the mutant phenotypes of dsp2-D. And we found that DSP2 encodes a LOB family transcript factor and negatively regulates plant height and panicle length in rice. Our findings will open new perspectives on DSP2-based improvement of plant height and panicle type of rice.

Key words: rice, dwarf, panicle length, mutant, gene cloning

裘霖琳, 刘窍, 付亚萍, 刘文真, 胡国成, 翟玉凤, 庞礴, 汪得凯. 水稻矮化小穗基因DSP2的鉴定与克隆[J]. 中国水稻科学, 2022, 36(2): 150-158.

QIU Linlin, LIU Qiao, FU Yaping, LIU Wenzhen, HU Guocheng, ZHAI Yufeng, PANG Bo, WANG Dekai. Identification and Gene Cloning of DSP2 in Rice (Oryza sativa L.)[J]. Chinese Journal OF Rice Science, 2022, 36(2): 150-158.

图/表 5

表1 PCR鉴定及定量PCR所用的引物

Table 1 Primers used for PCR identification and quantitative real-time PCR (qRT-PCR) analysis.

引物名称	正向引物序列	反向引物序列	实验目的
Primer name	Forward primer(5'-3')	Reverse primer(5'-3')	Purpose
ORF1-qPCR	AGATCAACCTCCAACTACTCTC	AAGCTCAGTAGTACACAGGC	RT-PCR
ORF2-qPCR	GTGGTTCCAGAAGATCGTGTC	TTGTCGATGGACAGGAGCTC	RT-PCR
ORF3-qPCR	CACGCCTACGACAACATGAAC	GTACTCGAACGCGTTGACATC	RT-PCR
ORF4-qPCR	CCGGTGATACAAAGAGGTGC	ATATGCTTCGGCCACCTTG	RT-PCR
ORF5-qPCR	AGCGGTTGTCTGGTGATATC	AGACAGAAAACCCCTTGACG	RT-PCR
ORF6-qPCR	CAGCTTCTGATCCTGCAGTAG	ATCTCCCTTACTGATGCTGAC	RT-PCR
UBQ5-qPCR	ACCACTTCGACCGCCACTACT	ACGCCTAAGCCTGCTGGTT	RT-PCR
HPTⅡ	CAGAAGAAGATGTTGGCGAC	ATGTCCTGCGGGTAAATAGC	共分离分析 Co-segregation
DSP2-ORF	ATGTCGACATGGCTGGTGCTACGGCTGC	ATCTGCAGCTGAGAGTAGTTGGAGGTTGAT	全长ORF Full length ORF
NPTⅡ	TATGTCCTGATAGCGGTCCG	GTGCCCTGAATGAACTCCAG	转化植株检测 Detection of transgenic plants

图1 突变体dsp2-D的表型 AA、Aa、aa分别代表野生型日本晴植株、杂合体和dsp2-D纯合体。不同字母代表差异极显著（P<0.01,Tukey检验）,n=10。

Fig. 1. Phenotypes of the dsp2-D mutant. AA, Wild-type ‘Nipponbare’ plants; Aa, dsp2-D heterozygous plants; aa, dsp2-D homozygous plants. Values are mean ± SD of ten biological replicates. Samples with different letters are significantly different (P < 0.01; Tukey’s test).

图2 DSP2基因在第2染色体上的定位和T-DNA共分离分析 A―DSP2基因的初步定位;B―T-DNA插入位点共分离分析。M, 1 kb ladder; P为质粒阳性对照; A1~A3为野生型;B1~B7为杂合体;C1~C4为纯合体。

Fig. 2. Location of DSP2 on chromosome 2 and co-segregation analysis of T-DNA. A, Preliminary localization of DSP2 gene; B, Co-segregation analysis of T-DNA and dsp2-D phenotypes. M, 1 kb ladder, P, Plasmid positive control; A1-A3, Wild type; B1-B7, Heterozygote; C1-C4, Homozygous mutant.

图3 候选基因DSP2的表达分析 A―T-DNA插入位点;B, C―6个基因在突变体dsp2-D和野生型中的表达水平,水稻OsUBQ5为内参基因。Bar值表示3个生物学重复的平均值±SD;**表示0.01极显著水平。

Fig. 3. Expression analysis of candidate gene DSP2. A, Schematic diagram of T-DNA insertion sites; B and C, Expression levels of six candidate genes in dsp2-D and wild type analyzed by semi-quantitative RT-PCR and real time PCR; OsUBQ5 was amplified as a control. Bars represent standard deviation (n=3). ** indicates significant difference between Nipponbare and dsp2-D by t-test (P<0.01).

图4 DSP2基因的过量表达和功能验证 A―DSP2基因在野生型和转基因植株中的表达水平,其中WT代表野生型日本晴,CK代表空载体转化植株,OE-1#~OE-3#代表DSP2-OE转化植株;Bar值表示3个生物学重复的平均值±SD;**表示0.01极显著水平。B, C―DSP2过表达转基因植株的表型,CK为空载体转化植株。B图中标尺表示10 cm; C图中标尺表示5 cm。

Fig. 4. Overexpression of DSP2 gene and functional identification. A, Expression levels of DSP2 gene in wild-type and transgenic plants. WT, Wild type Nipponbare; CK, Control plasmid transgenic plants; OE-1#-OE-3#, DSP2-OE transgenic plants; Bars represent standard deviation (n=3). **indicates significant difference between Nipponbare and dsp2-D by t-test (P<0.01). B and C, Phenotype of the dsp2-D mutant and transgenic plant. B, Bar=10 cm; C, Bar=5 cm.

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