抑制OsPUT5基因表达降低水稻低温抗性

doi:10.3969/j.issn.1004-1524.2023.04.05

浙江农业学报 ›› 2023, Vol. 35 ›› Issue (4): 780-788.DOI: 10.3969/j.issn.1004-1524.2023.04.05

抑制OsPUT5基因表达降低水稻低温抗性

张斌(), 冯晓庆, 郑芊, 陈稳, 滕杰

湖南科技学院湖南省银杏工程技术研究中心,湖南永州 425199

收稿日期:2022-05-12 出版日期:2023-04-25 发布日期:2023-05-05
作者简介:张斌(1981—),男,湖南永州人,博士,副教授,研究方向为植物发育生物学。E-mail: zhangbin27104@163.com
基金资助:
湖南省自然科学基金(2022JJ30274);湖南省自然科学基金(2021JJ30292)

OsPUT5 silencing reduced low temperature resistance in rice

ZHANG Bin(), FENG Xiaoqing, ZHENG Qian, CHEN Wen, TENG Jie

Hunan Provincial Engineering Research Center for Ginkgo biloba, Hunan University of Science and Engineering, Yongzhou 425199, Hunan, China

Received:2022-05-12 Online:2023-04-25 Published:2023-05-05

摘要/Abstract

摘要：

为研究水稻(Oryza sativa)多胺转运蛋白(polyamine uptake transporters, PUT)的功能,利用生物信息学方法对水稻中OsPUT家族成员进行鉴定,并对OsPUT5蛋白结构进行分析,采用实时荧光定量PCR(qRT-PCR)检测OsPUT5基因在水稻不同组织中的表达情况,最后利用水稻超表达(overexpress,OE)株系、日本晴(Nipponbare,Nip)株系和RNAi株系初步研究OsPUT5基因的功能。结果表明,水稻中含有6个OsPUT,其中OsPUT5是一种含有10个跨膜结构域,且N端和C端均位于细胞膜外,并具有腐胺-鸟氨酸逆向转运蛋白PotE特有结构域的膜蛋白。基因表达模式显示,OsPUT5基因在叶片中表达量最高,在根、茎和花中无显著差异(P>0.05)。在生命周期内,正常条件下,OE株系、Nip株系和RNAi株系无明显的表型差异。在水稻幼苗期,4 ℃处理30 h后,RNAi株系、Nip株系和OE株系的相对电导率、丙二醛含量和脯氨酸含量均显著(P<0.05)升高,且表型都受到一定程度的影响。与Nip株系和OE株系相比,RNAi株系受影响最大,叶片枯萎,失水严重,植株鲜重和脯氨酸含量显著降低(P<0.05),相对电导率和丙二醛含量显著升高(P<0.05)。低温胁迫处理后继续培养10 d,RNAi株系存活率显著(P<0.05)低于Nip株系和OE株系。表明超表达或抑制OsPUT5基因表达对水稻的正常生长发育没有明显影响,但抑制该基因表达降低了水稻对低温胁迫的抗性。

关键词: 水稻, 多胺转运蛋白, 多胺

Abstract:

In order to study the function of polyamine uptake transporters (PUT) in rice, the members of OsPUT family in rice were identified by bioinformatics methods, and the structure of OsPUT5 protein was analyzed. The expression of OsPUT5 gene in different tissues of rice was detected by qRT-PCR, and the function of OsPUT5 gene was preliminarily studied by using rice overexpress (OE) lines, Nipponbare (Nip) lines and RNAi lines. The results showed that there were 6 OsPUTs in rice, among which, OsPUT5 was a membrane protein with 10 transmembrane domains and with the unique domain of putrescine ornithine antiporter PotE, it’s N-terminal and C-terminal were located outside the membrane. The gene expression pattern showed that OsPUT5 gene had the highest expression in leaves, but there was no significant(P>0.05) difference among roots, stems and flowers. In the life cycle, under normal conditions, there was no obvious phenotypic difference among RNAi lines, Nip lines and OE lines. At the seedling stage of rice, the relative conductivity, malondialdehyde content and proline content of RNAi lines, Nip lines and OE lines were significantly (P<0.05) increased after treatment at 4 ℃ for 30 h. Moreover, phenotypes were affected, compared with Nip lines and OE lines, RNAi lines were most affected, with withered leaves, serious water lost, significantly (P<0.05) reduced plant fresh weight and proline content, and significantly (P<0.05) increased relative conductivity and malondialdehyde content. Normal culture for 10 days after cold stress treatment, the survival rate of RNAi lines was significantly (P<0.05) lower than that of Nip lines and OE lines. It showed that overexpression or inhibition of OsPUT5 gene expression had no significant effect on the normal growth and development of rice, but inhibition of OsPUT5 gene expression reduced the resistance of rice to cold stress.

