Agronomic Traits of Marker-free Transgenic japonica Rice with Overexpression of OsPHF1 Under Low Phosphorus Environment

doi:10.16819/j.1001-7216.2018.8034

Abstract

Abstract:

【Objective】PHF1 (phosphate transporter assistance factor) regulates specific phosphorous transporters after transcriptional regulation and affects the utilization efficiency of phosphate. The main purpose is to develop selective-marker-free OsPHF1-overexpressed japonica rice (Kongyu 131), and study the agronomic traits of transgenic rice at different low phosphorus concentrations. It contributes to the cultivation of transgenic rice varieties with high phosphorus utilization efficiency.【Method】Marker-free OsPHF1-overexpressed transgenic lines were obtained by using double T-DNA methods, Agrobacterium-mediated infection and subsequent screening. The field experiments for T₃ and T₄ generations of transgenic plants were carried out under low phosphorus concentration (75 or 112.5 kg /hm² calcium superphosphate) medium-to-low phosphorus concentrations (225 or 300 kg /hm² calcium superphosphate) and normal phosphorus concentration (450 kg /hm² calcium superphosphate).【Result】Three marker-free homozygous transgenic lines F18-18, F22-32 and F25-6 were obtained. The expression levels of OsPHF1 in lines F22-32 and F25-6 were much higher than that of wild type. Field experiments showed that compared with the wild type, the tiller number of F22-32 and F25-6 (T₃ generation) were increased by 55% and 25%, respectively and the yield per plant of F22-32 and F25-6 were increased by 38%–34%, respectively under medium-to-low phosphorus environment (300 kg/hm² calcium superphosphate). The yield of F22-32 and F25-6 transgenic lines (T₄) increased by 30% to 35%, the biggest improvement under low phosphorus (112.5 kg /hm² calcium superphosphate). Meantime, the tiller number and yield of F22-32 and F25-6 (T₄) also showed obvious boost under medium-to-low phosphorus (225 kg /hm² calcium superphosphate) levels. 【Conclusion】Marker-free OsPHF1 transgenic lines were cultivated using double T-DNA method. Two generation of homozygous transgenic lines showed significantly increased tiller number and yield at medium-to-low phosphorus level (112.5, 225 or 300 kg /hm² calcium superphosphate).

Key words: OsPHF1, overexpression, marker-free transgenic rice, low phosphorus concentration, yield

摘要：

【目的】 磷酸盐转运体运输协助因子(PHF1)通过转录后调节特定磷转运蛋白,影响磷酸盐的利用效率。本研究通过培育过表达OsPHF1的无选择标记转基因粳稻空育131,研究在不同磷浓度环境中OsPHF1的过表达对粳稻空育131产量的影响,为培育可商品化的磷高效转基因水稻品种提供依据。方法利用双T-DNA方法构建OsPHF1的过表达载体,通过农杆菌侵染法和后续筛选获得了无选择标记的转基因空育131纯合株系,通过对T₃和T₄代转基因植株的田间试验,研究转基因品系在低磷浓度(75或112.5 kg/hm²过磷酸钙)、中低磷浓度(225或300 kg/hm²过磷酸钙)和正常磷浓度(450 kg/hm²过磷酸钙)下的农艺性状。结果获得了3个无筛选标记的纯合OsPHF1过表达转基因空育131株系F18-18、F22-32和F25-6。其中,F22-32和F25-6的OsPHF1的表达量远高于野生型。大田试验显示,F22-32和F25-6株系的T₃代在中低磷(300 kg/hm²过磷酸钙)环境中,分蘖数比对照分别增加了55%和25%,增产幅度分别为38%和34%;F22-32和F25-6株系T₄代在低磷条件下(112.5 kg/hm²过磷酸钙)产量的增幅最大,增产了30%~35%;在中低磷条件下(225 kg/hm²过磷酸钙)分蘖数和产量也有明显增加。结论双T-DNA法能用于培育过表达OsPHF1的无筛选标记转基因水稻。田间试验显示,高表达OsPHF1的转基因株系在中低磷条件下(112.5、225或300 kg/hm²过磷酸钙)分蘖数和产量稳定增加。

关键词: OsPHF1, 过表达, 无选择标记的转基因水稻, 低磷浓度, 产量

CLC Number:

Zhanghua HU, Xiuyan LIU, Yufeng WANG, Yu PENG, Xiaoliang SHI, Sheng TENG. Agronomic Traits of Marker-free Transgenic japonica Rice with Overexpression of OsPHF1 Under Low Phosphorus Environment[J]. Chinese Journal OF Rice Science, 2018, 32(6): 557-564.

