Construction of Yeast One-hybrid Bait Vector of Tobacco NtCBT Gene Promoter and Screening of Interacted Proteins

doi:10.13560/j.cnki.biotech.bull.1985.2021-1597

Abstract

Abstract:

To screen the upstream regulatory transcription factors of cembratrienol synthase（NtCBT）in the cembranoids synthesis pathway of tobacco（Nicotiana tabacum L.），the promoter of NtCBT gene was cut into P1 to P6 regions，six yeast one-hybrid bait vectors pAbAi-Px were constructed and transformed into Y1H competent yeast cells，bait strains were constructed，and a self-activation experiment was completed. Furthermore，a screening was finished from yeast cDNA library of tobacco trichomes in order to obtain the transcription factors that interacted with P5 regions（-279 - -119 bp）. The results showed that，the growths of the strains containing P1 to P5 regions bait strain were inhibited on the medium added with 200 ng/mL AbA. A total of 49 positive colonies were obtained through Y1H screening when P5 regions as bait strain，among them，35 colonies were non-repeating sequences，and 3 of colonies were annotated as transcription factors of ANL2，ML1 and NF-Y. Above results lay a foundation for further study of gene expression and regulation mechanism of NtCBT.

Key words: yeast one-hybrid, transcription factors, Nicotiana tabacum L., promoter, NtCBT gene, cembranoids

YU Jing, YANG Hui, YU Shi-zhou, ZHAO Hui-na, ZHENG Qing-xia, WANG Bing, LEI Bo. Construction of Yeast One-hybrid Bait Vector of Tobacco NtCBT Gene Promoter and Screening of Interacted Proteins[J]. Biotechnology Bulletin, 2022, 38(10): 73-79.

Figures/Tables 8

Fig.1 Linearization of recombinant bait vector pAbAi-Px M：Marker. Lane 1，3，5，7，9，and 11：Undigested plasmid pAbAi-P1，pAbAi-P2，pAbAi-P3，pAbAi-P4，pAbAi-P5 and pAbAi-P6，respectively. Lane 2，4，6，8，10，and 12：Plasmid pAbAi-P1，pAbAi-P2，pAbAi-P3，pAbAi-P4，pAbAi-P5 and pAbAi-P6，digested with BstB I restricted enzyme，respectively

Fig.2 Self-activation of pAbAi-Px bait vector A-E：Yeast transformants of Y1H[pAbAi-P1]，Y1H[pAbAi-P2]，Y1H[pAbAi-P3]，Y1H[pAbAi-P4] and Y1H[pAbAi-P5]in SD/-Ura medium，respectively. F-J：Yeast transformants of Y1H[pAbAi-P1]，Y1H[pAbAi-P2]，Y1H[pAbAi-P3] ，Y1H[pAbAi-P4] and Y1H[pAbAi-P5]in SD/-Ura/AbA（200ng/mL）medium，respectively

Fig.3 Self-activation of pAbAi-P6 bait vector Yeast transformants of Y1H[pAbAi-P6]in A：SD/-Ura，B：SD/-Ura/AbA（200 ng/mL），C：SD/-Ura/AbA（500 ng/mL），D：SD/-Ura/AbA（800 ng/mL）and E：SD/-Ura/AbA（1 000 ng/mL）medium，respectively

Fig.4 Schematic diagram of P1-P6 regions of NtCBT promoter and prediction of transcription factor binding motif in the P5 region Downward arrow indicates the transcription start site

Fig.5 Identification of Y1H[pAbAi -P5]yeast colonies by PCR M：Marker. Lane 1 and 2：PCR-amplified products

Fig.6 Preliminary screening of binding protein interacting with P5 regions of NtCBT promoter A and B：Partial colonies during the preliminary screening

Fig.7 Secondary screening of binding protein interacting with P5 regions of NtCBT promoter 1#：ANL2；41#：NF-YA3；44#：ML1 1-50：Colonies of 1#-50# yeasts. +：Positive control. -：Negative control

Fig.8 Identification of partial positive colonies by PCR M：DL2000 DNA marker. Lane 1-24：PCR-amplified products