Key words: rice, polyamine transporter, polyamines

中图分类号:

S511

张斌, 冯晓庆, 郑芊, 陈稳, 滕杰. 抑制OsPUT5基因表达降低水稻低温抗性[J]. 浙江农业学报, 2023, 35(4): 780-788.

ZHANG Bin, FENG Xiaoqing, ZHENG Qian, CHEN Wen, TENG Jie. OsPUT5 silencing reduced low temperature resistance in rice[J]. Acta Agriculturae Zhejiangensis, 2023, 35(4): 780-788.

图/表 5

表1 基因信息

Table 1 Gene information

多胺转运蛋白基因名称 Name of polyamine transporter gene	多胺转运蛋白基因编号 Locus of polyamine transporter gene	物种 Species
OsPUT1	Os02g0700500	水稻 Oryza sativa L.
OsPUT2	Os03g0374900	水稻 Oryza sativa L.
OsPUT3	Os03g0375300	水稻 Oryza sativa L.
OsPUT4	Os03g0375966	水稻 Oryza sativa L.
OsPUT5	Os03g0576900	水稻 Oryza sativa L.
OsPUT6	Os12g0580400	水稻 Oryza sativa L.
ATPUT1	AT1G31820	拟南芥 Arabidopsis thaliana
ATPUT2	AT1G31830	拟南芥 Arabidopsis thaliana
ATPUT3	AT5G05630	拟南芥 Arabidopsis thaliana
ATPUT4	AT3G13620	拟南芥 Arabidopsis thaliana
ATPUT5	AT3G19553	拟南芥 Arabidopsis thaliana

图1 OsPUT5蛋白的生物信息学分析 A,水稻和其他物种PUT家族蛋白系统进化树;B,OsPUT5蛋白含10个跨膜结构域且N端和C端均位于细胞质侧;C,OsPUT5蛋白保守的结构域,黑条显示氨基酸序列的长度。

Fig.1 Bioinformatics analysis of OsPUT5 protein A, Phylogenetic tree of PUT family protein in rice and other plants; B, A cartoon representation showing the 10 transmembrane domains in the OsPUT5 protein with both C-and N-termini in the cytoplasmic side; C, Domains conserved in the OsPUT5 protein. The black bar shows the length of the amino acid sequence.

图2 转基因苗鉴定和基因相对表达水平检测 A,RNAi株系DNA水平检测,1~6为抗性苗,7为pFGC5941-OsPUT5-RNAi质粒;B,OE株系DNA水平检测,1~4为抗性苗,5为pCAM1390-OsPUT5质粒;C,OE株系和RNAi株系RNA水平检测;D,OsPUT5基因表达模式。柱上无相同字母代表差异显著(P<0.05),误差线代表标准差,下同。

Fig.2 Identification of transgenic seedlings and detection of gene expression level A, DNA level detection of RNAi lines, 1-6 were resistant plantlets, 7 was pFGC5941-OsPUT5-RNAi plasmid; B, DNA level detection of OE lines, 1-4 were resistant plantlets, 5 was pCAM1390-OsPUT5 plasmid; C, RNA level detection in OE lines and RNAi lines; D, OsPUT5 expression pattern. Different lowercase letters indicated significant difference at P <0.05, the error bars represented the standard deviation. The same as below.

图3 OE株系、Nip株系和RNAi株系幼苗耐低温性比较 G,植株低温处理后继续正常培养10 d后的表型;H,植株低温处理后继续正常培养10 d后的存活率。

Fig.3 Comparison of cold tolerance of seedlings of OE lines, Nip lines and RNAi lines G, Phenotype of plant after 10 days of normal culture after low temperature treatment; H, The survival rate of plants after 10 days of normal culture after low temperature treatment.

表2 OE株系、Nip株系和RNAi株系主要农艺性状的比较

Table 2 Comparison of main agronomic characters of OE lines, Nip lines and RNAi lines

株系 Line	株高 Plant height/cm	穗长 Ear length/cm	每穗粒数 Seed numbers per panicle	初级枝梗数 Primary branch number	每穗实粒数 Plump grains per panicle	千粒重 1 000-grain weight/g	结实率 Seed setting rate/%
OE株系OE lines	81.2±5.8 a	19.9±2.4 a	103.1±23.3 a	10.2±0.9 a	83.1±18.4 a	24.5±0.45 a	80.6±8.2 a
Nip株系Nipponbare lines	81.7±4.6 a	20.3±2.1 a	102.8±20.5 a	10.4±0.7 a	84.4±19.7 a	24.3±0.47 a	82.1±9.0 a
RNAi株系RNAi lines	79.8±6.2 a	19.7±1.8 a	101.9±21.6 a	10.1±0.7 a	83.6±18.2 a	24.1±0.33 a	82.0±10.2 a

