Overexpressing Analysis of the Flowering Time Gene OsDTH10 in Rice

doi:10.16819/j.1001-7216.2016.5140

Abstract

Abstract:

Flowering time (heading date) is an important agronomic trait of crops, and it is closely linked with the yield of crops. To reveal the function of rice flowering time gene OsDTH10 in rice, we constructed an overexpressing vector of OsDTH10 and analyzed the function of OsDTH10 by the reverse genetics. The results showed that the OsDTH10-overexpressing (OsDTH10-OV) transgenic plants displayed a late heading phenotype, suggesting that the overexpression of OsDTH10delayed the heading-date of rice. RT-PCR expression analysis showed that the expression level of OsDTH10was increased obviously in OsDTH10-OV lines with phenotype compared with the transgenic lines without phenotype and wild type plants, indicating that late heading phenotype in OsDTH10-OV lines was caused by the overexpression of OsDTH10. The tissue-specific expression showed that OsDTH10 was expressed in different organs, with the higher expression levels in stem and leaf sheath. Also, we further analyzed the expression of OsDTH10 in leaves at various leaf-ages, and the result showed that OsDTH10 exhibited higher expression in unexpanded flag leaf and the second leaf from the top than these in the third and the fourth leaf from the top. In addition, we also analyzed the expression of OsDTH10 in different photoperiods, and the result indicated that OsDTH10displayed a higher expression during the day time (light stage) and a lower expression at night (dark stage) whatever short-day and long-day conditions, suggesting that OsDTH10 might be involved in the photoperiodic pathways to regulate the heading-date in rice.

Key words: rice, OsDTH10, overexpression, expression analysis

摘要：

开花时间(抽穗期)是农作物一个重要的农艺性状,与农作物的产量息息相关。调节农作物的开花抽穗时间是提高农作物产量的一条重要途径。为了揭示水稻开花时间基因OsDTH10在水稻抽穗期调控中的功能,利用反向遗传学方法构建了水稻OsDTH10基因过量表达载体,获得转基因植株,分析目的基因在过量表达条件下的功能。结果表明,过量表达OsDTH10导致水稻的抽穗期推迟;RT-PCR检测结果表明OsDTH10的表达量在晚抽穗的转基因株系中明显提高,但在没有表型的转基因株系及野生型中表达量较低,说明晚抽穗的表型是由于OsDTH10过量表达所致。组织特异性表达结果表明OsDTH10在植物的不同器官中都有表达,但在水稻的茎、叶鞘中表达量较高。分析OsDTH10在不同叶龄叶片中的表达,结果表明OsDTH10在未完全展开的剑叶叶片及倒2叶中的表达量较高,但在下部较老的倒3叶及倒4叶中的表达量下降。另外,分析OsDTH10在不同光周期条件下的昼夜节律性表达,结果表明无论在短日照还是长日照条件下,OsDTH10都在白天(光期)有表达高峰,在夜晚(暗期)表达降低,表明OsDTH10可能涉及光周期调控通路调节水稻的抽穗期。

关键词: 水稻, OsDTH10, 过量表达, 表达分析

CLC Number:

Q786
S511.01

Ying-ying LI, Wei XIONG, Ting SONG, Zhen-hua CAO, Wei-jiang LUAN. Overexpressing Analysis of the Flowering Time Gene OsDTH10 in Rice[J]. Chinese Journal OF Rice Science, 2016, 30(2): 121-126.

李莹莹, 熊炜, 宋婷, 曹振华, 栾维江. 水稻开花时间基因OsDTH10的过表达分析[J]. 中国水稻科学, 2016, 30(2): 121-126.

Figures/Tables 4

Fig. 1. Construction of overexpression vetor. A, Constructure of overexpression vetor; LB, Left border of T-DNA; NPT Ⅱ, Neomycin phosphotransferaseⅡ; p2×35S, Double cauliflower mosaic virus (CaMV) 35S promoter; RB, Right border of T-DNA; B, PCR amplification of OsDTH10 full open reading frame; C, Digesting identification of recombinant plasmid; 1-3, Three independent clones of recombinant plasmid; M, DNA marker.

Fig. 2. Phenotype and expression analysis of OsDTH10 -overexpressing transgenic lines(OsDTH10-OV). A, Phenotype of transgenic plants; WT, Transgenic plants with empty vector; OsDTH10-OV, OsDTH10-Overexpressing transgenic plants. B, qRT-PCR and RT-PCR analysis of OsDTH10 expression level in different OsDTH10-OV lines; 1 to 5 are five independent OsDTH10-OV lines; C, Heading date of OsDTH10-OV line(mean ± SD,n≥15).

Fig. 3. Tissue-specific expression of OsDTH10. A,Tissue-specific expression of OsDTH10; M, Shoot apical meristem; R, Root; L, Leaf; LS, Leaf sheath; S, Stem; P1, 14 cm panicle; P2, 4 cm panicle; P3, 25 cm panicle. B, Expression of OsDTH10 in leaves at various leaf-ages. L1, The unexpanded flag leaf; L2, The second leaf from the top; L3, The third leaf from the top; L4, The fourth leaf from the top.

Fig. 4. Circadian rhythm expression of OsDTH10 under different photoperiods. A, Expression level of OsDTH10 in short-day conditions; B, Expression level of OsDTH10 in long-day conditions.

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