Identification and Expression Analysis of HD‑ZIP Family in Salvia miltiorrhiza

doi:10.15933/j.cnki.1004-3268.2024.07.008

Abstract

Abstract: To screen HD‑ZIP genes（SmHD‑ZIPs）in Salvia miltiorrhiza in response to high temperature stress，SmHD‑ZIPs were identified and analyzed by bioinformatics，and the expression pattern of SmHD‑ZIPs under high temperature stress was detected by qPCR. The results showed that there were 44 HD‑ZIP genes in S.miltiorrhiza，most of which were unevenly distributed on 8 chromosomes. SmHD‑ZIPs could be divided into four subfamilies：HD‑ZIP Ⅰ，HD‑ZIP Ⅱ，HD‑ZIP Ⅲ，HD‑ZIP Ⅳ. The sequence analysis results showed that the amino acid residues of these proteins ranged from 180 to 982，and the relative molecular weight of these proteins ranged from 20. 947 ku to 109. 620 ku. SmHD‑ZIPs were hydrophilic proteins without transmembrane domain，and most of them did not contain signal peptides.The isoelectric points of SmHD‑ZIPs were between 4. 48 and 10. 91，and they were almost expressed in the nucleus with the poor protein stability. Among the 44 SmHD‑ZIP genes，there were 10 gene duplication events，all of which were purified selection. The results of structure and motif analysis showed that the exon number of members of the same subfamily was basically the same，and the members of HD‑ZIP Ⅲ subfamily had the largest exon number and the largest motif number. Motifs 1 and 6 were conserved motifs of SmHD‑ZIP，motifs 10，11，12，13 and 15 was unique to the HD‑ZIP Ⅲ subfamily，while motif 5 were unique to the HD‑ZIP IV subfamily. By analyzing the expression patterns of SmHD‑ZIPs under high temperature（37℃）stress，it was found that the number of genes with increased and decreased expression was basically the same. Among the genes with up‑regulated expression，the expression levels of SmHD‑ZIP1.11，SmHD‑ZIP1.13，SmHD‑ZIP2.2，SmHD‑ZIP2.5，SmHD‑ZIP3.1，SmHD‑ZIP3.4，SmHD‑ZIP4.9，SmHD‑ZIP4.10 and SmHD‑ZIP4.12 were increased by more than 10 times，which could be used as candidate gene resources to improve the heat resistance of S.miltiorrhiza.

Key words: Salvia miltiorrhiza, HD?ZIP family, Phylogenetic tree, Quantitative PCR, High temperature stress

摘要： 为筛选丹参（Salvia miltiorrhiza）中响应高温胁迫的HD-ZIP基因，利用生物信息学研究方法，对丹参全基因组的HD-ZIP基因进行了鉴定和分析，并以天丹1号为材料，通过qPCR技术检测了丹参HDZIP基因在高温胁迫下的表达模式。结果表明，丹参全基因组中包含44个HD-ZIP基因（SmHD-ZIP），不均匀地分布于8条染色体上。SmHD-ZIP可分为HD-ZIP Ⅰ、HD-ZIP Ⅱ、HD-ZIP Ⅲ和HD-ZIP Ⅳ 4个亚家族，序列分析结果表明，这些蛋白质的氨基酸残基数为180~982 个，相对分子质量为20.947~109.620 ku。SmHD-ZIP均为亲水蛋白，无跨膜结构域，绝大多数不含信号肽，等电点为4.48~10.91，且几乎都在细胞核表达，蛋白质稳定性较差。44个SmHD-ZIP 基因中有10个基因复制事件，均为纯化选择。结构和模体分析结果表明，同一亚家族成员的外显子数目基本一致，HD-ZIP Ⅲ亚家族成员的外显子数目最多，模体数也最多。模体1、6是SmHD-ZIP的保守模体，模体10、11、12、13、15为HD-ZIP Ⅲ亚家族特有，模体5为HD-ZIP Ⅳ亚家族特有。通过分析高温（37 ℃）胁迫下SmHD-ZIP 的表达模式发现，表达量升高和下降的基因数基本一致，在上调表达的基因中，SmHD-ZIP1.11、SmHD-ZIP1.13、SmHD-ZIP2.2、SmHD-ZIP2.5、SmHD-ZIP3.1、SmHD-ZIP3.4、SmHD-ZIP4.9、SmHD-ZIP4.10 和SmHD-ZIP4.12 的表达量提高了10倍以上，可作为提高丹参耐热性的候选基因资源。

