Identification of PeNAC121 Gene Promoter and Stress Response Pattern Analysis in Populus euphratica

doi:10.7525/j.issn.1673-5102.2022.02.008

Abstract

Abstract:

To explore the stress response and regulatory mechanism of NAC transcription factor family members in Populus euphratica， the promoter sequence of PeNAC121 gene was cloned from leaves of Populus euphrachiae by PCR and its structural characteristics were analyzed using bioinformatics tools. The PeNAC121 promoter was used to drive the expression of the GUS reporter gene in the triploid Populus tomentosa， and then GUS staining and enzyme activity quantitative analysis were performed in the obtained transgenic plants after different stress treatments. The cloned promoter sequence of PeNAC121 gene was 1 997 bp（located in the upstream of start codon ATG） in length， included a large number of light-responsive elements， and also contained a number of abiotic stress and phytohormone responsive elements， such as low temperature response element LTR， drought response element MBS， abscisic acid（ABA） response element， as well as gibberellin（GA） response elements， respectively. The result of tissue expression pattern showed that the PeNAC121 gene was mainly expressed in stemsof P. euphratica， with low expression in roots and leaves. The results of GUS histochemical staining and enzyme activity test of transgenic plants showed that the PeNAC121 promoter was significantly induced by NaCl， mannitol， ABA and 4 ℃ low temperature respectively. It was speculated from the above results that PeNAC121 gene was closely related to the response to stress. The results showed that the promoter of PeNAC121 gene was an inducible promoter responding to multiple stresses. The study provid a theoretical reference for elucidating the role of PeNAC121 gene in stress response and regulation of P. euphratica.

Key words: Populus euphratica, PeNAC121 promoter, stress response, inducible promoter

CLC Number:

S792.11

Wang Xie, Tianjing Li, Xinyao Li, Fenming Yang, Yin’an Yan, Yongfeng Gao. Identification of PeNAC121 Gene Promoter and Stress Response Pattern Analysis in Populus euphratica[J]. Bulletin of Botanical Research, 2022, 42(2): 234-242.

Figures/Tables 10

Table 1

Fig.1

Fig.2

Table 2

The Cis-acting elements in the isolated PeNAC121 promoter region

元件 Element	功能 Function	核心序列 Core sequence	数量 Amount	位置 Position
非生物胁迫响应元件 Abiotic stress responsive elements
LTR	低温响应元件Low temperature response element	CCGAAA	1	-1 802（+）
MBS	参与干旱诱导MYB结合位点 Participate in drought induced MYB binding site	CAACTG	1	-63（-）
ARE	抗氧化反应元件 Antioxidant reaction element	AAACCA	2	-7（-） -1 401（-）
TC-rich repeats	防卫和胁迫响应顺式作用元件 Cis-acting element involved in defense and stress responsiveness	ATTCTCTAAC GTTTTCTTAC	2	-1 325（+） -1 782（-）
WUN-motif	创伤响应元件 Wound-responsive element	TTATTACAT TTATTACAT	2	-1 421（-） -1 365（-）
光响应元件 Light responsive elements
Box 4	参与光响应部分保守DNA组件 Part of a conserved DNA module involved in light responsiveness	ATTAAT	4	-1 050（+） -887（+） -845（+） -613（+）
G-Box	参与光响应的顺式调控元件 Cis-acting regulatory element involved in light responsiveness	CACGTT	1	-1 732（+）
G-box	参与光响应的顺式调控元件 Cis-acting regulatory element involved in light responsiveness	TACGTG	1	-1 104（+）
GT1-motif	光响应元件Light responsive element	GGTTAA	1	-1 806（-）
TCT-motif（光反应元件）	光响应元件Light responsive element	TCTTAC	2	-1 321（+） -1 327（-）
GATA-motif	光响应元件Light responsive element	GATAGGG	1	-1 549（+）
MRE（涉及光反应的MYB结合位点）	涉及光反应的MYB结合位点 MYB binding sites involved in light reactions	AACCTAA	1	-834（-）
chs-CMA1a（光反应元件）	光响应元件Light responsive element	TTACTTAA	1	-1 919（+）
植物激素响应元件 Phytohormone responsive elements
TATC-box（赤霉素反应作用元件）	赤霉素响应顺式作用元件 Cis-acting element involved in gibberellin responsiveness	TATCCCA	1	-1 031（+）
ABRE	脱落酸响应顺式作用元件 Cis-acting element involved in the abscisic acid responsiveness	ACGTG	2	-1 103（+） -1 727（-）

Table 2

Fig.3

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Fig.8

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名称 Name	序列 Sequence（5′—3′）	功能 Function
PeNAC121 Pro-F0	GGATTTATCAAGATTCAAGCCT	克隆PeNAC121启动子 Cloning NAC121 gene promoter
PeNAC121 Pro-R0	TTACAGGGTGGAGGTGAGC
PeNAC121 Pro-F1	AAGCTTAAGAGTGGGGCAGATGATACC
PeNAC121 Pro-R1	GGATCCATCGTTGCAGTCATTGCACC
qRT-PeUBQ-F	CCAAGCCCAAGAAGATCAAGC	检测PeNAC121组织表达表达模式 Detecting the tissue dexpression patterns of PeNAC121 gene
qRT-PeUBQ-R	GCACCGCACTCAGCATTAGG
qRT-PeNAC121-F	CTCACCTCCACCCTGTAATGC
qRT-PeNAC121-R	CCTTCCCCTCCAAATGCTC
NPTII-F	TCTCATGCTGGAGTTCTTCGC	鉴定转基因阳性植株 Checking transgenic positive plants
NPTII-R	GTCACCGACTTGAGCCATTTG	鉴定转基因阳性植株 Checking transgenic positive plants

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