大麦非特异性磷脂酶C基因家族全基因组鉴定及苗期胁迫表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0201

摘要/Abstract

摘要：

【目的】非特异性磷脂酶C（non-specific phospholipase C, NPC）是磷脂酶C的一种，在植物生长发育和响应非生物胁迫等方面发挥重要作用。在大麦全基因组范围内鉴定NPC基因家族成员，分析相关基因表达特点，为大麦NPC基因功能研究及遗传改良提供理论依据。【方法】基于EnsemblPlants数据库收录的大麦全基因组数据，利用生物信息学的方法对大麦NPC基因家族进行鉴定，并对大麦NPC基因家族的理化特性、亚细胞定位、系统进化关系、基因结构、蛋白结构域和启动子顺势元件等进行分析。利用转录组数据和实时荧光定量PCR分析HvNPC基因在不同组织以及苗期在低温、干旱、盐胁迫下的表达特征。【结果】在大麦基因组中，共鉴定了5个非特异性磷脂酶C基因家族成员，其编码蛋白与其他植物的NPC基因结构相似，均具有Phosphoesterase结构域；HvNPC蛋白序列长度为477-553 aa，等电点为5.22-8.83，分子量为53.12-61.33 kD；亚细胞定位预测HvNPC基因定位于叶绿体、液泡膜、细胞质中；HvNPC启动子区含有大量与非生物胁迫有关的顺势作用元件。实时荧光定量PCR分析证实HvNPC基因受干旱、高盐及低温胁迫诱导表达，其中HvNPC2、HvNPC3和HvNPC5受低温和干旱胁迫诱导表达；HvNPC3和HvNPC5受盐胁迫诱导表达。【结论】在大麦基因组中共鉴定5个HvNPC基因，HvNPC基因成员具有器官表达特异性，并且不同基因对非生物胁迫响应不同。

关键词: 大麦, 非特异性磷脂酶C, 全基因组鉴定, 非生物胁迫, 表达分析

Abstract:

【Objective】Non-specific phospholipase C（NPC）is a type of phospholipase C, which plays an important role in plant growth and development and response to abiotic stresses. Identifying the NPC gene family members from the whole barley genome, and analyzing the expression characteristics of the related genes would provide a theoretical basis for the study of barley NPC gene function and genetic improvement.【Method】Based on the barley genome-wide data included in the EnsemblPlants database, bioinformatics methods were used to identify NPC gene family in barley and analyze their physicochemical properties, subcellular localization, phylogenetic relationship, gene structure, protein structural domains, and promoter cis-acting elements. Transcriptome data and RT- qPCR were used to analyse the expression characteristics of HvNPCs in different tissues as well as at seedling stage under low temperature, drought and salt stress.【Result】Five NPC members were identified in the barley genome, and the encoded proteins were structurally similar to NPC genes from other plants, all of which had a phosphoesterase structural domain ; the sequence lengths of the HvNPC proteins ranged from 477 to 553 aa, the isoelectric points ranged from 5.22 to 8.83, and the molecular weights ranged from 53.12 to 61.33 kD. Subcellular localization showed that the HvNPC gene was localized in chloroplasts, membrane-intrinsic proteins, and cytoplasm. The HvNPC promoter region contained a large number of cis-acting elements associated with adversity stresses, and real-time PCR analysis confirmed that the HvNPC gene was induced to express by drought, high salt and low temperature. Among them, HvNPC2, HvNPC3 and HvNPC5 were induced to be expressed by low-temperature stress and drought stress; and HvNPC3 and HvNPC5 were induced to be expressed by salt stress.【Conclusion】A total of five HvNPC genes were identified in the barley genome, and members of the HvNPC genes were organ-specofc expression and different genes responded differently to abiotic stresses.

Key words: Hordeum vulgare L., non-specific phospholipase C, genome-wide identification, abiotic stress, expression analysis

崔原瑗, 王昭懿, 白双宇, 任毓昭, 豆飞飞, 刘彩霞, 刘凤楼, 王掌军, 李清峰. 大麦非特异性磷脂酶C基因家族全基因组鉴定及苗期胁迫表达分析[J]. 生物技术通报, 2024, 40(8): 74-82.

CUI Yuan-yuan, WANG Zhao-yi, BAI Shuang-yu, REN Yu-zhao, DOU Fei-fei, LIU Cai-xia, LIU Feng-lou, WANG Zhang-jun, LI Qing-feng. Genome-wide Identification of Non-specific Phospholipase C Gene Family in Hordeum vulgare L. and Stress Expression Analysis at Seedling Stage[J]. Biotechnology Bulletin, 2024, 40(8): 74-82.