关键词: 丹参, HD-ZIP家族, 系统进化树, qPCR, 高温胁迫

CLC Number:

S567.23

LI Wan, CHENG Ruixing, DANG Xin. Identification and Expression Analysis of HD‑ZIP Family in Salvia miltiorrhiza[J]. Journal of Henan Agricultural Sciences, 2024, 53(7): 66-78.

李万, 成瑞星, 党鑫. 丹参HD-ZIP 基因家族的鉴定与表达分析[J]. 河南农业科学, 2024, 53(7): 66-78.

References

［1］HU W，HUANG C，DENG X M，et al.TaASR1，a transcription factor gene in wheat，confers drought stress tolerance in transgenic tobacco［J］.Plant，Cell & Environment，2013，36（8）：1449‑1464.
［2］FAN W，HAI M R，GUO Y L，et al.The ERF transcription factor family in cassava：Genome‑wide characterization and expression analyses against drought stress［J］.Scientific Reports，2016，6：37379.
［3］XU Y，HU W，SONG S，et al.MaDREB1F confers cold and drought stress resistance through common regulation
of hormone synthesis and protectant metabolite contents in banana［J］.Horticulture Research，2023，10（2）：227‑240.

［4］SCHENA M，DAVIS R W.HD‑Zip proteins：Members of an Arabidopsis homeodomain protein superfamily［J］.Proceedings of the National Academy of Sciences of the United States of America，1992，89（9）：3894‑3898.

