石仙桃中4种转录因子鉴定及其组织表达的研究

doi:10.13560/j.cnki.biotech.bull.1985.2023-1096

摘要/Abstract

摘要：

【目的】石仙桃Pholidota chinensis Lindl为珍稀濒危的附生兰，附生较地生而言进化出了许多特殊的形态结构和生理生化特性，而转录因子对调控植物的生长发育具有作用，旨在探讨和发掘石仙桃中调控其生长发育的转录因子。【方法】利用PacBio和Illumina测序技术获得石仙桃的全长转录组数据以及在根、根状茎、假鳞茎和叶片中的基因表达量，通过进一步生信分析，探讨石仙桃4种转录因子家族成员、理化特性及其组织表达特异性，并用氯化钠（NaCl）和茉莉酸甲酯（MeJA）诱导逆境胁迫，用RT-qPCR分析了C2H2家族和bHLH家族的表达量。【结果】石仙桃中鉴定的4种转录因子及其家族成员转录本为：C2H2家族21个、bHLH家族27个、WRKY家族30个、bZIP家族19个，这4个家族及其成员主要为亲水的不稳定性蛋白，亚细胞定位主要位于细胞核内，bHLH家族的部分成员位于细胞质、叶绿体等细胞器中，各基因家族均具有典型的保守结构域特征，在石仙桃的根、根状茎、假鳞茎和叶片呈现出明显的组织特异性。21个C2H2家族和27个bHLH家族中所有基因表达均受NaCl和MeJA影响，但家族成员受到促进或抑制表达及其表达量存在差异。【结论】鉴定了石仙桃中4个转录因子家族成员的数量，各家族成员主要是位于细胞核内的亲水不稳定性蛋白，并具有保守结构域特征和组织表达特异性。

关键词: 石仙桃, C2H2, bHLH, WRKY, bZIP, 理化特性, 组织表达

Abstract:

【Objective】Pholidota chinensis Lindl is a rare and endangered epiphytic orchid. The epiphytes have evolved many special morphological structures and physiological and biochemical characteristics compared to geophytes. The transcription factors play a role in regulating plant growth and development. The aim of this paper is to explore and discover transcription factors in P. chinensis that regulate its growth and development.【Method】This study used PacBio and Illumina sequencing techniques to obtain the full-length transcriptome data of P. chinensis and the gene expressions in the roots, rhizomes, pseudobulbs and leaves. After further bioinformatics analysis, the four transcription factor family members, physicochemical properties and tissue expression specificity of P. chinensis were revealed. The expression of C2H2 family and bHLH family was analyzed by RT-qPCR using sodium chloride（NaCl）and methyl jasmonate（MeJA）induced adversity stresses.【Result】The four identified transcription factors and their families members in P. chinensis were: 21 members in C2H2 family, 27 members in bHLH family, 30 members in WRKY family, and 19 members in bZIP family. These four families and their members were mainly hydrophilic unstable proteins, and their subcellular localization was mainly located in the nucleus. Some members of bHLH families were located in cytoplasm, chloroplast and other organelles. The gene family had typical conserved domain characteristics, and showed obvious tissue specificity in the roots, rhizomes, pseudobulbs and leaves of P. chinensis. Expressions of all genes in C2H2 families and bHLH families were affected by NaCl and MeJA, but family members differed in whether they were promoted or repressed in the expression and in the amount of their expression.【Conclusion】The number of four transcription factor family members in P. chinensis was identified, and the members of each family are mainly hydrophilic unstable proteins located in the nucleus with conserved structural domain features and tissue expression specificity.

Key words: Pholidota chinensis, C2H2, bHLH, WRKY, bZIP, physical and chemical properties, tissue expression

刘保财, 张武君, 赵云青, 黄颖桢, 陈菁瑛. 石仙桃中4种转录因子鉴定及其组织表达的研究[J]. 生物技术通报, 2024, 40(5): 141-152.

LIU Bao-cai, ZHANG Wu-jun, ZHAO Yun-qing, HUANG Ying-zhen, CHEN Jing-ying. Identification and Tissue Expression of Four Transcription Factors in Pholidota chinensis[J]. Biotechnology Bulletin, 2024, 40(5): 141-152.

