水稻幼苗酵母单杂文库构建及LAZY1上游调控因子筛选

doi:10.13560/j.cnki.biotech.bull.1985.2023-0001

摘要/Abstract

摘要：

LAZY1通过促进生长素的横向极性运输来调控水稻分蘖角，形成紧密直立的株型，从而提高水稻产量。为了研究LAZY1表达的分子调控机制，本研究利用酵母单杂筛库技术筛选水稻LAZY1的上游调控因子。首先以水稻粳稻品种日本晴基因组DNA为材料，经PCR扩增得到LAZY1的启动子序列，接着连接到pHIS2构建诱饵载体，并转化到AH109酵母菌株得到pHIS2-LAZY1诱饵菌。同时以萌发3 d的日本晴幼苗为材料，采用SMART技术构建cDNA文库，再将纯化的cDNA文库和pGADT7-Rec共转化酵母菌株Y187获得酵母单杂文库菌，接着将上述诱饵菌和文库菌进行接合完成酵母单杂筛库，共筛选得到21个候选的上游调控因子，进一步对其中的转录因子多蛋白桥梁因子OsMBF1进行双荧光素酶实验验证，结果显示OsMBF1可在体内促进LAZY1的转录。本研究得到LAZY1的上游正调控因子OsMBF1，为揭示LAZY1介导的生长素极性运输和分蘖角决定的调控网络提供了理论基础，将有助于揭示水稻由散生变为直立生长的奥秘，具有重要的理论和实践意义。

关键词: 水稻, LAZY1, 酵母单杂筛库, 转录因子

Abstract:

LAZY1 regulates rice tiller angle by promoting the lateral auxin transport to grow erectly in rice, thereby increasing its production. To explore the molecular regulatory mechanism of LAZY1 expression, a yeast one-hybrid library screening system was used to screen the upstream regulated factors of LAZY1. Firstly, the genome DNA of Nipponbare and amplified the promoter sequences of LAZY1 were extracted. Then, this fragment was ligated into the pHIS2 bait vector and transformed into Saccharomyces cerevisiae AH109 to obtain pHIS2-LAZY1 bait yeast. Meanwhile, a cDNA library of 3 DAG seeding of Nipponbare was constructed by SMART technology. Then, the purified cDNA library was co-transformed into S. cerevisiae Y187 with pGADT7-Rec vector. The candidate transcription factors of LAZY1 were identified by mating bait yeast and library yeast. A total of 21 cDNA sequences were determined. Finally, the dual-luciferase transient assay showed that the transcription factor multiprotein-bridging factor（OsMBF1）activated the transcription of LAZY1 in vivo. In this study, more positive regulators of LAZY1 were identified, which will provide a theoretical basis for uncovering the network of auxin polar transport mediated by LAZY1 and tiller angle determination, and revealing the mystery of plant architecture from prostrate to erect.

Key words: Oryza sativa, LAZY1, yeast one-hybrid（Y1H）screening, transcription factor

黄小龙, 孙贵连, 马丹丹, 闫慧清. 水稻幼苗酵母单杂文库构建及LAZY1上游调控因子筛选[J]. 生物技术通报, 2023, 39(9): 126-135.

HUANG Xiao-long, SUN Gui-lian, MA Dan-dan, YAN Hui-qing. Construction of Yeast One-hybrid Library and Screening of Factors Regulating LAZY1 Expression in Rice[J]. Biotechnology Bulletin, 2023, 39(9): 126-135.

