Characterization and transcriptional regulation analysis of the LpPIL5 gene in perennial ryegrass

doi:10.11686/cyxb2021382

Abstract

Abstract:

Phytochrome-interacting factors （PIFs）， a subfamily of the bHLH family， play important roles in plant light and hormone signaling， and stress tolerance. In the current study， a PIF gene was cloned from perennial ryegrass （Lolium perenne）， and its genetic sequence was most closely related to Arabidopsis thaliana AtPIF5， therefore， it was named LpPIL5-like （LpPIL5）. The coding region sequence （CDS） of this LpPIL5 gene is 1410 bp， containing 5 exons， encoding 470 amino acids. The protein it codes for has a typical bHLH domain and APB motif and is localized in the nucleus. The promoter region （1500 bp） of LpPIL5 has various cis-elements， including ABRE， MBS， G-box， and others， related to light， hormone， and stress responses. The results of transcription analysis showed that relative expression of LpPIL5 was high in leaves， but low in roots， stems， and leaf sheaths. The transcription of LpPIL5 was also regulated by circadian rhythm， and was significantly higher in light than in dark. In addition， its relative expression levelin leaves was significantly inhibited under polyethylene glycol （PEG）， NaCl， CdCl₂， high temperature stresses， and by N-（phenylmethyl）-9H-purin-6-amine （6-BA） and abscisic acid （ABA） treatments. In roots， the relative expression of the LpPIL5 gene increased significantly after 12 h of these treatments， and became significantly higher than in control plants after 24 h of treatment.

Key words: perennial ryegrass, basic helix-loop-helix, phytochrome-interacting factors, subcellular localization, transcription analysis

Jia-ming YAO, Yue HE, Huan-huan HAO, Xin-ru HUANG, Jing ZHANG, Bin XU. Characterization and transcriptional regulation analysis of the LpPIL5 gene in perennial ryegrass[J]. Acta Prataculturae Sinica, 2022, 31(9): 155-167.

Figures/Tables 12

References 44

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引物名称Primer name	上游引物序列Forward primer sequence （5′-3′）	下游引物序列Reverse primer sequence （5′-3′）
LpPIL5-CDS	CGCggatccATGAACCAATTCGTGCCT	CCCaagcttTCTCTTGTTCGGATGCG
LpPIL5-qPCR	CCGACTCGCTCGACAAGGAC	GGAAGACGTCCGACAGCTCG
LpeLF4A	AACTCAACTTGAAGTGTTGGAGTG	AGATCTGGTCCTGGAAAGAATATG

引物名称Primer name	上游引物序列Forward primer sequence （5′-3′）	下游引物序列Reverse primer sequence （5′-3′）
LpPIL5-CDS	CGCggatccATGAACCAATTCGTGCCT	CCCaagcttTCTCTTGTTCGGATGCG
LpPIL5-qPCR	CCGACTCGCTCGACAAGGAC	GGAAGACGTCCGACAGCTCG
LpeLF4A	AACTCAACTTGAAGTGTTGGAGTG	AGATCTGGTCCTGGAAAGAATATG

溶液名称Solution name	成分Ingredient
酶解液Enzyme solution	10 mmol·L^-1 MES，1.5%纤维素酶R10（Cellulase R10），0.75%离析酶R10（Macerozyme R10）， 20 mmol·L^-1 KCl，10 mmol·L^-1 CaCl₂，0.1% BSA，0.6 mol·L^-1甘露醇Mannitol
W5 溶液W5 solution	154 mmol·L^-1 NaCl，125 mmol·L^-1 CaCl₂，5 mmol·L^-1 KCl，2 mmol·L^-1 MES
MMg溶液MMg solution	0.4 mol·L^-1甘露醇Mannitol，15 mmol·L^-1 MgCl₂，4 mmol·L^-1 MES
PEG 4000溶液PEG 4000 solution	20% PEG 4000，10 mmol·L^-1 CaCl₂，0.3 mol·L^-1甘露醇Mannitol

溶液名称Solution name	成分Ingredient
酶解液Enzyme solution	10 mmol·L^-1 MES，1.5%纤维素酶R10（Cellulase R10），0.75%离析酶R10（Macerozyme R10）， 20 mmol·L^-1 KCl，10 mmol·L^-1 CaCl₂，0.1% BSA，0.6 mol·L^-1甘露醇Mannitol
W5 溶液W5 solution	154 mmol·L^-1 NaCl，125 mmol·L^-1 CaCl₂，5 mmol·L^-1 KCl，2 mmol·L^-1 MES
MMg溶液MMg solution	0.4 mol·L^-1甘露醇Mannitol，15 mmol·L^-1 MgCl₂，4 mmol·L^-1 MES
PEG 4000溶液PEG 4000 solution	20% PEG 4000，10 mmol·L^-1 CaCl₂，0.3 mol·L^-1甘露醇Mannitol

名称 Name	序列 Sequence	功能 Function	个数Number
A-box	CCGTCC	顺式调控元件Cis-acting regulatory element	1
ABRE	TACGGTC	脱落酸响应顺式作用元件Cis-acting element involved in the abscisic acid responsiveness	2
ABRE	ACGTG	脱落酸响应顺式作用元件Cis-acting element involved in the abscisic acid responsiveness	2
MBS	CAACTG	MYB结合位点参与干旱诱导MYB binding site involved in drought-inducibility	1
CGTCA-motif	CGTCA	茉莉酸甲酯顺式作用元件Cis-acting regulatory element involved in the methyl jasmonate-responsiveness	1
TGACG-motif	TGACG	茉莉酸甲酯顺式作用元件Cis-acting regulatory element involved in the methyl jasmonate-responsiveness	1
G-box	CACGAC	光响应顺式作用元件Cis-acting regulatory element involved in light responsiveness	1
G-box	TAACACGTAG	光响应顺式作用元件Cis-acting regulatory element involved in light responsiveness	1
G-box	CACGTC	光响应顺式作用元件Cis-acting regulatory element involved in light responsiveness	1
G-box	TACGTG	光响应顺式作用元件Cis-acting regulatory element involved in light responsiveness	1
CAAT-box	CAAT	启动子和增强子区域的共同顺式作用元件Common cis-acting element in promoter and enhancer regions	9
CAAT-box	CAAAT	启动子和增强子区域的共同顺式作用元件Common cis-acting element in promoter and enhancer regions	6
CAAT-box	CCAAT	启动子和增强子区域的共同顺式作用元件Common cis-acting element in promoter and enhancer regions	5
CAAT-box	TGCCAAC	启动子和增强子区域的共同顺式作用元件Common cis-acting element in promoter and enhancer regions	1
I-box	cCATATCCAAT	部分光响应元件Part of a light responsive element	1
BoxⅡ	ACACGTAGA	部分光响应元件Part of a light responsive element	1
TCCC-motif	TCTCCCT	部分光响应元件Part of a light responsive element	2
TCT-motif	TCTTAC	部分光响应元件Part of a light responsive element	2
TATA-box	ATATAT	转录开始的核心启动子元件Core promoter element around -30 of transcription start	1
TATA-box	TATAA	转录开始的核心启动子元件Core promoter element around -30 of transcription start	2
TATA-box	TATAAAA	转录开始的核心启动子元件Core promoter element around -30 of transcription start	2
TATA-box	TATAAA	转录开始的核心启动子元件Core promoter element around -30 of transcription start	2
TATA-box	TATA	转录开始的核心启动子元件Core promoter element around -30 of transcription start	3