References 24

[1]	Tian J, Peng Z, Zhang J, et al. McMYB10 regulates coloration via activating McF3’H and later structural genes in ever-red leaf crabapple[J]. Plant Biotechnol J, 2015, 13(7):948-961. doi: 10.1111/pbi.12331 pmid: 25641214
[2]	靳进朴, 郭安源, 何坤, 等. 植物转录因子分类、预测和数据库构建[J]. 生物技术通报, 2015, 31(11):68-77. doi: 10.13560/j.cnki.biotech.bull.1985.2015.11.004
	Jin JP, Guo AY, He K, et al. Classification, prediction and database construction of plant transcription factors[J]. Biotechnol Bull, 2015, 31(11):68-77. doi: 10.13560/j.cnki.biotech.bull.1985.2015.11.004
[3]	李圣彦, 郎志宏, 黄大昉. 真核生物启动子研究概述[J]. 生物技术进展, 2014, 4(3):158-164.
	Li SY, Lang ZH, Huang DF. Research progress on eukaryotic promoter[J]. Curr Biotechnol, 2014, 4(3):158-164.
[4]	王冬, 张小全, 杨铁钊, 等. 类西柏烷二萜代谢机理及调控研究进展[J]. 中国烟草学报, 2014, 20(3):113-118.
	Wang D, Zhang XQ, Yang TZ, et al. Research progress on metabolic mechanism of cembranoid diterpenes and its regulation[J]. Acta Tabacaria Sin, 2014, 20(3):113-118.
[5]	Yan N, Du YM, Liu XM, et al. A review on bioactivities of tobacco cembranoid diterpenes[J]. Biomolecules, 2019, 9(1):30. doi: 10.3390/biom9010030 URL
[6]	Yan N, Du YM, Liu XM, et al. Chemical structures, biosynthesis, bioactivities, biocatalysis and semisynthesis of tobacco cembranoids:an overview[J]. Ind Crops Prod, 2016, 83:66-80. doi: 10.1016/j.indcrop.2015.12.031 URL
[7]	张谦, 薛宇, 贺凌霄, 等. 密度、施氮量和留叶数对渠首1号香气特性彰显的影响[J]. 生物技术通报, 2021, 37(6):24-35. doi: 10.13560/j.cnki.biotech.bull.1985.2020-1245
	Zhang Q, Xue Y, He LX, et al. Effects of planting density, amount of nitrogen application and left leaf number on the highlighting of flavor style in qushou 1[J]. Biotechnol Bull, 2021, 37(6):24-35.
[8]	Guo ZH, Wagner GJ. Biosynthesis of cembratrienols in cell-free extracts from trichomes of Nicotiana tabacum[J]. Plant Sci, 1995, 110(1):1-10. doi: 10.1016/0168-9452(95)04174-S URL
[9]	Ennajdaoui H, Vachon G, Giacalone C, et al. Trichome specific expression of the tobacco(Nicotiana sylvestris)cembratrien-ol synthase genes is controlled by both activating and repressing cis-regions[J]. Plant Mol Biol, 2010, 73(6):673-685. doi: 10.1007/s11103-010-9648-x pmid: 20495852
[10]	Wang EM, Gan SS, Wagner GJ. Isolation and characterization of the CYP71D16 trichome-specific promoter from Nicotiana tabacum L[J]. J Exp Bot, 2002, 53(376):1891-1897. doi: 10.1093/jxb/erf054 URL
[11]	王琪, 朱延明, 王冬冬. 酵母单杂交系统在植物基因工程研究中的应用[J]. 北京林业大学学报, 2008, 30(1):141-147.
	Wang Q, Zhu YM, Wang DD. Application of yeast one hybrid system in plant gene engineering[J]. J Beijing For Univ, 2008, 30(1):141-147.
[12]	郇蕾, 王旭旭, 等. 桃ABA信号关键基因PpABI5酵母单杂交文库构建及其上游转录因子的筛选[J]. 植物生理学报, 2017, 53(7):1259-1266.
	Huan L, Wang XX, et al. Constructing yeast one-hybrid library and screening the potential regulator of PpABI5 in peach(Prunus persica)[J]. Plant Physiol J, 2017, 53(7):1259-1266.
