水稻<i>OsSPL3</i>启动子克隆及表达分析

doi:10.3785/j.issn.1008-9209.2022.05.091

浙江大学学报（农业与生命科学版）

2023, Vol. 49

Issue (3): 319-327 DOI: 10.3785/j.issn.1008-9209.2022.05.091

作物科学

水稻OsSPL3启动子克隆及表达分析

曾慧玲¹(

),莫祖意¹,蒲巧贤¹,王家澍¹,范凯²,李兆伟¹(

)

^1.福建农林大学生命科学学院，福建福州 350002
^2.福建农林大学农学院，福建福州 350002

Cloning and expression analysis of OsSPL3 promoter in rice

Huiling ZENG¹(

),Zuyi MO¹,Qiaoxian PU¹,Jiashu WANG¹,Kai FAN²,Zhaowei LI¹(

)

^1.College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
^2.College of Agriculture, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China

全文: PDF(3317 KB) HTML

摘要：

OsSPL转录因子在水稻（Oryza sativa）根系、叶片、花器官、穗等发育与逆境响应过程中起重要作用。本文通过对OsSPL3启动子的分析，探究了OsSPL3转录因子在水稻中的表达模式及其对干旱胁迫的响应方式。利用PLACE和Plant CARE在线软件分析OsSPL3启动子区的顺式作用元件，并构建OsSPL3启动子与β-葡糖醛酸糖苷酶（β-glucuronidase, GUS）基因的重组表达载体，转化‘中花11’（ZH11）水稻愈伤组织，筛选获得阳性转基因植株，且对pOsSPL3-GUS转基因植株的GUS表达活性以及在干旱胁迫与脱落酸（abscisic acid, ABA）处理下的表达方式进行检测。启动子分析结果表明，OsSPL3启动子区除包含必要的转录起始核心元件与光响应元件外，还包括3个MYB参与的干旱诱导元件、3个赤霉素响应元件、2个厌氧诱导必需作用元件、1个低温响应作用元件、1个胚乳表达调控元件、1个玉米醇溶蛋白代谢调控元件和1个分生组织表达相关调控元件。GUS染色结果显示，GUS基因在新生叶片、茎鞘、胚芽鞘等幼嫩组织及根冠、分生区、伸长区等根系旺盛生长部位中表达活性较高。此外，干旱胁迫能明显增强转基因水稻叶片与根系的GUS活性。本研究结果表明，OsSPL3转录因子在水稻种子萌发后的胚芽鞘生长、新叶发生、根系延伸等器官发育与茎鞘伸长等过程中发挥调控作用，同时，OsSPL3转录因子还参与水稻干旱胁迫响应过程。

关键词： 水稻; OsSPL3转录因子; 启动子; β-葡糖醛酸糖苷酶; 干旱胁迫

Abstract:

OsSPL transcription factor plays an important role in the development and stress response of rice (Oryza sativa) roots, leaves, floral organs, and ears. In this study, the OsSPL3 promoter was analyzed to explore the expression pattern of OsSPL3 transcription factor in rice and its response to drought stress. The Cis-acting elements in the OsSPL3 promoter region were analyzed by PLACE and Plant CARE online softwares, and the recombinant expression vector of OsSPL3 promoter and β-glucuronidase(GUS) gene was constructed, which was transformed into ZH11 rice callus, and positive transgenic plants were obtained by screening. The GUS expression activity of pOsSPL3-GUS transgenic plants and the expression patterns under drought stress and abscisic acid (ABA) treatments were detected. The results of promoter analysis showed that in addition to the necessary transcription initiation core elements and light-responsive elements, the OsSPL3 promoter region also included three MYB-involved drought-inducible elements, three gibberellin-responsive elements, two anaerobic induction essential elements, one low temperature response element, one endosperm expression regulatory element, one zein metabolism regulatory element and one meristem expression-related regulatory element. The results of GUS staining showed that the expression activity of GUS gene in young leaves, stem sheaths, coleoptiles and other young tissues was high, as well as in the vigorous growth parts of roots such as root cap, meristem zone, and elongation zone. In addition, the drought stress could significantly enhanced the GUS activity of transgenic rice leaves and roots. It shows that OsSPL3 transcription factor plays a regulatory role in the process of coleoptile growth, new leaf formation, root extension and stem sheath elongation after seed germination, and OsSPL3 transcription factor is also involved in the response process of rice drought stress.