图/表 7

表1 RT-qPCR 引物

Table 1 Primers used for RT-qPCR

引物名称 Primer name	引物序列 Primer sequence（5'-3'）	退火温度Annealing temperature/℃
HvNPC1-F	AACAAGCCCCCATCACCG	58.9
HvNPC1-R	CCACCTTGAAGGCGTCGT	58.6
HvNPC2-F	ACTACCAGTACCCGCCGT	58.9
HvNPC2-R	ACCACGTAGTTGGGCAGC	58.6
HvNPC3-F	GACGTGCTGGACCAGGTC	59.4
HvNPC3-R	CGGAGGAGAGCAGGGAGT	59.8
HvNPC4-F	GATGCCGACGAGGATCCG	59.2
HvNPC4-R	ATCCCCGTTCAGCACTGC	58.8
HvNPC5-F	CGTCTTCGACCGCTGGTT	58.4
HvNPC5-R	CGTTGAAGGTGAGCCCGT	58.6
HvActin-F	CAGGTATCGCTGACCGTATGAG	57.5
HvActin-R	TGGAAAGTGCTGAGTGAGGCT	58.9

表2 大麦NPC基因家族成员基本理化性质

Table 2 Basic physico-chemical properties of NPC family in barley（Hordeum vulgare L）

基因名称 Gene name	基因ID Transcript ID	物理位置 Physical position	相对分子量 Molecular weight/kD	等电点 pI	亲水性总平均值 Grand average of hydrophilicity	蛋白长度 Protein length/aa	亚细胞定位 Subcellular location
HvNPC1	HORVU.MOREX.r3.7HG0752530.1	7H:631001620-631003350	53.12	5.22	-0.317	477	细胞质
HvNPC2	HORVU.MOREX.r3.1HG0085590.1	1H:496122549-496124474	61.33	6.53	-0.364	553	叶绿体
HvNPC3	HORVU.MOREX.r3.3HG0232030.1	3H:28367740-28369602	58.77	8.83	-0.204	532	叶绿体
HvNPC4	HORVU.MOREX.r3.3HG0322080.1	3H:600541735-600546962	59.07	5.98	-0.424	533	液泡膜
HvNPC5	HORVU.MOREX.r3.5HG0527960.1	5H:566994044-566997667	60.76	6.80	-0.367	545	叶绿体

图1 不同物种NPC基因家族系统进化树

Fig. 1 Phylogenetic tree of NPC gene family in different species

图2 大麦NPC基因家族的结构特征 A：紫色条表示外显子，黑线表示内含子，灰色条表示第灰色条表示非翻译区（UTR）；B：黄色条表示特征域phosphoesterase（PF04185）

Fig. 2 Structural features of NPC gene family in barely A: Purple bars indicate the exon, lines indicate the intron while grey color bars indicate the un-translated region（UTR）. B: The yellow bar indicates the signature domain phosphoesterase（PF04185）

图3 HvNPC基因家族各成员启动子区顺式作用元件 ABRE：脱落酸响应元件；CGTCA-motif、TGACG-motif：茉莉酸甲酯响应元件；GARE-motif、P-box、TATC-box：赤霉素响应元件；TGA-element：生长素响应元件；TCA-element：水杨酸响应元件；MBS、MYC、MYB：干旱胁迫响应元件；TC-rich：防御和胁迫响应元件；LTR：低温胁迫响应元件；ARE：厌氧诱导响应元件；O2-site：玉米醇溶蛋白代谢调控元件；RY-element:种子特异性调控元件；Sp1、TCT-motif、AE-box、BoxII、I-box：光响应元件

Fig. 3 Cis-acting elements in the promoters of HvNPC gene family members ABRE: Abscisic acid responsive element. CGTCA-motif, TGACG-motif: MeJA responsive element. GARE-motif, P-box, TATC-box: Gibberellin responsive element. TGA-element: Auxin responsive element. TCA-element: Salicylic acid responsive element. MBS, MYC, MYB: Drought responsive element. TC-rich: Defense and stress responsive element. LTR: Low-temperature responsive element. ARE: Essential for the anaerobic responsive element. O2-site: Zein metabolism regulatory element. RY-element: Seed-specific regulatory element. Sp1, TCT-motif, AE-box, BoxII, I-box: Light responsive element

图4 大麦NPC基因在14个组织器官中的表达谱 A：胚（萌发后4 d）；B：种子根（茎长10 cm）；C：茎（10 cm）；D：黄化幼苗（黑暗条件下10 d）；E：分蘖（6叶期，第3节）；F：根（4周）； G：表皮（4周）；H：花序（5 mm）；I：花序（1-1.5 cm）；J：花轴（5周）；K：外稃（6周）；L：浆片（6周）；M：内稃（6周）；N：衰老叶片（2周）

Fig. 4 Expression profile of NPC gene in 14 tissues and organs of barley A: Embryo（4 d after germination）. B: Roots from the seedlings（shoot length 10 cm）. C: Shoots（10 cm）. D: Etiolated seedling （10 d old at dark）. E: Developing tillers（six leaf stge, 3rd internode）. F: Root（4 weeks）. G: Epidermis（4 weeks）. H: Florescences（5 mm）. I: Florescences（1-1.5 cm）. J: Rachis（5 weeks）. K: Lemma（6 weeks）. L: Lodicule（6 weeks）. M: Palea（6 weeks）. N: Senescing leaf（2 weeks）

图5 低温、氯化钠、干旱处理下HvNPCs基因的表达模式 A：4℃处理下HvNPCs的基因表达谱；B：NaCl处理下HvNPCs的基因表达谱；C：PEG处理下HvNPCs的基因表达谱；不同小写字母代表显著差异（P<0.05）

Fig. 5 Relative expression pattern of HvNPCs genes under low temperature, NaCl, and drought treatments A: Gene expression profiles of HvNPCs under 4℃ treatment. B: Gene expression profiles of HvNPCs under NaCl treatment. C: Gene expression profiles of HvNPCs under PEG treatment. Different lower letters indicate significant differences（P<0.05）

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