图/表 6

图1 数量前30的转录因子家族

Fig. 1 Number of top 30 transcription factor families

图2 石仙桃中C2H2基因家族特征 A: C2H2家族保守结构域; B: C2H2家族不同组织表达差异; C: C2H2家族进化

Fig. 2 Characteristics of C2H2 gene family in P. chinensis A: Conserved motifs in C2H2 family; B: differential expressions of C2H2 family in different tissues; C: C2H2 family evolution

图3 石仙桃中bHLH基因家族特征 A: bHLH家族保守结构域；B: bHLH家族不同组织表达差异；C: bHLH家族进化

Fig. 3 Characteristics of bHLH gene family in P. chinensis A: Conserved motifs in bHLH family. B: Differential expressions of bHLH family in different tissues. C: bHLH family evolution

图4 石仙桃中WRKY基因家族特征 A: WRKY家族保守结构域; B: WRKY家族不同组织表达差异; C: WRKY家族进化

Fig. 4 Characteristics of WRKY gene family in P. chinensis A: Conserved motifs of WRKY family. B: WRKY family evolution. C: Differential expressions of WRKY family in different tissues

图5 石仙桃中bZIP基因家族特征 A: bZIP家族保守结构域; B: bZIP家族进化; C: bZIP家族不同组织表达差异

Fig. 5 Characteristics of bZIP gene family in P. chinensis A: Conserved motifs of bZIP family; B: bZIP family evolution; C: differential expressions of bZIP family in different tissues

图6 石仙桃中C2H2（A）和bHLH（B）家族在NaCl和MeJA胁迫下表达分析

Fig. 6 Expression analysis of C2H2 (A) and bHLH (B) families in P. chinensis under NaCl and MeJA stresses