Key words: rice (Oryza sativa) OsSPL3 transcription factor promoter β-glucuronidase (GUS) drought stress

收稿日期: 2022-05-09 出版日期: 2023-06-25

CLC:

S511.22

基金资助: 福建省自然科学基金面上项目(2021J01098)

通讯作者: 李兆伟 E-mail: 18349325508@163.com;lizw197@163.com

作者简介: 曾慧玲（https://orcid.org/0000-0002-0670-9585），E-mail：18349325508@163.com

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	曾慧玲
	莫祖意
	蒲巧贤
	王家澍
	范凯
	李兆伟

引用本文:

曾慧玲,莫祖意,蒲巧贤,王家澍,范凯,李兆伟. 水稻OsSPL3启动子克隆及表达分析[J]. 浙江大学学报（农业与生命科学版）, 2023, 49(3): 319-327.

Huiling ZENG,Zuyi MO,Qiaoxian PU,Jiashu WANG,Kai FAN,Zhaowei LI. Cloning and expression analysis of OsSPL3 promoter in rice. Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(3): 319-327.

链接本文:

https://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2022.05.091 或 https://www.zjujournals.com/agr/CN/Y2023/V49/I3/319

图1 OsSPL3 启动子的PCR克隆M：DL5000 DNA标志物；1：OsSPL3启动子。

表1 OsSPL3启动子顺式作用元件分析

图2 阳性转基因植株的PCR鉴定M：DL2000 DNA标志物；1：阳性对照；2～22：潮霉素基因；23：阴性对照。

图3 转基因水稻苗期不同组织的GUS染色与野生型ZH11水稻不同组织中 OsSPL3 表达量检测A.新叶尖端；B.新叶与完全展开的倒2叶；C.新叶、倒2叶与倒3叶；D.倒2叶、倒3叶、叶鞘上部；E.叶鞘中部；F.叶鞘近基部；G.根系残留种子胚根；H～I.初生根、不定根、分枝根与侧根；J.叶片与叶鞘衔接处的叶耳组织；K.胚芽鞘与新生嫩叶；L.种子萌发后的不定根。标尺为0.5 cm。

图4 PEG6000胁迫与ABA处理下转基因水稻叶片与根系的GUS染色检测A、D.正常营养液培养；B、E. 20% PEG6000胁迫；C、F. 100 µmol/L ABA处理。标尺为0.5 cm。

图5 PEG6000胁迫与ABA处理下pOsSPL3-GUS转基因水稻叶片和根系中 GUS 基因表达量A、C. 叶片；B、D. 根系。短栅上不同小写字母表示在P＜0.05水平差异有统计学意义，下同。