图/表 7

参考文献 27

[1]	区树俊, 汪鸿儒, 储成才. 亚洲栽培稻主要驯化性状研究进展[J]. 遗传, 2012, 34(11): 1379-1389.
	Ou SJ, Wang HR, Chu CC. Major domestication traits in Asian rice[J]. Hereditas, 2012, 34(11): 1379-1389.
[2]	何芹. 水稻穗形基因OsAFB6和分蘖角度基因LAZY1的功能研究[D]. 武汉: 华中农业大学.
	He Q. Study on the function of ear shape gene OsAFB6 and tillering angle gene LAZY1 in rice[D]. Wuhan: Huazhong Agricultural University.
[3]	Jones JW, Adair CR. A “lazy” mutation in rice[J]. J Hered, 1938, 29(8): 315-318. doi: 10.1093/oxfordjournals.jhered.a104527 URL
[4]	Yoshihara T, Iino M. Identification of the gravitropism-related rice gene LAZY1 and elucidation of LAZY1-dependent and-independent gravity signaling pathways[J]. Plant Cell Physiol, 2007, 48(5): 678-688. doi: 10.1093/pcp/pcm042 URL
[5]	Abe J, Morita S. Growth direction of nodal roots in rice: its variation and contribution to root system formation[J]. Plant Soil, 1994, 165(2): 333-337. doi: 10.1007/BF00008078 URL
[6]	Godbolé R, Michalke W, Nick P, et al. Cytoskeletal drugs and gravity-induced lateral auxin transport in rice coleoptiles[J]. Plant Biol, 2000, 2(2): 176-181. doi: 10.1055/s-2000-9154 URL
[7]	Türkan I, Suge H. Survey of endogenous gibberellins in a barley mutant showing abnormal response to gravity[J]. Jpn J Genet, 1991, 66(1): 41-48. doi: 10.1266/jjg.66.41 URL
[8]	Van Overbeek J. Growth substance curvatures of avena in light and dark[J]. J Gen Physiol, 1936, 20(2): 283-309. doi: 10.1085/jgp.20.2.283 pmid: 19872993
[9]	Sang D, Chen D, Liu G, et al. Strigolactones regulate rice tiller angle by attenuating shoot gravitropism through inhibiting auxin biosynthesis[J]. PNAS, 2014, 111(30): 11199-11204. doi: 10.1073/pnas.1411859111 pmid: 25028496
[10]	Derbyshire P, Byrne ME. MORE SPIKELETS1 is required for spikelet fate in the inflorescence of Brachypodium[J]. Plant Physiol, 2013, 161(3): 1291-1302. doi: 10.1104/pp.112.212340 pmid: 23355632
[11]	Hollender CA, Hill JL Jr, Waite J, et al. Opposing influences of TAC1 and LAZY1 on lateral shoot orientation in Arabidopsis[J]. Sci Rep, 2020, 10: 6051. doi: 10.1038/s41598-020-62962-4 pmid: 32269265
[12]	Dong ZB, Jiang C, Chen XY, et al. Maize LAZY1 mediates shoot gravitropism and inflorescence development through regulating auxin transport, auxin signaling, and light response[J]. Plant Physiol, 2013, 163(3): 1306-1322. doi: 10.1104/pp.113.227314 pmid: 24089437
[13]	Zhang N, Yu H, Yu H, et al. A core regulatory pathway controlling rice tiller angle mediated by the LAZY1-dependent asymmetric distribution of auxin[J]. Plant Cell, 2018, 30(7): 1461-1475. doi: 10.1105/tpc.18.00063 URL
[14]	Gietz RD, Schiestl RH. High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method[J]. Nat Protoc, 2007, 2(1): 31-34. doi: 10.1038/nprot.2007.13 pmid: 17401334
[15]	Zong W, Tang N, Yang J, et al. Feedback regulation of ABA signaling and biosynthesis by a bZIP transcription factor targets drought-resistance-related genes[J]. Plant Physiol, 2016, 171(4): 2810-2825. doi: 10.1104/pp.16.00469 pmid: 27325665
[16]	Akiyama T, Pillai MA, Sentoku N. Cloning, characterization and expression of OsGLN2, a rice endo-1, 3-β-glucanase gene regulated developmentally in flowers and hormonally in germinating seeds[J]. Planta, 2004, 220(1): 129-139. doi: 10.1007/s00425-004-1312-8 URL
[17]	Kashiwagi T, Togawa E, Hirotsu N, et al. Improvement of lodging resistance with QTLs for stem diameter in rice(Oryza sativa L.)[J]. Theor Appl Genet, 2008, 117(5): 749-757. doi: 10.1007/s00122-008-0816-1 pmid: 18575836
[18]	Trivedi DK, Yadav S, Vaid N, et al. Genome wide analysis of Cyclophilin gene family from rice and Arabidopsis and its comparison with yeast[J]. Plant Signal Behav, 2012, 7(12): 1653-1666. doi: 10.4161/psb.22306 pmid: 23073011
[19]	Burd CG, Dreyfuss G. Conserved structures and diversity of functions of RNA-binding proteins[J]. Science, 1994, 265(5172): 615-621. doi: 10.1126/science.8036511 pmid: 8036511
[20]	Yang HJ, Zhou Y, Zhang YN, et al. Identification of transcription factors of nitrate reductase gene promoters and NRE2 cis-element through yeast one-hybrid screening in Nicotiana tabacum[J]. BMC Plant Biol, 2019, 19(1): 1-8. doi: 10.1186/s12870-018-1600-2
[21]	Dong CL, He F, Berkowitz O, et al. Alternative splicing plays a critical role in maintaining mineral nutrient homeostasis in rice(Oryza sativa)[J]. Plant Cell, 2018, 30(10): 2267-2285. doi: 10.1105/tpc.18.00051 URL
[22]	Suzuki N, Sejima H, Tam R, et al. Identification of the MBF₁ heat-response regulon of Arabidopsis thaliana[J]. Plant J, 2011, 66(5): 844-851. doi: 10.1111/j.1365-313X.2011.04550.x URL
[23]	Takemaru KI, Li FQ, Ueda H, et al. Multiprotein bridging factor 1(MBF1)is an evolutionarily conserved transcriptional coactivator that connects a regulatory factor and TATA element-binding protein[J]. Proc Natl Acad Sci USA, 1997, 94(14): 7251-7256. doi: 10.1073/pnas.94.14.7251 pmid: 9207077
[24]	Nobuhiro S, Ludmila R, Liang HJ, et al. Enhanced tolerance to environmental stress in transgenic plants expressing the transcriptional coactivator multiprotein bridging factor 1c[J]. Plant Physiol, 2005, 139(3): 1313-1322. doi: 10.1104/pp.105.070110 pmid: 16244138
[25]	Jaimes-Miranda F, Chávez Montes RA. The plant MBF₁ protein family: a bridge between stress and transcription[J]. J Exp Bot, 2020, 71(6): 1782-1791. doi: 10.1093/jxb/erz525 pmid: 32037452
[26]	Kim MJ, Lim GH, Kim ES, et al. Abiotic and biotic stress tolerance in Arabidopsis overexpressing the Multiprotein bridging factor 1a(MBF1a)transcriptional coactivator gene[J]. Biochem Biophys Res Commun, 2007, 354(2): 440-446. doi: 10.1016/j.bbrc.2006.12.212 URL
[27]	Qin DD, Wang F, Geng XL, et al. Overexpression of heat stress-responsive TaMBF1c, a wheat(Triticum aestivum L.) Multiprotein Bridging Factor, confers heat tolerance in both yeast and rice[J]. Plant Mol Biol, 2015, 87(1-2): 31-45. doi: 10.1007/s11103-014-0259-9 URL