［5］ARIEL F D，MANAVELLA P A，DEZAR C A，et al.The true story of the HD‑Zip family［J］.Trends in Plant Science，2007，12（9）：419‑426.
［6］SHAO J X，HAIDER I，XIONG L Z，et al.Functional analysis of the HD‑Zip transcription factor genes Oshox12 and Oshox14 in rice［J］.PLoS One，2018，13（7）：e0199248.
［7］ZHAO Y，ZHOU Y Q，JIANG H Y，et al.Systematic analysis of sequences and expression patterns of drought‑responsive members of the HD‑Zip gene family in maize［J］.PLoS One，2011，6（12）：e28488.
［8］CHEN X，CHEN Z，ZHAO H L，et al.Genome‑wide analysis of soybean HD‑Zip gene family and expression profiling under salinity and drought treatments［J］.PLoS One，2014，9（2）：e87156.
［9］JIANG H Y，JIN J，LIU H，et al.Genome‑wide analysis of HD‑Zip genes in grape（Vitis vinifera）［J］.Tree Genetics & Genomes，2014，11（1）：827.
［10］YANG Q S，NIU Q F，LI J Z，et al.PpHB22，a member of HD‑Zip proteins，activates PpDAM1 to regulate bud
dormancy transition in ‘Suli’ pear（Pyrus pyrifolia white pear group）［J］.Plant Physiology and Biochemistry，2018，127：355‑365.
［11］PALENA C M，TRON A E，BERTONCINI C W，et al.Positively charged residues at the N‑terminal arm of the homeodomain are required for efficient DNA binding by homeodomain‑leucine zipper proteins［J］.Journal of Molecular Biology，2001，308（1）：39‑47.
［12］DENG X，PHILLIPS J，MEIJER A H，et al.Characterization of five novel dehydration‑responsive homeodomain leucine zipper genes from the resurrection plant Craterostigma plantagineum［J］.Plant Molecular Biology，2002，49（6）：601‑610.
［13］DEZAR C A，GAGO G M，GONZÁLEZ D H，et al.Hahb⁃4，a sunflower homeobox‑leucine zipper gene，is a developmental regulator and confers drought tolerance to Arabidopsis thaliana plants［J］.Transgenic Research，2005，14（4）：429‑440.
［14］MANAVELLA P A，ARCE A L，DEZAR C A，et al.Cross‑talk between ethylene and drought signalling pathways is mediated by the sunflower Hahb‑4 transcription factor［J］.The Plant Journal，2006，48（1）：125‑137.
［15］ARIEL F， DIET A， VERDENAUD M， et al.Environmental regulation of lateral root emergence in Medicago truncatula requires the HD‑Zip Ⅰ transcription factor HB1［J］.The Plant Cell，2010，22（7）：2171‑2183.
［16］WANG Q Q，ZHA K Y，CHAI W B，et al.Functional analysis of the HD‑Zip Ⅰ gene ZmHDZ1 in ABA‑mediated salt tolerance in rice［J］.Journal of Plant Biology，2017，60（2）：207‑214.
［17］HU T X，YE J，TAO P W，et al.The tomato HD‑Zip Ⅰ transcription factor SlHZ24 modulates ascorbate accumulation through positive regulation of the D‑mannose/L‑galactose pathway［J］.The Plant Journal，2016，85（1）：16‑29.
［18］TRON A E，BERTONCINI C W，PALENA C M，et al.Combinatorial interactions of two amino acids with a single base pair define target site specificity in plant dimeric homeodomain proteins［J］.Nucleic Acids Research，2001，29（23）：4866‑4872.
［19］RUEDA E C，DEZAR C A，GONZALEZ D H，et al.Hahb‑10，a sunflower homeobox‑leucine zipper gene，is regulated by light quality and quantity，and promotes early flowering when expressed in Arabidopsis［J］.Plant and Cell Physiology，2005，46（12）：1954‑1963.
［20］SESSA G，STEINDLER C，MORELLI G，et al.The Arabidopsis Athb‑8，‑9 and ‑14 genes are members of a small gene family coding for highly related HD‑Zip proteins［J］.Plant Molecular Biology，1998，38（4）：609‑622.
［21］CÔTÉ C L，BOILEAU F，ROY V，et al.Gene family structure，expression and functional analysis of HD‑Zip Ⅲ genes in angiosperm and gymnosperm forest trees［J］.BMC Plant Biology，2010，10：273‑290.
［22］NAKAMURA M，KATSUMATA H，ABE M，et al.Characterization of the class Ⅳ homeodomain‑leucine zipper gene family in Arabidopsis［J］.Plant Physiology，2006，141（4）：1363‑1375.
［23］王瑞，鲁丽丽，刘拓，等.棉花中HD-Zip Ⅳ家族基因的全基因组鉴定及特征分析［J］.分子植物育种，2017，15（8）：2921‑2936.
WANG R，LU L L，LIU T，et al.Genome‑wide identification and characterization of the HD‑Zip Ⅳ gene family in cotton（Gossypium spp.）［J］.Molecular Plant Breeding，2017，15（8）：2921‑2936.
［24］孙云飞，巢建国，谷巍，等.高温胁迫对丹参光合生理指标及叶绿素荧光特性的影响［J］.北方园艺，2016（6）：142‑147.
SUN Y F，CHAO J G，GU W，et al.Effect of high temperature stress on photosynthetic physiological indexes and chlorophyll fluorescence characteristics of Salvia miltiorrhiza bga［J］.Northern Horticulture，2016（6）：142‑147.
［25］HUALA E，DICKERMAN A W，GARCIA‑HERNANDEZ M，et al.The Arabidopsis information resource（TAIR）：A comprehensive database and web‑based information retrieval，analysis，and visualization system for a model plant［J］.Nucleic Acids Research，2001，29（1）：102‑105.