图6 PEG6000胁迫与ABA处理下野生型ZH11水稻叶片中 OsSPL3 表达量

1	陈开,唐瑭,张冬平,等.生长素和细胞分裂素参与构建水稻根系的研究进展[J].植物生理学报,2020,56(12):2495-2509. DOI:10.13592/j.cnki.ppj.2020.0398 CHEN K, TANG T, ZHANG D P, et al. Recent advances in auxin-cytokinin interactions involved in shaping architecture of rice root system[J]. Plant Physiology Journal, 2020, 56(12): 2495-2509. (in Chinese with English abstract) doi: 10.13592/j.cnki.ppj.2020.0398
2	郭韬,余泓,邱杰,等.中国水稻遗传学研究进展与分子设计育种[J].中国科学(生命科学),2019,49(10):1185-1212. DOI:10.1360/SSV-2019-0209 GUO T, YU H, QIU J, et al. Advances in rice genetics and breeding by molecular design in China[J]. Scientia Sinica Vitae, 2019, 49(10): 1185-1212. (in Chinese with English abstract) doi: 10.1360/SSV-2019-0209
3	CHEN X B, ZHANG Z L, LIU D M, et al. SQUAMOSA promoter-binding protein-like transcription factors: star players for plant growth and development[J]. Journal of Integrative Plant Biology, 2010, 52(11): 946-951. DOI: 10.1111/j.1744-7909.2010.00987.x doi: 10.1111/j.1744-7909.2010.00987.x
4	LAN T, ZHENG Y L, SU Z L, et al. OsSPL10, a SBP-box gene, plays a dual role in salt tolerance and trichome formation in rice (Oryza sativa L.)[J]. Genes, Genomes, Genetics (G3), 2019, 9(12): 4107-4114. DOI: 10.1534/g3.119.400700 doi: 10.1534/g3.119.400700
5	LEE J W, PARK J J, KIM S L, et al. Mutations in the rice liguleless gene result in a complete loss of the auricle, ligule, and laminar joint[J]. Plant Molecular Biology, 2007, 65(4): 487-499. DOI: 10.1007/s11103-007-9196-1 doi: 10.1007/s11103-007-9196-1
6	YANG R X, LI P C, MEI H L, et al. Fine-tuning of MiR528 accumulation modulates flowering time in rice[J]. Molecular Plant, 2019, 12(8): 1103-1113. DOI: 10.1016/j.molp.2019.04.009 doi: 10.1016/j.molp.2019.04.009
7	SI L Z, CHEN J Y, HUANG X H, et al. OsSPL13 controls grain size in cultivated rice[J]. Nature Genetics, 2016, 48(4): 447-456. DOI: 10.1038/ng.3518 doi: 10.1038/ng.3518
8	WANG S K, WU K, YUAN Q B, et al. Control of grain size, shape and quality by OsSPL16 in rice[J]. Nature Genetics, 2012, 44(8): 950-954. DOI: 10.1038/ng.2327 doi: 10.1038/ng.2327
9	WANG S K, LI S, LIU Q, et al. The OsSPL16-GW7 regulatory module determines grain shape and simultaneously improves rice yield and grain quality[J]. Nature Genetics, 2015, 47(8): 949-954. DOI: 10.1038/ng.3352 doi: 10.1038/ng.3352
10	YUAN H, QIN P, HU L, et al. OsSPL18 controls grain weight and grain number in rice[J]. Journal of Genetics and Genomics, 2019, 46(1): 41-51. DOI: 10.1016/j.jgg.2019.01.003 doi: 10.1016/j.jgg.2019.01.003
11	HUANG X Z, QIAN Q, LIU Z B, et al. Natural variation at the DEP1 locus enhances grain yield in rice[J]. Nature Genetics, 2009, 41(4): 494-497. DOI: 10.1038/ng.352 doi: 10.1038/ng.352
12	SHAO Y L, ZHOU H Z, WU Y R, et al. OsSPL3, an SBP-domain protein, regulates crown root development in rice[J]. The Plant Cell, 2019, 31(6): 1257-1275. DOI: 10.1105/tpc.19.00038 doi: 10.1105/tpc.19.00038
13	ZHOU M Q, TANG W. MicroRNA156 amplifies transcription factor-associated cold stress tolerance in plant cells[J]. Molecular Genetics and Genomics, 2019, 294(2): 379-393. DOI: 10.1007/s00438-018-1516-4 doi: 10.1007/s00438-018-1516-4
14	BIRKENBIHL R P, JACH G D, SAEDLER H, et al. Functional dissection of the plant-specific SBP-domain: overlap of the DNA-binding and nuclear localization domains[J]. Journal of Molecular Biology, 2005, 352(3): 585-596. DOI: 10.1016/j.jmb.2005.07.013 doi: 10.1016/j.jmb.2005.07.013