胡张华, 刘秀艳, 王玉锋, 彭瑜, 史晓亮, 滕胜. 低磷条件下过表达OsPHF1基因对粳稻农艺性状的影响[J]. 中国水稻科学, 2018, 32(6): 557-564.

Figures/Tables 7

References 20

[1]	Schachtman D P, Reid R J, Ayling S M.Phosphorus uptake by plants: From soil to cell.Plant Physiol, 1998, 116(2): 447-453.
[2]	Wang F, Deng M J, Xu J M, Zhu X L, Mao C Z.Molecular mechanisms of phosphate transport and signaling in higher plants.Sem Cell & Dev Biol, 2018, 74: 114-122.
[3]	Muchhal U S, Pardo J M, Raghothama K G.Phosphate transporters from the higher plantArabidopsis thaliana. Proc Natl Acad Sci USA, 1996, 93(19): 10519-10523.
[4]	Rae A L, Cybinski D H, Jarmey J M, Smith F W.Characterization of two phosphate transporters from barley: Evidence for diverse function and kinetic properties among members of the Pht1 family.Plant Mol Biol, 2003, 53(1): 27-36.
[5]	Raghothama K G.Phosphate transport and signaling.Curr Opin Plant Biol, 2000, 3(3): 182-187.
[6]	Mudge S R, Rae A L, Diatloff E, Smith F W.Expression analysis suggests novel roles for members of the Pht1 family of phosphate transporters inArabidopsis. Plant J, 2002, 31(3): 341-353.
[7]	Jia H, Zhang S, Wang L, Yang Y, Zhang H, Cui H, Shao H, Xu G.OsPht1;8, a phosphate transporter, is involved in auxin and phosphate star vation response in rice.J Exp Bot, 2017, 68(18): 5057-5068.
[8]	Shin H, Shin H S, Dewbre G R, Harrison M J.Phosphate transport in Arabidopsis: Pht1;1 and Pht1;4 play a major role in phosphate acquisition from both low- and high- phosphate environments. Plant J, 2004, 39(4): 629-642.
[9]	Wang C, Yue W, Ying Y, Wang S, Secco D, Liu Y, Whelan J, Tyerman S D, Shou H.Rice SPX-Major facility superfamily3, a vacuolar phosphate efflux transporter, is involved in maintaining phosphate homeostasis in rice.Plant Physiol, 2015, 169(4): 2822-2831.
[10]	Karthikeyan A S, Varadarajan D K, Mukatira U T,D'Urzo M P,Damsz B, Raghothama K G. Regulated expression of Arabidopsis phosphate transporters. Plant Physiol, 2002, 130(1): 221-233.
[11]	Barlowe C, Schekman R.SEC12 encodes a guanine- nucleotide-exchange factor essential for transport vesicle budding from the ER. Nature, 1993, 365(6444): 347-349.
[12]	Barlowe C.Signals for COPII-dependent export from the ER: What’s the ticket out?Trends Cell Biol, 2003, 13(6): 295-300.
[13]	Gonzalez E, Solano R, Rubio V, Leyva A, Paz-Ares J.PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1 is a plant-specific SEC12-related protein that enables the endoplasmic reticulum exit of a high-affinity phosphate transporter inArabidopsis. Plant Cell, 2005, 17(12): 3500-3512.
[14]	Chen J, Liu Y, Ni J, Wang Y, Bai Y, Shi J, Gan J, Wu Z, Wu P.OsPHF1 regulates the plasma membrane localization of low- and high-affinity inorganic phosphate transporters and determines inorganic phosphate uptake and translocation in rice. Plant Physiol, 2011, 157(1): 269-278.
[15]	Wu P, Shou H X, Xu G H, Lian X M.Improvement of phosphorus efficiency in rice on the basis of understanding phosphate signaling and homeostasis.Curr Opin Plant Biol, 2013, 16(2): 205-212.
[16]	Gilbert N.Environment: The disappearing nutrient.Nature, 2009, 461(7265): 716-718.
[17]	Komari T, Hiei Y, Saito Y, Murai N, Kumashiro T.Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers. Plant J, 1996, 10(1): 165-74.
[18]	韩兆雪, 曹墨菊, 朱祯, 荣廷昭. DREB基因双T-DNA植物表达载体的构建及验证. 分子植物育种, 2004, 2(1): 7-12.
	Han Z X, Cao M J, Zhu Z, Rong T Z.Construction and verification of double T-DNA plant expression vector of the DREB gene. Mol Plant Breed, 2004, 2(1): 7-12. (in Chinese with English abstract)
[19]	胡张华, 吴关庭, 金卫, 王伏林, 陈锦清. 农杆菌介导的水稻转化及bar基因稳定遗传. 浙江农业学报, 2003, 15(6): 327-331.
	Hu Z H, Wu G T, Jin W, Wang F L, Chen J Q.Agrobacterium-mediated transformation of rice and stable inheritance of bar gene. Acta Agric Zhejiang, 2003, 15(6): 327-331. (in Chinese with English abstract)
[20]	于恒秀, 陆美芳, 陈秀花, 龚志云, 刘巧泉, 顾铭洪. 不同转化方法培育无抗性选择标记转基因水稻效率的比较. 中国水稻科学, 2009, 23(2): 120-126.
	Yu H X, Lu M F, Che X H, Gong Z Y, Liu Q Q, Gu M H.Comparison on efficiency of generating selectable marker-free transgenic rice by different transformation methods.Chin J Rice Sci, 2009, 23(2): 120-126. (in Chinese with English abstract)