参考文献 32

[1]	IGARASHI K, KASHIWAGI K. Functional roles of polyamines and their metabolite acrolein in eukaryotic cells[J]. Amino Acids, 2021, 53(10): 1473-1492. DOI PMID
[2]	YU J, WANG B A, FAN W Q, et al. Polyamines involved in regulating self-incompatibility in apple[J]. Genes, 2021, 12(11): 1797. DOI URL
[3]	DAS K C, MISRA H P. Hydroxyl radical scavenging and singlet oxygen quenching properties of polyamines[J]. Molecular and Cellular Biochemistry, 2004, 262(1): 127-133. DOI URL
[4]	ZARZA X, SHABALA L, FUJITA M, et al. Extracellular spermine triggers a rapid intracellular phosphatidic acid response in Arabidopsis, involving PLDδ activation and stimulating ion flux[J]. Frontiers in Plant Science, 2019, 10: 601. DOI URL
[5]	SODA K. Overview of polyamines as nutrients for human healthy long life and effect of increased polyamine intake on DNA methylation[J]. Cells, 2022, 11(1): 164. DOI URL
[6]	CHEN D D, SHAO Q S, YIN L H, et al. Polyamine function in plants: metabolism, regulation on development, and roles in abiotic stress responses[J]. Frontiers in Plant Science, 2018, 9: 1945. DOI PMID
[7]	TAKÁCS Z, POÓR P, TARI I. Interaction between polyamines and ethylene in the response to salicylic acid under normal photoperiod and prolonged darkness[J]. Plant Physiology and Biochemistry, 2021, 167: 470-480. DOI PMID
[8]	GERLIN L, BAROUKH C, GENIN S. Polyamines: double agents in disease and plant immunity[J]. Trends in Plant Science, 2021, 26(10): 1061-1071. DOI PMID
[9]	何超, 沈登荣, 花蕾, 等. 梨小食心虫滞育过程中多胺代谢的变化[J]. 浙江农业学报, 2015, 27(11): 1960-1964.
	HE C, SHEN D R, HUA L, et al. Changes of polyamine metabolism in larvae of oriental fruit moth, Grapholita molesta, during diapause development[J]. Acta Agriculturae Zhejiangensis, 2015, 27(11): 1960-1964. (in Chinese with English abstract)
[10]	胡俊杰, 张古文, 胡齐赞, 等. 低温胁迫对菜用大豆生长、叶片活性氧及多胺代谢的影响[J]. 浙江农业学报, 2011, 23(6): 1113-1118.
	HU J J, ZHANG G W, HU Q Z, et al. Effects of chilling stress on growth, metabolism of reactive oxygen species and polyamines in vegetable soybean seedlings[J]. Acta Agriculturae Zhejiangensis, 2011, 23(6): 1113-1118. (in Chinese with English abstract)
[11]	JIMÉNEZ-BREMONT J F, CHÁVEZ-MARTÍNEZ A I, ORTEGA-AMARO M A, et al. Translational and post-translational regulation of polyamine metabolic enzymes in plants[J]. Journal of Biotechnology, 2022, 344: 1-10. DOI URL
[12]	KHAZAAL S, AL SAFADI R, OSMAN D, et al. Investigation of the polyamine biosynthetic and transport capability of Streptococcus agalactiae: the non-essential PotABCD transporter[J]. Microbiology (Reading, England), 2021, 167(12): 001124.
[13]	FUJITA M, FUJITA Y, IUCHI S, et al. Natural variation in a polyamine transporter determines paraquat tolerance in Arabidopsis[J]. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(16): 6343-6347.
[14]	CHAI H X, GUO J F, ZHONG Y L, et al. The plasma-membrane polyamine transporter PUT3 is regulated by the Na(+)/H(+) antiporter SOS1 and protein kinase SOS2[J]. The New Phytologist, 2020, 226(3): 785-797. DOI URL
[15]	MULANGI V, CHIBUCOS M C, PHUNTUMART V, et al. Kinetic and phylogenetic analysis of plant polyamine uptake transporters[J]. Planta, 2012, 236(4): 1261-1273. DOI PMID
[16]	WIPF D, LUDEWIG U, TEGEDER M, et al. Conservation of amino acid transporters in fungi, plants and animals[J]. Trends in Biochemical Sciences, 2002, 27(3): 139-147. PMID
[17]	OKUMOTO S, PILOT G. Amino acid export in plants: a missing link in nitrogen cycling[J]. Molecular Plant, 2011, 4(3): 453-463. DOI PMID
[18]	BEGAM R A, GOOD A G. The Arabidopsis paraquat resistant1 mutant accumulates leucine upon dark treatment[J]. Botany, 2017, 95(7): 751-761. DOI URL