15	SHALOM L, SHLIZERMAN L, ZUR N, et al. Molecular characterization of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL)gene family from Citrus and the effect of fruit load on their expression[J]. Frontier in Plant Science, 2015, 6: 389. DOI: 10.3389/fpls.2015.00389 doi: 10.3389/fpls.2015.00389
16	NANDA S, HUSSAIN S. Genome-wide identification of the SPL gene family in Dichanthelium oligosanthes [J]. Bioinfor-mation, 2019, 15(3): 165-171. DOI: 10.6026/97320630015165 doi: 10.6026/97320630015165
17	XIONG J S, ZHENG D, ZHU H Y, et al. Genome-wide identification and expression analysis of the SPL gene family in woodland strawberry Fragaria vesca [J]. Genome, 2018, 61(9): 675-683. DOI: 10.1139/gen-2018-0014 doi: 10.1139/gen-2018-0014
18	CAI C P, GUO W Z, ZHANG B H. Genome-wide identification and characterization of SPL transcription factor family and their evolution and expression profiling analysis in cotton[J]. Scientific Reports, 2018, 8(1): 762. DOI: 10.1038/s41598-017-18673-4 doi: 10.1038/s41598-017-18673-4
19	LI J, HOU H M, LI X Q, et al. Genome-wide identification and analysis of the SBP-box family genes in apple (Malus×domestica Borkh.)[J]. Plant Physiology and Biochemistry, 2013, 70: 100-114. DOI: 10.1016/j.plaphy.2013.05.021 doi: 10.1016/j.plaphy.2013.05.021
20	PAN F, WANG Y, LIU H L, et al. Genome-wide identification and expression analysis of SBP-like transcription factor genes in moso bamboo (Phyllostachys edulis)[J]. BMC Genomics, 2017, 18: 486. DOI: 10.1186/s12864-017-3882-4 doi: 10.1186/s12864-017-3882-4
21	ZHANG B, XU W N, LIU X, et al. Functional conservation and divergence among homoeologs of TaSPL20 and TaSPL21, two SBP-box genes governing yield-related traits in hexaploid wheat[J]. Plant Physiology, 2017, 174(2): 1177-1191. DOI: 10.1104/pp.17.00113 doi: 10.1104/pp.17.00113
22	LI L, SHI F, WANG Y Q, et al. TaSPL13 regulates inflorescence architecture and development in transgenic wheat (Triticum aestivum L.)[J]. Plant Science, 2020, 296: 110516. DOI: 10.1016/j.plantscience.2020.110516 doi: 10.1016/j.plantscience.2020.110516
23	TRIPATHI R K, BREGITZER P, SINGH J. Genome-wide analysis of the SPL/miR156 module and its interaction with the AP2/miR172 unit in barley[J]. Scientific Reports, 2018, 8(1): 7085. DOI: 10.1038/s41598-018-25349-0 doi: 10.1038/s41598-018-25349-0
24	LI S K, LI L, JIANG Y, et al. SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) gene family: transcriptome-wide identification, phylogenetic relationship, expression patterns and network interaction analysis in Panax ginseng C. A. Meyer[J]. Plants, 2020, 9(3): 354. DOI: 10.3390/plants9030354 doi: 10.3390/plants9030354
25	PRESTON J C, JORGENSEN S A, OROZCO R, et al. Paralogous SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes differentially regulate leaf initiation and reproductive phase change in petunia[J]. Planta, 2016, 243(2): 429-440. DOI: 10.1007/s00425-015-2413-2 doi: 10.1007/s00425-015-2413-2
26	BRAY E A. Molecular responses to water deficit[J]. Plant Physiology, 1993, 103(4): 1035-1040. DOI: 10.1104/pp.103.4.1035 doi: 10.1104/pp.103.4.1035
27	YUE B, XUE W Y, XIONG L Z, et al. Genetic basis of drought resistance at reproductive stage in rice: separation of drought tolerance from drought avoidance[J]. Genetics, 2006, 172(2): 1213-1228. DOI: 10.1534/genetics.105.045062 doi: 10.1534/genetics.105.045062

