低磷条件下过表达OsPHF1基因对粳稻农艺性状的影响

doi:10.16819/j.1001-7216.2018.8034

中国水稻科学 ›› 2018, Vol. 32 ›› Issue (6): 557-564.DOI: 10.16819/j.1001-7216.2018.8034

低磷条件下过表达OsPHF1基因对粳稻农艺性状的影响

胡张华¹, 刘秀艳², 王玉锋³, 彭瑜³, 史晓亮³, 滕胜^3,^*()

¹浙江省农业科学院病毒学与生物技术研究所浙江省植物有害生物防控重点实验室-省部共建国际重点实验室培育中心,杭州 310021
²杭州电子科技大学材料与环境工程学院,杭州 310018
³中国科学院上海植物生理生态研究所/分子植物卓越中心光合作用与环境生物学实验室,上海 200032

收稿日期:2018-03-23 修回日期:2018-06-04 出版日期:2018-11-27 发布日期:2018-11-10
通讯作者: 滕胜
基金资助:
国家重点研发计划资助项目(2016YFD0100700);农业部转基因专项(2016ZX08009003-004, 2014ZX08001-005);国家自然科学基金资助项目(31570269, 31570279)

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

Zhanghua HU¹, Xiuyan LIU², Yufeng WANG³, Yu PENG³, Xiaoliang SHI³, Sheng TENG^3,^*()

¹ State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, Institute of Virology and Biotechnology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
² College of Material and Environmental Engineering, Hangzhou Dianzi University, Hangzhou 310018, China
³ Laboratory of Photosynthesis and Environmental Biology, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai 200032, China

Received:2018-03-23 Revised:2018-06-04 Online:2018-11-27 Published:2018-11-10
Contact: Sheng TENG

摘要/Abstract

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

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

中图分类号:

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.

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.

图/表 7

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′)
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

图1 重组载体pCDMAR-OsPHF1的构建^ LB1和LB2为T-DNA左边界, RB1和RB2为T-DNA右边界; Hygromycin(R)为潮霉素磷酸转移酶基因, 潮霉素抗性筛选标记; MAR为核基质附着区序列; CaMV 35S为花椰菜花叶病毒启动子。

Fig. 1. Construction of overexpression vector of pCDMAR- OsPHF1.^ LB1 and LB2, Left border of T-DNA; RB1 and RB2, Right border of T-DNA; Hygromycin(R), Hygromycin phosphotransferase (HPT), resistant to hygromycin B; MAR, Matrix attachment regions; CaMV 35S, 35S promoter of CaMV.

图2 pCDMAR-OsPHF1过表达载体的双酶切验证^重组载体pCDMAR-OsPHF1的双酶切鉴定(XbaⅠ和SalⅠ)。泳道1~5为5个不同单克隆, 其中,M为DNA标记; 克隆1、2、4、5酶切后能得到约1 kb的片段, 为阳性克隆。

Fig. 2. Confirmation of recombinant plasmid pCDMAR- OsPHF1 by double enzyme digestion.^ Confirmation of recombinant plasmid pCDMAR-OsPHF1 by double enzyme digestion. Lanes 1-5, Five independent clones of recombinant plasmid. M represents DNA marker; A band about 1 kb appears at lanes 1, 2, 4 and 5, indicating that they are positive cloneion.

表2 T1代转基因水稻中插入基因OsPHF1分离比鉴定

Table 2 Identification of separation ratio for the inserted gene OsPHF1 in T1 generation transgenic rice.

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

图3 T1水稻中潮霉素抗性筛选标记HPT和插入基因OsPHF1的双基因PCR鉴定^ M–DNA标记;1–空育131(对照);2~10–不同转OsPHF1基因T1代株系。上排1~10为OsPHF1基因扩增情况, 插入的OsPHF1的扩增产物为215 bp(箭头所指), 基因组中扩增产物为600 bp。下排为相同模板对应的筛选标记HPT基因扩增情况, 产物为1000 bp(箭头所指)。泳道2, 3, 5, 6为转基因品系F18, F22, F25, F32。

Fig. 3. PCR analysis of OsPHF1 gene and selective marker HPT in T1 transgenic plants.^ M, DNA marker. Lane 1, Kongyu 131(control); 2-10, PCR results for different OsPHF1 transgenic T1 lines’ genomic DNA. Upper row is the amplification of OsPHF1 gene, and the amplified product of the inserted OsPHF1 is 215 bp, while the endogenous OsPHF1 in the genome is 600 bp. The bottom row represents the amplification of HPT gene, and the product is 1000 bp. Lanes 2, 3, 5 and 6 represent transgenic lines of F18, F22, F25 and F32, respectively.

表3 荧光定量PCR检测不同转基因株系中OsPHF1的总表达量

Table 3 The total relative expression of OsPHF1 in three independent overexpression transgenic plants by real-time PCR.

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

表4 T3代OsPHF1转基因植株在上海基地的农艺性状

Table 4 Agronomic traits of OsPHF1 transgenic plants (T3 generation) at Shanghai base.

施磷肥量 P application /(kg∙hm^-2)	株系名称 Line name	T₃植株, 上海基地 T₃ at Shanghai base				T₄植株, 海南省三亚基地 T₄ at Sanya base, Hainan Province
施磷肥量 P application /(kg∙hm^-2)	株系名称 Line name	分蘖数 Tiller number	单株产量 Yield per plant /g	千粒重 1000-grain weight /g	结实率 Seed-setting rate /%	分蘖数 Tiller number	单株产量 Yield per plant /g
112.5	F22-32	16.44±4.16 a	13.26±2.71 b	27.64±2.29 a	94±4 a	23.33±2.94 ab	40.36±6.32 b
	F25-6	16.20±5.16 a	12.61±2.50 b	26.98±2.71 a	93±6 a	22.83±3.49 ab	38.62±8.21 b
	空育131 Kongyu 131	15.20±4.62 a	10.90±2.75 a	27.29±2.36 a	95±4 a	19.83±1.47 a	29.70±5.31 a
225.0	F22-32	23.60±6.33 a	19.18±5.13 b	30.13±2.39 a	91±8 a	23.60±2.61 ab	36.91±4.02 b
	F25-6	21.50±4.30 a	17.89±3.43 b	29.23±2.10 a	92±6 a	22.67±4.18 ab	37.29±7.91 b
	空育131 Kongyu 131	15.15±3.20 b	14.27±2.00 a	28.41±2.33 a	94±5 a	20.67±3.08 a	31.74±6.39 a
450.0	F22-32	21.75±2.14 a	20.83±3.76 a	29.37±2.03 a	92±6 a	25.60±4.33 a	40.91±4.52 a
	F25-6	21.91±3.44 a	20.02±3.06 a	29.97±2.84 a	93±7 a	24.67±3.78 a	39.29±5.31 a
	空育131 Kongyu 131	22.89±3.76 a	22.28±3.61 a	30.52±2.54 a	95±5 a	29.67±3.28 a	42.70±6.11 a

参考文献 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)

低磷条件下过表达OsPHF1基因对粳稻农艺性状的影响

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

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