水稻OsENO2-2基因过表达对水稻抽穗期的影响

doi:10.16819/j.1001-7216.2018.7055

摘要/Abstract

摘要：

【目的】 OsENO2-2是OsENO2通过可变剪切产生的短转录本,其cDNA序列从粳稻中花11中分离获得。本研究的主要目的是初步分析OsENO2-2在水稻抽穗期调控中的作用。【方法】构建了OsENO2-2的超量表达载体,获得转基因植株,通过对表型的观察和统计,分析目的基因在过量表达条件下的功能,并利用反向遗传学方法验证该基因的功能。【结果】 OsENO2-2过量表达导致水稻在长日照条件下的抽穗期推迟,而短日照条件下抽穗期无明显变化。通过qRT-PCR方法对水稻开花关键基因的检测发现,在长日照条件下,RFT1的表达量在超表达材料中显著下调,其他开花重要基因表达量在野生型和超表达材料中没有显著变化;而在短日照条件下,所有检测基因的表达量在野生型和超表达材料中均没有显著变化。【结论】在长日照条件下,OsENO2-2主要通过调控RFT1基因的表达来调控水稻抽穗期。

关键词: 水稻, 过量表达, OsENO2-2, 烯醇化酶基因, 抽穗期

Abstract:

【Objective】 OsENO2-2 was isolated from japonica cultivar Zhonghua 11 as a short transcript produced by alternative splicing of OsENO2 which is homologous to AtENO2. To reveal the function of OsENO2-2,【Method】 we constructed an overexpression vector of OsENO2-2 and obtained the transgenetic plants. Based on the observation and statistics of phenotype in overexpressed plant and wild-type, we validated the function of OsENO2-2 using reverse genetics method. 【Result】 Overexpression of OsENO2-2 transgenetic plants delayed heading date only under long-day conditions but not short-day conditions. qRT-PCR expression analysis of the critical flowering time genes showed that in long-day conditions, the expression of RFT1 in overexpressed plants was downregulated, and the expression levels of the other tested genes did not change significantly in wild-type and overexpressed plants; but in short-day conditions, the expression of all the tested genes in wild-type and over-expression plants did not change significantly. 【Conclusion】 OsENO2-2 might be involved in the photoperiodic pathways to regulate the heading date in rice by regulating the expression of RFT1 gene.

Key words: rice, overexpression, OsENO2-2, enolase gene, heading date

中图分类号:

刘小云, 李晓, 李腾飞, 苏鲁方. 水稻OsENO2-2基因过表达对水稻抽穗期的影响[J]. 中国水稻科学, 2018, 32(1): 12-22.

Xiaoyun LIU, Xiao LI, Tengfei LI, Lufang SU. Overexpression of OsENO2-2 Affects Heading Date in Rice[J]. Chinese Journal OF Rice Science, 2018, 32(1): 12-22.

图/表 10

图1 超表达载体和RNAi载体的构建 A-超表达载体结构。LB为T-DNA左边界;Hn-潮霉素抗性筛选标记;Pubiqutin为玉米ubiqutin基因启动子;GUS为β-葡萄糖苷酸酶基因;RB为T-DNA右边界。B-RNAi载体结构。35S-花椰菜花叶病毒启动子;R-f为OsENO2-2 RNAi片段。

Fig. 1. Construction of overexpression vector and RNAi vector. A, Structure of overexpression vector; LB, Left border of T-DNA; Hn, Hygromycin; Pubiqutin, Promoter of ubiqutin; GUS, beta-glucuronidase; RB, Right border of T-DNA. B, Structure of RNAi vector. 35S, 35S promoter of CaMV; R-f, RNAi fragment of OsENO2-2.

表1 本研究所用引物

Table 1 Primers used in the study.

引物名称 Primer name	引物序列(5′-3′) Primer sequence(5′-3′)
OsENO2-2-F	GGGGGTACCATGGTTCAACAGCTTGATGGAA
OsENO2-2-R	GGGGGTACCTTAGTACGGCTCCACAGGGG
R-OsENO2-2-F	AGAACTAGTGGTACCTGCTTCCACCGGAATATATGAG
R-OsENO2-2-R	AGAGAGCTCGGATCCCCCTCTTTGTTTTCCTGAATGTTA
Hn-F	TACACAGCCATCGGTCCAGA
Hn-R	TAGGAGGGCGTGGATATGTC
OsENO2-2-QF	GGCCAAGATGCCACAAATGT
OsENO2-2-QR	TTGCCTGTGTAGCCAGCCTTA
Actin-QF	TGTATGCCAGTGGTCGTACCA
Actin-QR	CCAGCAAGGTCGAGACGAA
RFT1-QF	TGGTGTTCGTGCTGTTCCA
RFT1-QR	TTGTAGAGCTCGGCGAAGTTC
RID1-QF	CGACGACAATAGCTCGATCGC
RID1-QR	GTGCATGGTCACGGAGCCTT
Hd3a-QF	GCTCACTATCATCATCCAGCATG
Hd3a-QR	CCTTGCTCAGCTATTTAATTGCATAA
Hd1-QF	TCAG CAACAGCATATCT TTCTCATCA
Hd1-QR	TCTGGAATTTGGCTATACTATCACC
Ehd1-QF	GGATGCAAGGAAATCATGGA
Ehd1-QR	AATCCCATCGGAAATCTTGG
OsGI-QF	TGGAGAAAGGTTGTGGATGC
OsGI-QR	GATAGACGGCACTTCAGCAGAT
Ghd7-QF	AAATCCGGTACGCGTCCAG
Ghd7-QR	GACATAGGTGGATGGCGGTG