用途Application	引物名称Primer name	序列 Sequences（5'-3'）
启动子扩增	LAZY1-F	ATTACCCACATTCATTTAGTTG
启动子扩增	LAZY1-R	CTTGGAAGCCGGTTTAGCGT
pHIS2-LAZY1的构建	pHIS2-LAZY1-F	GAATTCATTACCCACATTCATTTAGT
pHIS2-LAZY1的构建	pHIS2-LAZY1-R	ACGCGTCTTGGAAGCCGGTTTAGCGT
pAbAi-LAZY1的构建	pAbAi-LAZY1-F	CGAGCTCGTGTAAATTCGCGGTTAATT
pAbAi-LAZY1的构建	pAbAi-LAZY1-R	CCCGGGGTGAGTCGTATTACAATTC
pGADT7-OsMBF1的构建	pGADT7-OsMBF1-F	CGGGATCCGGCCGGGATTGGTCCGATC
pGADT7-OsMBF1的构建	pGADT7-OsMBF1-R	CGAGCTCGCCCCCCCTCGAGTTTCTT
文库检测	T7引物	TAATACGACTCACTATAGGGC
文库检测	AD引物	GTGAACTTGCGGGGTTTTTC
190LUC-LAZY1的构建	LUC -LAZY1-F	TTTGGAGAGGACACGCTGGATCCATTACCCACATTCAT
190LUC-LAZY1的构建	LUC- LAZY1-R	ATAGTAATTGTAATGGATCTGCTTGGAAGCCGGTTTA
none-OsMBF1	OsMBF1-F	ACTAGTGGATCCCCCGGGCTGATGGCCGGGATTGGTCC
none-OsMBF1	OsMBF1-R	GGTACCGGGCCCCCCCTCGAGTTTCTTGCCGCGCAGCT