［26］JIANG S Y，GONZÁLEZ J M，RAMACHANDRAN S.Comparative genomic and transcriptomic analysis of tandemly and segmentally duplicated genes in rice［J］.PLoS One，2013，8（5）：e63551.
［27］LIU B L，ZHANG G D，MURPHY A，et al.Differences between the bud end and stem end of potatoes in dry matter content，starch granule size，and carbohydrate metabolic gene expression at the growing and sprouting stages［J］.Journal of Agricultural and Food Chemistry，2016，64（5）：1176‑1184.
［28］MONIZ DE SÁ M，DROUIN G.Phylogeny and substitution rates of angiosperm actin genes［J］.Molecular Biology and Evolution，1996，13（9）：1198‑1212.
［29］庾琪唯，刘宇琦，苏文欣，等.MFSD8结构与功能的预测分析研究［J］.医药卫生，2022（11）：1‑3.
YU Q W，LIU Y Q，SU W X，et al.Predictive analysis of MFSD8 structure and function［J］.Medicine and Hygiene，2022（11）：1‑3.
［30］DREWS O，REIL G，PARLAR H，et al.Setting up standards and a reference map for the alkaline proteome of the Gram‑positive bacterium Lactococcus lactis［J］.Proteomics，2004，4（5）：1293‑1304.
［31］李欣宇，徐启宇，卫宇翔，等.高粱LKR基因结构及在种子发育中的表达特征分析［J］.山西农业科学，2022，50（12）：1591‑1598.
LI X Y，XU Q Y，WEI Y X，et al.Analysis of the structure and expression characteristics of Sorghum LKR gene in seed development［J］.Journal of Shanxi Agricultural Sciences，2022，50（12）：1591‑1598.
［32］刘俊，李龙，陈玉龙，等.杜仲LBD基因家族全基因组鉴定与进化表达分析［J］.中草药，2022，53（10）：3142‑3155.
LIU J，LI L，CHEN Y L，et al.Identification，evolution and expression analysis of LBD gene family in Eucommia ulmoides［J］.Chinese Traditional and Herbal Drugs，2022，53（10）：3142‑3155.
［33］LI W，DONG J Y，CAO M X，et al.Genome‑wide identification and characterization of HD‑ZIP genes in potato［J］. Gene，2019，697：103‑117.
［34］AOYAMA T，DONG C H，WU Y，et al.Ectopic expression of the Arabidopsis transcriptional activator Athb‑1 alters leaf cell fate in tobacco［J］.The Plant Cell，1995，7（11）：1773‑1785.
［35］CIARBELLI A R，CIOLFI A，SALVUCCI S，et al.The Arabidopsis homeodomain‑leucine Zipper Ⅱ gene family：Diversity and redundancy［J］.Plant Molecular Biology，2008，68（4）：465‑478.
［36］BAIMA S，POSSENTI M，MATTEUCCI A，et al.The Arabidopsis ATHB‑8 HD‑zip protein acts as a differentiation‑promoting transcription factor of the vascular meristems［J］.Plant Physiology，2001，126（2）：643‑655.
［37］ITOH J I，HIBARA K I，SATO Y，et al.Developmental role and auxin responsiveness of class Ⅲ homeodomain leucine zipper gene family members in rice［J］.Plant Physiology，2008，147（4）：1960‑1975.
［38］YAMASAKI K， KIGAWA T， SEKI M， et al.DNA‑binding domains of plant‑specific transcription factors：Structure，function，and evolution［J］.Trends in Plant Science，2013，18（5）：267‑276.
［39］LYNCH M，CONERY J S.The evolutionary fate and consequences of duplicate genes［J］.Science，2000，290（5494）：1151‑1155.
［40］ZHANG L S，CHEN F，ZHANG X T，et al.The water lily genome and the early evolution of flowering plants［J］.Nature，2020，577：79‑84.
［41］OH S K，YOON J，CHOI G J，et al.Capsicum annuum homeobox 1（CaHB1）is a nuclear factor that has roles in plant development，salt tolerance，and pathogen defense［J］.Biochemical and Biophysical Research Communications，2013，442（1/2）：116‑121.
［42］WANG H，LIN J，LI X G，et al.Genome‑wide identification of pear HD‑Zip gene family and expression patterns under stress induced by drought，salinity， and pathogen［J］.Acta Physiologiae Plantarum，2015，37（9）：189‑208.
［43］孙瑞芬，张艳芳，郭树春，等.向日葵盐诱导HD-Zip类转录因子HB-12的克隆及表达分析［J］.分子植物育种，2017，15（6）：2077‑2087.
SUN R F，ZHANG Y F，GUO S C，et al.Cloning and expression analysis of a salt‑stress‑induced HD‑Zip transcription factor HB⁃12 from sunflower（Helianthus annuus L.）［J］.Molecular Plant Breeding，2017，15（6）：2077‑2087.
［44］YANG Y F，LUANG S，HARRIS J，et al.Overexpression of the class Ⅰ homeodomain transcription factor TaHDZipI‑5 increases drought and frost tolerance in transgenic wheat［J］.Plant Biotechnology Journal，2018，16（6）：1227‑1240.
［45］CHANG X X，HAN S K，MENG K，et al.Expression level of a transcription factor gene mdhd‑zip up‑regulated during apple fruit senescence［J］.Agricultural Science & Technology，2016，17（5）：1065‑1069.
［46］ZHOU C，ZHU L，MA Z Y，et al.A homolog of Class Ⅳ HD‑Zip transcription factors，EsHdzip1，confers drought resistance in tobacco via enhanced the capacity of water conserving and absorbing［J］.Acta Physiologiae Plantarum，2015，37（7）：124.