[13]	廖文彬, 李雅韵, 等. 木薯干旱胁迫下离区发育相关基因MeAP2-2酵母单杂交文库构建及其上游调控基因的筛选[J]. 中国农学通报, 2015, 31(9):119-127.
	Liao WB, Li YY, et al. Yeast one-hybrid library construction and upstream gene analysis of MeAP2-2 in cassava under drought condition[J]. Chin Agric Sci Bull, 2015, 31(9):119-127.
[14]	余玉珍, 等. 烟草尼古丁去甲基酶基因CYP82E4启动子结合蛋白的鉴定与分析[J]. 云南大学学报:自然科学版, 2015, 37(1):140-146.
	Yu YZ, et al. Identification and analysis of regulatory proteins bin-ding to the promoter of nicotine demethylase gene CYP82E4 in tobacco[J]. J Yunnan Univ Nat Sci Ed, 2015, 37(1):140-146.
[15]	宋国琦, 王兴军, 赵传志, 等. 胚柄特异性表达顺式元件结合蛋白酵母单杂交文库的构建[J]. 农业生物技术学报, 2011, 19(5):953-959.
	Song GQ, Wang XJ, Zhao CZ, et al. Construction of yeast one-hybrid library for screening suspensor specific cis-element binding protein genes[J]. J Agric Biotechnol, 2011, 19(5):953-959.
[16]	Spyropoulou EA, Haring MA, Schuurink RC. Expression of Terpenoids 1, a glandular trichome-specific transcription factor from tomato that activates the terpene synthase 5 promoter[J]. Plant Mol Biol, 2014, 84(3):345-357. doi: 10.1007/s11103-013-0142-0 pmid: 24142382
[17]	余婧, 等. 烟草NtCBT基因启动子克隆及腺毛特异表达区域缺失分析[J]. 植物生理学报, 2020, 56(5):1006-1014.
	Yu J, et al. Cloning of NtCBT gene promoter and deletion analysis of glandular trichomes specific expression in Nicotiana tabacum[J]. Plant Physiol J, 2020, 56(5):1006-1014.
[18]	Kubo H, et al. ANTHOCYANINLESS2, a homeobox gene affecting anthocyanin distribution and root development in Arabidopsis[J]. Plant Cell, 1999, 11(7):1217-1226. pmid: 10402424
[19]	Nadakuduti SS, Pollard M, Kosma DK, et al. Pleiotropic phenotypes of the sticky peel mutant provide new insight into the role of CUTIN DEFICIENT2 in epidermal cell function in tomato[J]. Plant Physiol, 2012, 159(3):945-960. doi: 10.1104/pp.112.198374 pmid: 22623518
[20]	Zhang F, Zuo KJ, Zhang JQ, et al. An L1 box binding protein, GbML1, interacts with GbMYB25 to control cotton fibre development[J]. J Exp Bot, 2010, 61(13):3599-3613. doi: 10.1093/jxb/erq173 pmid: 20667961
[21]	丁慧霞, 刘凤, 等. 植物中NF-Y转录因子的结构和功能研究进展[J]. 分子植物育种, 2017, 15(5):1691-1701.
	Ding HX, Liu F, et al. The structure and function of NF-Y in plants[J]. Mol Plant Breed, 2017, 15(5):1691-1701.
[22]	Siriwardana CL, Gnesutta N, Kumimoto RW, et al. NUCLEAR FACTOR Y, subunit A(NF-YA)proteins positively regulate flowering and act through FLOWERING LOCUS T[J]. PLoS Genet, 2016, 12(12):e1006496. doi: 10.1371/journal.pgen.1006496 URL
[23]	Li S, Li K, et al. Genome-wide analysis of tomato NF-Y factors and their role in fruit ripening[J]. BMC Genomics, 2016, 17:36. doi: 10.1186/s12864-015-2334-2 pmid: 26742635
[24]	Ren C, Zhang Z, Wang Y, et al. Genome-wide identification and characterization of the NF-Y gene family in grape(vitis vinifera L.)[J]. BMC Genomics, 2016, 17(1):605. doi: 10.1186/s12864-016-2989-3 URL