生物技术通报 ›› 2024, Vol. 40 ›› Issue (8): 74-82.doi: 10.13560/j.cnki.biotech.bull.1985.2024-0201
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()
收稿日期:
2024-03-14
出版日期:
2024-08-26
发布日期:
2024-07-30
通讯作者:
李清峰,男,博士,副教授,研究方向:麦类作物遗传育种;E-mail: liqingfeng2017@nxu.edu.cn作者简介:
崔原瑗,女,硕士研究生,研究方向:麦类作物遗传育种;E-mail: 18695124679@163.com
基金资助:
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()
Received:
2024-03-14
Published:
2024-08-26
Online:
2024-07-30
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。
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.
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 |
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 |
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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