[19]	DONG S C, HU H Z, WANG Y M, et al. A pqr2 mutant encodes a defective polyamine transporter and is negatively affected by ABA for paraquat resistance in Arabidopsis thaliana[J]. Journal of Plant Research, 2016, 129(5): 899-907. DOI URL
[20]	SAGOR G H M, BERBERICH T, KOJIMA S, et al. Spermine modulates the expression of two probable polyamine transporter genes and determines growth responses to cadaverine in Arabidopsis[J]. Plant Cell Reports, 2016, 35(6): 1247-1257. DOI URL
[21]	MARTINIS J, GAS-PASCUAL E, SZYDLOWSKI N, et al. Long-distance transport of thiamine (vitamin B₁) is concomitant with that of polyamines[J]. Plant Physiology, 2016, 171(1): 542-553. DOI PMID
[22]	SHEN Y, RUAN Q X, CHAI H X, et al. The Arabidopsis polyamine transporter LHR1/PUT3 modulates heat responsive gene expression by enhancing mRNA stability[J]. The Plant Journal, 2016, 88(6): 1006-1021. DOI URL
[23]	AHMED S, ARIYARATNE M, PATEL J, et al. Altered expression of polyamine transporters reveals a role for spermidine in the timing of flowering and other developmental response pathways[J]. Plant Science, 2017, 258: 146-155. DOI PMID
[24]	LI J Y, MU J Y, BAI J T, et al. PARAQUAT RESISTANT1, a golgi-localized putative transporter protein, is involved in intracellular transport of paraquat[J]. Plant Physiology, 2013, 162(1): 470-483. DOI PMID
[25]	张斌, 刘询, 耿雅楠, 等. 水稻多胺转运蛋白OsPUT1基因RNAi载体的构建及遗传转化[J]. 分子植物育种, 2016, 14(10): 2653-2658.
	ZHANG B, LIU X, GENG Y N, et al. Construction and genetic transformation of RNAi vector for OsPUT1 gene in rice[J]. Molecular Plant Breeding, 2016, 14(10): 2653-2658. (in Chinese with English abstract)
[26]	张斌, 阮颖. 水稻多胺转运蛋白OsPUT1基因过表达载体构建及遗传转化[J]. 基因组学与应用生物学, 2018, 37(1): 386-392.
	ZHANG B, RUAN Y. Construction of overexpression vector and genetic transformation of polyamine transporter OsPUT1 gene in rice[J]. Genomics and Applied Biology, 2018, 37(1): 386-392. (in Chinese with English abstract)
[27]	王长龙, 张力, 罗立新, 等. 水稻NAL11基因对苗期非生物逆境的响应分析[J]. 华北农学报, 2020, 35(4): 120-128. DOI
	WANG C L, ZHANG L, LUO L X, et al. Response analysis of rice gene NAL11 to abiotic stresses at the stage of seedling[J]. Acta Agriculturae Boreali-Sinica, 2020, 35(4): 120-128. (in Chinese with English abstract)
[28]	张斌, 黄亚玲, 彭英姿, 等. 拟南芥突变体atput3过表达水稻OsPUT1基因对百草枯敏感性的研究[J]. 农业生物技术学报, 2023, 30(2): 242-249.
	ZHANG B, HUANG Y L, PENG Y Z, et al. Study on the sensitivity of Arabidopsis thaliana mutant atput3 which overexpressing rice(Oryza sativa) OsPUT1 gene to paraquat[J]. Journal of Agricultural Biotechnology, 2023, 30(2): 242-249. (in Chinese with English abstract)
[29]	ZHAO H M, MA H L, YU L, et al. Genome-wide survey and expression analysis of amino acid transporter gene family in rice (Oryza sativa L.)[J]. PLoS One, 2012, 7(11): e49210. DOI URL
[30]	IGARASHI K, KASHIWAGI K. Characteristics of cellular polyamine transport in prokaryotes and eukaryotes[J]. Plant Physiology and Biochemistry, 2010, 48(7): 506-512. DOI PMID
[31]	ALHAG A, SONG J, DAHRO B, et al. Genome-wide identification and expression analysis of polyamine uptake transporter gene family in sweet orange (Citrus sinensis)[J]. Plant Biology (Stuttgart, Germany), 2021, 23(6): 1157-1166. DOI PMID
[32]	LI M, WANG C H, SHI J L, et al. Abscisic acid and putrescine synergistically regulate the cold tolerance of melon seedlings[J]. Plant Physiology and Biochemistry, 2021, 166: 1054-1064. DOI PMID

抑制OsPUT5基因表达降低水稻低温抗性

OsPUT5 silencing reduced low temperature resistance in rice

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