[1]	LI Peng, SUN Yanju, WANG Yinbiao, JIN Qianyue, LIANG Xiaoxiao, YIN Mei, WANG Xuannian, LIU Xingyou, WANG Liping. Development and Application of TB Green Ⅱ Real‑time PCR Method for Detection of Porcine Circovirus Type 3 [J]. Journal of Henan Agricultural Sciences, 2023, 52(3): 135-142.
[2]	WANG Hongkun, WANG Yihua, ZHOU Danyin, DONG Kun, ZHANG Xuan. Establishment of Two⁃step Fluorescence Quantitative PCR Method for Detection of Deformed Wing Virus in Honeybee [J]. Journal of Henan Agricultural Sciences, 2022, 51(3): 154-161.
[3]	WANG Fei, LI Xuemeng, YANG Jin, WEN Yi, ZHAO Chenchen, ZHAO Ying, LIU Yuxia, GAO Suxia, QI Wenping, QIN Yanhong, LU Chuantao. Screening，Identification and Effect Evaluation of Three Plant Growth‑Promoting Rhizobacteria from Salvia miltiorrhiza [J]. Journal of Henan Agricultural Sciences, 2022, 51(12): 81-89.
[4]	YANG Yang, LIU Yuanyuan. Identification and Sequence Analysis of the MdLAC Gene Family Members in Apple [J]. Journal of Henan Agricultural Sciences, 2021, 50(7): 125-135.
[5]	YANG Jin, WEN Yi, GAO Suxia, LIU Yuxia, LU Chuantao, WANG Fei, LIU Hongyan. Identification of Pathogens Causing Salvia miltiorrhiza Root Rot Disease in Henan Province [J]. Journal of Henan Agricultural Sciences, 2021, 50(10): 92-98.
[6]	ZHOU Xinyu, CHEN Xinxin, QIAO Songlin, GUO Zhenhua, LI Rui, DENG Ruiguang, ZHANG Gaiping. Establishment and Application of Real-time PCR for Differentiation of Porcine Reproductive and Respiratory Syndrome Virus Strains NADC30-like and JXA1-R [J]. Journal of Henan Agricultural Sciences, 2021, 50(1): 142-151.
[7]	HUANG MingjieZHANG Yong, CHEN Xiang, CHEN Wei, MO Xianting, GUO Yong, WANG Tiansong. Bioinformatics Analysis of the Protein Encoded by the SLC13 Gene Family in Bovine [J]. Journal of Henan Agricultural Sciences, 2020, 49(3): 157-166.