[1]

附图1

Download

[1]	杨临泽,寿惠霞. 水稻根系分泌物脱氧麦根酸对根际和根内细菌群落组成的影响[J]. 浙江大学学报（农业与生命科学版）, 2023, 49(3): 376-388.
[2]	吕梦琪, Sunghwan JUNG, 麻志宏, 万亮, 孙大伟, 岑海燕. 基于无损力学平台的水稻倒伏表型分析(英文)[J]. 浙江大学学报（农业与生命科学版）, 2023, 49(1): 129-140.
[3]	张健男,王依名,张洁净,陈磊,罗金燕,易建平,李斌,安千里. 利用实时荧光定量PCR和数字PCR检测鉴定水稻细菌性条斑病菌[J]. 浙江大学学报（农业与生命科学版）, 2023, 49(1): 55-64.
[4]	巩龙达,陈凯,李丹,蔡梅,王京文,张奇春. 复合钝化剂施用水平对镉污染农田土壤的修复效果[J]. 浙江大学学报（农业与生命科学版）, 2022, 48(3): 359-368.
[5]	洪叶,张国平. 大麦灌浆期干旱胁迫对麦芽主要品质性状的影响[J]. 浙江大学学报（农业与生命科学版）, 2022, 48(2): 135-140.
[6]	周认,蔡宇,林恬逸,柴明良. 模拟干旱胁迫下褪黑素和表油菜素内酯对沟叶结缕草长期继代培养愈伤组织再生的影响[J]. 浙江大学学报（农业与生命科学版）, 2022, 48(1): 36-44.
[7]	黄贝,王鹏,温明霞,吴韶辉,徐建国. 不同程度干旱对温州蜜柑树势和成花生理的影响[J]. 浙江大学学报（农业与生命科学版）, 2021, 47(5): 557-565.
[8]	谢鹏尧,富昊伟,唐政,麻志宏,岑海燕. 基于RGB图像的冠层尺度水稻叶瘟病斑检测与抗性评估[J]. 浙江大学学报（农业与生命科学版）, 2021, 47(4): 415-428.
[9]	吴伊鑫,黄奇伟,叶木军,梁永超,彭红云. 农药减施条件下追施硅肥对水稻抗逆性及产量的影响[J]. 浙江大学学报（农业与生命科学版）, 2021, 47(4): 507-516.
[10]	李宏宇,李坤,杨知. 基于时间序列全极化合成孔径雷达的水稻物候期反演[J]. 浙江大学学报（农业与生命科学版）, 2021, 47(3): 404-414.
[11]	刘钰莹,张妍,汪哲远,李廷强. 硝化抑制剂与生物炭配施对水稻土氮素转化及氮肥利用率的影响[J]. 浙江大学学报（农业与生命科学版）, 2021, 47(2): 223-232.
[12]	邹文娴,周于宁,顾思婷,黄涂海,支裕优,孟龙,施加春,陈謇,徐建明. 关键时期淹水对不同土壤上水稻镉累积和转运的影响[J]. 浙江大学学报（农业与生命科学版）, 2021, 47(1): 74-88.
[13]	怀燕,陈照明,张耿苗,姜铭北,许剑锋,王强. 水稻侧深施肥技术的氮肥减施效应[J]. 浙江大学学报（农业与生命科学版）, 2020, 46(2): 217-224.
[14]	郑好,吕夏晨,谭赛琼,路雪丽,张弦,张晓勤,薛大伟. 干旱胁迫下大麦蜡质缺失突变体的生理生化指标及蜡质基因表达[J]. 浙江大学学报（农业与生命科学版）, 2019, 45(1): 8-13.
[15]	高晓月,董彬,张超,付建新,胡绍庆,赵宏波,梁立军. *桂花扩展蛋白基因OfEXPA2、OfEXPA4和OfEXLA1启动子克隆及活性分析*[J]. 浙江大学学报（农业与生命科学版）, 2019, 45(1): 23-29.

Viewed

Full text

Abstract

Cited

Shared

Discussed