参考文献 39

[1]	Liu BC, Chen JY, Zhang WJ, et al. The gastrodin biosynthetic pathway in Pholidota chinensis Lindl. revealed by transcriptome and metabolome profiling[J]. Front Plant Sci, 2022, 13: 1024239.
[2]	刘保财, 黄颖桢, 赵云青, 等. 野生石仙桃立地环境与生长特性研究[J]. 福建农业学报, 2017, 32(8): 864-869.
	Liu BC, Huang YZ, Zhao YQ, et al. Natural habitat and growth characteristics of wild Pholidota chinensis lindl[J]. Fujian J Agric Sci, 2017, 32(8): 864-869.
[3]	Wang DL, Lin FX, Tang MM, et al. Two new phenanthrene derivatives from Pholidota chinensis[J]. Phytochem Lett, 2021, 43: 123-125.
[4]	Li DL, Zheng XL, Duan L, et al. Ethnobotanical survey of herbal tea plants from the traditional markets in Chaoshan, China[J]. J Ethnopharmacol, 2017, 205: 195-206.
[5]	Ti HH, Zhuang ZX, Li Y, et al. Three new phenanthrenes from Pholidota chinensis Lindl. and their antibacterial activity[J]. Nat Prod Res, 2022, 36(8): 2056-2062.
[6]	袁佳, 张苑雯, 遆慧慧, 等. 石仙桃化学成分及其抗菌抗炎活性研究[J]. 广东药科大学学报, 2021, 37(4): 1-7.
	Yuan J, Zhang YW, Ti HH, et al. Study on the chemical constituents, antimicrobial and anti-inflammatory activities of Pholidota chinensis Lindl[J]. J Guangdong Pharm Univ, 2021, 37(4): 1-7.
[7]	Luo DH, Wang ZJ, Li ZM, et al. Structure of an entangled heteropolysaccharide from Pholidota chinensis Lindl and its antioxidant and anti-cancer properties[J]. Int J Biol Macromol, 2018, 112: 921-928.
[8]	Xu M, Wang AD. Recent advances in the regulation of climacteric fruit ripening: hormone, transcription factor and epigenetic modifications[J]. Front Agric Sci Eng, 2021, 8(2): 314-334. doi: 10.15302/J-FASE-2021386
[9]	Hong JC. General aspects of plant transcription factor families[M]// Plant Transcription Factors. Amsterdam: Elsevier, 2016: 35-56.
[10]	Wang C, Hao XL, Wang Y, et al. Identification of WRKY transcription factors involved in regulating the biosynthesis of the anti-cancer drug camptothecin in Ophiorrhiza pumila[J]. Hortic Res, 2022, 9: uhac099.
[11]	Yang LY, Miao LY, Gong Q, et al. Advances in studies on transcription factors in regulation of secondary metabolites in Chinese medicinal plants[J]. Plant Cell Tissue Organ Cult PCTOC, 2022, 151(1): 1-9.
[12]	刘保财, 胡学博, 张武君, 等. 石仙桃与细叶石仙桃叶绿体基因组解析及其系统发育[J]. 草业学报, 2024, 33(4): 171-185. doi: 10.11686/cyxb2023192
	Liu BC, Hu XB, Zhang WJ, et al. Chloroplast genome assembly and phylogenetic analysis of Pholidota chinensis and Pholidota cantonensis[J]. Acta Prataculturae Sinica, 2024, 33(4): 171-185.
[13]	Zheng Y, Jiao C, Sun HH, et al. iTAK: a program for genome-wide prediction and classification of plant transcription factors, transcriptional regulators, and protein kinases[J]. Mol Plant, 2016, 9(12): 1667-1670. doi: S1674-2052(16)30223-4 pmid: 27717919
[14]	刘美琦, 孙伟, 孟祥霄, 等. 药用植物大麻C2H2基因家族鉴定与表达分析[J]. 药学学报, 2021, 56(5): 1486-1496.
	Liu MQ, Sun W, Meng XX, et al. Identification and expression analysis of the C2H2 gene family in Cannabis sativa L[J]. Acta Pharm Sin, 2021, 56(5): 1486-1496.
[15]	陈凤琼, 陈秋森, 林佳昕, 等. 番茄DIR基因家族鉴定及其对非生物胁迫响应的分析[J]. 中国农业科学, 2022, 55(19): 3807-3824. doi: 10.3864/j.issn.0578-1752.2022.19.010
	Chen FQ, Chen QS, Lin JX, et al. Genome-wide identification of DIR family genes in tomato and response to abiotic stress[J]. Sci Agric Sin, 2022, 55(19): 3807-3824.
[16]	赵玎玲, 王梦璇, 孙天杰, 等. 大豆单锌指蛋白基因GmSZFP的克隆及其在SMV与寄主互作中的功能[J]. 中国农业科学, 2022, 55(14): 2685-2695. doi: 10.3864/j.issn.0578-1752.2022.14.001
	Zhao DL, Wang MX, Sun TJ, et al. Cloning of the soybean single zinc finger protein gene GmSZFP and its functional analysis in SMV-host interactions[J]. Sci Agric Sin, 2022, 55(14): 2685-2695.
[17]	赵霞, 李元敏, 李依民, 等. 基于全长转录组测序的掌叶大黄R1-MYB基因家族鉴定分析[J]. 药学学报, 2023, 58(5): 1354-1363.
	Zhao X, Li YM, Li YM, et al. Identification and analysis of R1-MYB gene family in Rheum palmatum L. based on full-length transcriptome sequencing[J]. Acta Pharm Sin, 2023, 58(5): 1354-1363.
[18]	Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method[J]. Methods, 2001, 25(4): 402-408. doi: 10.1006/meth.2001.1262 pmid: 11846609
[19]	钟婵娟, 彭伟业, 王冰, 等. 植物逆境响应相关的C2H2型锌指蛋白研究进展[J]. 植物生理学报, 2020, 56(11): 2356-2366.
	Zhong CJ, Peng WY, Wang B, et al. Advances of plant C2H2 zinc finger proteins in response to stresses[J]. Plant Physiol J, 2020, 56(11): 2356-2366.
[20]	Bai QX, Niu ZM, Chen QY, et al. The C2H2-type zinc finger transcription factor OSIC1 positively regulates stomatal closure under osmotic stress in poplar[J]. Plant Biotechnol J, 2023, 21(5): 943-960.