引物名称 Primer name	引物序列(5′-3′) Primer sequence(5′-3′)
PHF1-full-S	GCTCTAGAATGGCAGGCGGCGGAGGTGGCGAG(XbaⅠ)
PHF1-full-A	GCGTCGACTCACCAGGGGTTCTGGTCCTCAGG(SalⅠ)
HYB-3S	TGAAAAAGCCTGAACTCACCG
HYB-4A	TATTTCTTTGCCCTCGGACG
PHF1-7S	GATGGGAAGTATCTGGCTTTGGG
PHF1-10A	AACAGGATGGCTGACACTAGGAA
PHF-418S	CTCAGAATATTTCATTGGCCGAGC
PHF-633A	CCTAGAAAAGCGGCAACATTCAATCTTC
Ubi-S	GACGGACGCACCCTGGCTGACTAC
Ubi-A	TGCTGCCAATTACCATATACCACGAC

引物名称 Primer name	引物序列(5′-3′) Primer sequence(5′-3′)
PHF1-full-S	GCTCTAGAATGGCAGGCGGCGGAGGTGGCGAG(XbaⅠ)
PHF1-full-A	GCGTCGACTCACCAGGGGTTCTGGTCCTCAGG(SalⅠ)
HYB-3S	TGAAAAAGCCTGAACTCACCG
HYB-4A	TATTTCTTTGCCCTCGGACG
PHF1-7S	GATGGGAAGTATCTGGCTTTGGG
PHF1-10A	AACAGGATGGCTGACACTAGGAA
PHF-418S	CTCAGAATATTTCATTGGCCGAGC
PHF-633A	CCTAGAAAAGCGGCAACATTCAATCTTC
Ubi-S	GACGGACGCACCCTGGCTGACTAC
Ubi-A	TGCTGCCAATTACCATATACCACGAC

株系名称 Line name	检测的总株数 Total number of detected plants	含有插入的OsPHF1株数 Plant number containing inserted OsPHF1	不含插入的OsPHF1株数 Plant number without inserted OsPHF1	χ²_(3：1)
F18	57	39	18	1.32
F22	56	45	11	0.86
F25	56	40	16	0.38
F32	60	43	17	0.36

株系名称 Line name	检测的总株数 Total number of detected plants	含有插入的OsPHF1株数 Plant number containing inserted OsPHF1	不含插入的OsPHF1株数 Plant number without inserted OsPHF1	χ²_(3：1)
F18	57	39	18	1.32
F22	56	45	11	0.86
F25	56	40	16	0.38
F32	60	43	17	0.36

株系名称 Line name	OsPHF1相对表达量（叶） Relative expression of OsPHF1 (Leaf)	OsPHF1相对表达量（根） Relative expression of OsPHF1 (Root)
F18-18	0.38±0.03 b	0.05±0.00 a
F22-32	16.17±4.57 d	1.42±0.94 b
F25-6	12.92±1.11 c	1.28±0.89 b
空育131 Kongyu 131	0.20±0.03 a	0.07±0.01 a