用途Application	引物名称Primer name	序列 Sequences（5'-3'）
启动子扩增	LAZY1-F	ATTACCCACATTCATTTAGTTG
启动子扩增	LAZY1-R	CTTGGAAGCCGGTTTAGCGT
pHIS2-LAZY1的构建	pHIS2-LAZY1-F	GAATTCATTACCCACATTCATTTAGT
pHIS2-LAZY1的构建	pHIS2-LAZY1-R	ACGCGTCTTGGAAGCCGGTTTAGCGT
pAbAi-LAZY1的构建	pAbAi-LAZY1-F	CGAGCTCGTGTAAATTCGCGGTTAATT
pAbAi-LAZY1的构建	pAbAi-LAZY1-R	CCCGGGGTGAGTCGTATTACAATTC
pGADT7-OsMBF1的构建	pGADT7-OsMBF1-F	CGGGATCCGGCCGGGATTGGTCCGATC
pGADT7-OsMBF1的构建	pGADT7-OsMBF1-R	CGAGCTCGCCCCCCCTCGAGTTTCTT
文库检测	T7引物	TAATACGACTCACTATAGGGC
文库检测	AD引物	GTGAACTTGCGGGGTTTTTC
190LUC-LAZY1的构建	LUC -LAZY1-F	TTTGGAGAGGACACGCTGGATCCATTACCCACATTCAT
190LUC-LAZY1的构建	LUC- LAZY1-R	ATAGTAATTGTAATGGATCTGCTTGGAAGCCGGTTTA
none-OsMBF1	OsMBF1-F	ACTAGTGGATCCCCCGGGCTGATGGCCGGGATTGGTCC
none-OsMBF1	OsMBF1-R	GGTACCGGGCCCCCCCTCGAGTTTCTTGCCGCGCAGCT

名称Name	序列Sequence	功能Functions
TATA-box	TATATA（AA）	-30转录起始结合
CAAT-box	CC（A）AAT/ TGCCAAC	启动子和增强子共同作用的顺式作用元件
ABRE	ACGTG/CACGTG	脱落酸响应
ATCT-motif	AATCTAATCC	光响应元件
Box 4	ATTAAT	光响应元件
TCCC-motif	TCTCCCT	光响应元件
GATA-motif	GATAGGA/ GATAGGG	光响应元件
G-box	CACGAC/ CACGTC/ CACGTG	光响应元件
CAT-box	GCCACT	与分生组织表达相关的顺式作用的调控元件
circadian	CAAAGATATC	光周期调控元件
CGTCA-motif	CGTCA	茉莉酸甲酯响应元件
TGACG-motif	TGACG	茉莉酸甲酯响应元件
MBS	CAACTG	参与干旱诱导的MYB结合位点
MRE	AACCTAA	参与光响应的MYB结合位点
MSA-like	TCAAACGGT	参与细胞周期调节的顺式作用元件
TGA-element	AACGAC	生长素响应元件
MYC	CATGTG	功能未知
WRE3	CCACCT	功能未知
W box	TTGACC	功能未知