[21]	Feng Y, Zhang SR, Li J, et al. Dual-function C2H2-type zinc-finger transcription factor GmZFP7 contributes to isoflavone accumulation in soybean[J]. New Phytol, 2023, 237(5): 1794-1809.
[22]	Mohammad, Hurrah IM, Kumar A, et al. Synergistic interaction of two bHLH transcription factors positively regulates artemisinin biosynthetic pathway in Artemisia annua L[J]. Physiol Plant, 2023, 175(1): e13849.
[23]	Wang WY, Yu JQ, Du MC, et al. Basic helix-loop-helix(bHLH)transcription factor MdbHLH3 negatively affects the storage performance of postharvest apple fruit[J]. Hortic Plant J, 2022, 8(6): 700-712.
[24]	田韦韦, 严志祥, 王成, 等. 基于全长转录组测序的多花黄精WRKY转录因子家族分析[J]. 中国中药杂志, 2023, 48(4): 939-950.
	Tian WW, Yan ZX, Wang C, et al. Analysis of WRKY transcription factor family based on full-length transcriptome sequencing in Polygonatum cyrtonema[J]. China J Chin Mater Med, 2023, 48(4): 939-950.
[25]	叶炜, 王培育, 颜沛沛, 等. 文心兰WRKY转录因子在印度梨形孢诱导的抗软腐病作用中的响应分析[J]. 植物生理学报, 2023, 59(1): 113-126.
	Ye W, Wang PY, Yan PP, et al. Response analysis of WRKY transcription factors in resistance to soft rot induced by Piriformospora indica in Oncidium[J]. Plant Physiol J, 2023, 59(1): 113-126.
[26]	Ren LP, Wan WY, Yin DD, et al. Genome-wide analysis of WRKY transcription factor genes in Toona sinensis: an insight into evolutionary characteristics and terpene synthesis[J]. Front Plant Sci, 2023, 13: 1063850.
[27]	崔荣秀, 张议文, 陈晓倩, 等. 植物bZIP参与胁迫应答调控的最新研究进展[J]. 生物技术通报, 2019, 35(2): 143-155. doi: 10.13560/j.cnki.biotech.bull.1985.2018-0562
	Cui RX, Zhang YW, Chen XQ, et al. The latest research progress on the stress responses of bZIP involved in plants[J]. Biotechnol Bull, 2019, 35(2): 143-155.
[28]	Niu SK, Gu XY, Zhang Q, et al. Grapevine bZIP transcription factor bZIP45 regulates VvANN1 and confers drought tolerance in Arabidopsis[J]. Front Plant Sci, 2023, 14: 1128002.
[29]	刘保财, 陈菁瑛, 黄颖桢, 等. 石仙桃人工栽培技术[J]. 福建农业科技, 2017(12): 40-42.
	Liu BC, Chen JY, Huang YZ, et al. Artificial cultivation techniques of Pholidota chinensis Lindl[J]. Fujian Agric Sci Technol, 2017(12): 40-42.
[30]	张桐, 李智强, 伍国强. WRKY转录因子在植物逆境响应中的作用[J]. 生物技术通报, 2021, 37(10): 203-215. doi: 10.13560/j.cnki.biotech.bull.1985.2020-1481
	Zhang T, Li ZQ, Wu GQ. Role of WRKY transcription factor in plant response to stresses[J]. Biotechnol Bull, 2021, 37(10): 203-215. doi: 10.13560/j.cnki.biotech.bull.1985.2020-1481
[31]	Kumar R, Das S, Mishra M, et al. Emerging roles of NAC transcription factor in medicinal plants: progress and prospects[J]. 3 Biotech, 2021, 11(10): 425. doi: 10.1007/s13205-021-02970-x pmid: 34567930
[32]	Li C, Gong QQ, Liu P, et al. Co-expressed network analysis based on 289 transcriptome samples reveals methyl jasmonate-mediated gene regulatory mechanism of flavonoid compounds in Dendrobium catenatum[J]. Plant Physiol Biochem, 2024, 206: 108226.
[33]	Zeng X, Ling H, Chen XM, et al. Genome-wide identification, phylogeny and function analysis of GRAS gene family in Dendrobium catenatum(Orchidaceae)[J]. Gene, 2019, 705: 5-15. doi: S0378-1119(19)30391-9 pmid: 30999026
[34]	Wang MJ, Ou Y, Li Z, et al. Genome-wide identification and analysis of bHLH transcription factors related to anthocyanin biosynthesis in Cymbidium ensifolium[J]. Int J Mol Sci, 2023, 24(4): 3825.
[35]	Yang K, Hou YB, Wu M, et al. DoMYB5 and DobHLH24, transcription factors involved in regulating anthocyanin accumulation in Dendrobium officinale[J]. Int J Mol Sci, 2023, 24(8): 7552.
[36]	Chuang YC, Hung YC, Tsai WC, et al. PbbHLH4 regulates floral monoterpene biosynthesis in Phalaenopsis orchids[J]. J Exp Bot, 2018, 69(18): 4363-4377.
[37]	Hsieh MH, Pan ZJ, Lai PH, et al. Virus-induced gene silencing unravels multiple transcription factors involved in floral growth and development in Phalaenopsis orchids[J]. J Exp Bot, 2013, 64(12): 3869-3884.
[38]	邢丙聪, 苏立样, 万思琦, 等. 金线莲中调控胚胎发育WRKY转录因子筛选及克隆分析[J]. 中草药, 2022, 53(12): 3745-3754.
	Xing BC, Su LY, Wan SQ, et al. Screening and cloning analysis of WRKY transcription factor regulating embryo development from Anoectochilus roxburghii[J]. Chin Tradit Herb Drugs, 2022, 53(12): 3745-3754.
[39]	Zeng DQ, Teixeira da Silva JA, Zhang MZ, et al. Genome-wide identification and analysis of the APETALA2(AP2)transcription factor in Dendrobium officinale[J]. Int J Mol Sci, 2021, 22(10): 5221.