图2 OsENO2-2全长开放阅读框的PCR扩增和过表达载体的鉴定 A-OsENO2-2全长开放阅读框的PCR扩增。B-过表达载体的酶切鉴定。1~4为4个不同单克隆,其中1、3、4为阳性克隆;2-阴性对照;M为DNA标记。

Fig. 2. PCR amplification of OsENO2-2 and identification of recombinant plasmid. A, PCR amplification of OsENO2-2 full open reading frame. B, Digesting identification of recombinant plasmid. Lanes 1-4, Four independent clones of recombinant plasmid. Lane 1, 3, 4, Positive clones; Lane 2, Negative control.

图3 OsNEO2-2组织特异性表达 S-幼苗;FL-剑叶;L-抽穗期倒2叶;LS-叶鞘;ST-茎秆;R-根;PA-幼穗;EN-胚乳。

Fig. 3. Tissue-specific expression of OsENO2-2. S, Seedling; FL, Flag leaf; L, The second leaf from the top at the heading stage; LS, Leaf sheath; ST, Stem; R, Root; PA, Panicle; EN, Endosperm.

图4 T0代转基因阳性植株阳性鉴定以及表达量检测 A-转基因阳性植株Hn基因PCR鉴定,自上到下依次为超表达载体、空载体对照组、RNAi载体的转基因植株。B-转基因植株OsENO2-2的表达量(平均值±标准差)。WT-野生型;OX-1~OX-8,8个超表达转基因植株;R-1~R-7,7个RNAi转基因植株。

Fig. 4. PCR analysis of Hn gene in T0 transgenic positive plants and expression analysis of OsENO2-2 in transgenic lines. A, PCR identification of Hn gene of the transgenic plants. The transgenic plants of the overexpressed lines, the transgenic plants of the control group lines, the transgenic plants of the RNAi lines, from up to down, respectively. B, The relative expression level of OsENO2-2 in transgenic plants(Mean±SD). WT, Wild type; OX-1 to OX-8, Eight independent overexpression transgenic plants. R-1 to R-7, Seven independent RNAi transgenic plants.

图5 转基因阳性植株的抽穗期(平均值±标准差) A-野生型和超表达植株的抽穗期表型。WT-野生型植株,OX-超表达植株; B-野生型和超表达植株在长日照大田的抽穗期(n=30)。WT-野生型植株,OX-1, OX-2, OX-3为超表达阳性株系,OX-7为转基因对照株系。LD-长日照条件。C-在长日照条件下(光照14 h/黑暗10 h)超表达,RNAi株系以及野生型株系的抽穗期(n=10)。R-1,R-2,R-5为RNAi抑制阳性株系。D-在短日照条件下(光照10 h/黑暗14 h)超表达,RNAi株系以及野生型株系的抽穗期(n=10)。SD-短日照条件。**表示与野生型植株相比差异达极显著水平(P<0.01,t检验)。

Fig. 5. Heading date of the transgenic plants(Mean±SD). A, Heading phenotypes of wild-type and overexpression plants. WT, Wild type; OX, Overexpression lines. B, Heading date of wild-type and overexpressing plants(n=30). WT, Wild type; OX-1, OX-2, OX-3, Overexpressed transgenetic positive plants; OX-7, The transgenic plants of the control group. C, Statistical analysis of heading date of wild-type, overexpressed plants and RNAi plants under long-day condition(14h light/10 h dark) (n=10). D, Statistical analysis of heading date of wild-type, overexpressed plants and RNAi plants under short-day condition(10h light/14h dark) (n=10). **The differences between wild type and transgenic plants are significant at 0.01 level(Student's t-test).

图6 OsENO2-2基因在不同光周期条件下的昼夜节律性表达(平均值±标准差) LD-长日照条件(光照14 h/黑暗10 h); SD-短日照条件(光照10 h/黑暗14 h); WT-野生型植株; OX-OsENO2-2超表达植株。

Fig. 6. Circadian rhythm expression of OsENO2-2 under different photoperiods(Mean±SD). LD, Long-day condition(light 14 h/dark 10 h); SD, Short-day condition(light 10 h/dark 14 h); WT, Wild type; OX, OsENO2-2 overexpressed plants.

图7 长日照条件下野生型植株(WT)与OsENO2-2超表达水稻植株(OX)中抽穗期关键基因的表达量(平均值±标准差)

Fig. 7. Expression levels of key heading regulatory genes in leaves of OsENO2-2 overexpression plants(OX) compared to wild type(WT) under long-day condition(Mean±SD).

图8 短日照条件下野生型植株(WT)与OsENO2-2超表达水稻植株(OX)中抽穗期关键基因的表达量(平均值±标准差)

Fig. 8. Expression levels of key heading regulatory genes in leaves of OsENO2-2 overexpression plants(OX) compared to wild type(WT) under short-day condition(Mean±SD).

图9 OsENO2-2基因在水稻长日照条件下调控开花途径中的作用

Fig. 9. Summary of OsENO2-2 function in LD pathways of rice flowering control.

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