名称Name	序列Sequence	功能Functions
TATA-box	TATATA（AA）	-30转录起始结合
CAAT-box	CC（A）AAT/ TGCCAAC	启动子和增强子共同作用的顺式作用元件
ABRE	ACGTG/CACGTG	脱落酸响应
ATCT-motif	AATCTAATCC	光响应元件
Box 4	ATTAAT	光响应元件
TCCC-motif	TCTCCCT	光响应元件
GATA-motif	GATAGGA/ GATAGGG	光响应元件
G-box	CACGAC/ CACGTC/ CACGTG	光响应元件
CAT-box	GCCACT	与分生组织表达相关的顺式作用的调控元件
circadian	CAAAGATATC	光周期调控元件
CGTCA-motif	CGTCA	茉莉酸甲酯响应元件
TGACG-motif	TGACG	茉莉酸甲酯响应元件
MBS	CAACTG	参与干旱诱导的MYB结合位点
MRE	AACCTAA	参与光响应的MYB结合位点
MSA-like	TCAAACGGT	参与细胞周期调节的顺式作用元件
TGA-element	AACGAC	生长素响应元件
MYC	CATGTG	功能未知
WRE3	CCACCT	功能未知
W box	TTGACC	功能未知

基因ID Gene ID	功能注释 Functional annotation	基因名称 Gene name
LOC_Os11g07020	果糖-二磷酸醛缩酶，参与糖酵解和糖异生的生物学过程	--
LOC_Os01g54940	线粒体互作蛋白，具有脱氢酶作用	--
LOC_Os02g55140	亮氨酸氨基肽酶，参与细胞内蛋白质的加工和正常转运	--
LOC_Os01g71670	内切1,3-β-葡聚糖酶，调节发芽种子的发育和激素	OsGLN2^[16]
LOC_Os12g19470	单加氧酶和二磷酸核酮糖羧化酶活性	OsRBCS4^[17]
LOC_Os11g44810	生长素抑制蛋白，影响腋芽的生长和分支	--
LOC_Os02g15310	富含丝氨酸/精氨酸的SC35类剪接因子	--
LOC_Os07g05360	光合系统蛋白pH依赖的PSII稳定蛋白	--
LOC_Os04g20990	催化还原tRNA上尿苷残基的5,6-双键	--
LOC_Os03g59310	40S核糖体S2，催化mRNA定向蛋白质合成	--
LOC_Os12g08260	含有脱氢酶E1结构域的蛋白，2-氧异戊酸脱氢酶	--
LOC_Os01g05670	脂质代谢过程中烯酰辅酶还原酶	--
LOC_Os03g59320	具有伴侣蛋白类功能	--
LOC_Os04g41620	几丁质家族蛋白质前体，催化几丁质聚合物中的水解	--
LOC_Os05g01270	细胞色素，增强多种非生物胁迫耐受性	OsCYP20-2^[18]
LOC_Os08g33820	颗粒膜的黏附和磷酸化的光调控	--
LOC_Os01g41710	颗粒膜的黏附和磷酸化的光调控	--
LOC_Os12g07210	果糖-二磷酸醛缩酶，在糖酵解和糖异生中起关键作用	--
LOC_Os08g27850	连接激素受体和TATA元素结合蛋白MBF1	--
LOC_Os04g53620	通过其翻译后附着（泛素化）作用于其他蛋白质	--
LOC_Os01g69950	核糖体蛋白L2，催化mRNA导向的蛋白质合成	--