Cloning and Expression Analysis of the Promoter of Vacuolar H+-ATPase E1 Subunit AtTUF Gene in Arabidopsis thaliana

doi:10.7668/hbnxb.20193384

Abstract

Abstract:

AtTUF gene encodes E1 subunit of vacuolar H⁺-ATP enzyme,and is closely related to the material transport across the tonoplast.In order to explore the tissue expression characteristics and molecular regulatory mechanism of AtTUF,and provide a theoretical basis for the utilization of AtTUF gene promoter and the in-depth study of the regulation characteristics of AtTUF gene expression,the promoter of AtTUF (pAtTUF)was cloned by PCR amplification with Columbia wild-type Arabidopsis as material,the cis-acting elements were predicted through PlantCARE and PLACE,the plant expression vector pBI121-pAtTUF,in which the galactosidase gene(GUS)was driven by pAtTUF,was constructed and transformed into wild-type Arabidopsis to obtain transgenic plants.Through GUS staining,the tissue expression pattern of pAtTUF and its response to hormone and stress were analyzed,and the expression of AtTUF in different tissues was quantitatively analyzed.The results showed that 2 000 bp promoter sequence upstream of the open reading frame of AtTUF gene was successfully cloned.Sequence analysis results showed that pAtTUF contained several cis-acting elements in response to light,abscisic acid(ABA),auxin,methyl jasmonate(MeJA)and drought.The staining results of transgenic lines showed that GUS driven by pAtTUF mainly expressed in the leaves and roots of Arabidopsis seedlings,and in rosette leaves,stems,cauline leaves,sepals,petals,anthers,stigmas,young siliques and immature seeds of adult plants.However, no GUS expression was detected in the hypocotyl at seedling stage,neither in the lateral branches of rosette leaves,mature pods or seeds at adult plant stage.GUS expression intensity was inhibited by ABA,indole acetic acid(IAA),salt,dark treatment and PEG treatment,which was consistent with its characteristics containing a variety of light regulatory elements,hormone regulatory elements and abiotic stress response elements.The Real-time quantitative RT-PCR results showed that AtTUF expressed in roots,stems,leaves,flowers,siliques and seeds,especially higher in siliques at the developmental stage.AtTUF also expressed higher in flowers and dry seeds,but lowest in roots.In conclusion,AtTUF gene plays an important role in the growth and development of plant organs(especially in the reproductive system)and in the response to ABA,IAA,salt,light and drought signals.

Key words: Arabidopsis thaliana, AtTUF, Promoter, Cis-acting elements, Tissue expression

SUN Haili, LIANG Jing, WANG Wenjia, DING Weihua, WANG Nina, LIU Kaiheng, LI Chengwei. Cloning and Expression Analysis of the Promoter of Vacuolar H⁺-ATPase E1 Subunit AtTUF Gene in Arabidopsis thaliana[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(6): 82-90. doi: 10.7668/hbnxb.20193384.

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Figures/Tables 8

Fig.1 PCR amplification of AtTUF promoter in Arabidopsis 1.PCR products of AtTUF promoter;M.Marker.

Fig.2 The construction of expression vector pBI121-pAtTUF A.Products of pBI121 digested by restriction enzyme digestion: 1.Products of pBI121 digested by Hind Ⅲ and BamH Ⅰ;M.Marker.B.PCR identification of pBI121-pAtTUF recombinant vector in E.coli: 1—5.Individual colonies;M.Marker;6.Positive control(PCR recovery product of AtTUF promoter);7.Negative control.C.Restriction enzyme digestion of the recombinant plasmid pBI121-pAtTUF:1.The enzyme digestion result of pBI121-pAtTUF by Hind Ⅲ and BamHⅠ;M.Marker.

Fig.3 The sites analysis of key cis-acting elements in AtTUF promoter in Arabidopsis

Tab.1 Key cis-acting elements in AtTUF promoter in Arabidopsis

元件类型 Element type	名称 Name	数量 Number	核心序列 Core sequence	功能 Function
光调控元件	MRE	3	AACCTAA	参与光响应的MYB结合位点
Light regulatory	G-Box	2	CACGTT	参与光响应的顺式作用元件
element	G-box	2	CACGTC/CACGAC	参与光响应的顺式作用元件
	Box Ⅱ	1	ACACGTTGT	光响应元件的一部分
	Ⅰ-box	1	GTATAAGGCC	光响应元件的一部分
	TCT-motif	1	TCTTAC	光响应元件的一部分
	GTGGC-motif	1	GATTCTGTGGC	光响应元件的一部分
	Box 4	5	ATTAAT	参与光响应的保守DNA组件的一部分
	AE-box	1	AGAAACAA	部分光响应组件
激素调控元件	TGA-element	1	AACGAC	生长素反应元件
Phytohormone regulatory	AuxRR-core	2	GGTCCAT	参与生长素反应的顺式作用调控元件
element	ABRE	4	ACGTG/TACGGTC	ABA响应顺式作用元件
	CGTCA-motif	2	CGTCA	参与MeJA反应的顺式作用调控元件
	TGACG-motif	2	TGACG	参与MeJA反应的顺式作用调控元件
胁迫调控元件	MBS	2	CAACTG	参与干旱诱导的MYB结合位点
Stress regulatory element	ARE	2	AAACCA	厌氧诱导必需的顺式作用元件
其他	GCN4-motif	1	TGAGTCA	参与胚乳表达的顺式调控元件
Other	CAT-box	1	GCCACT	分生组织表达相关顺式作用调控元件
	O₂-site	1	GATGA(C/T)(A/G)TG(A/G)	玉米醇溶蛋白代谢调节顺式作用调控元件

Fig.4 Screening(A)and PCR identification(B)of T₁ positive plants of pBI121-pAtTUF transgenic Arabidopsis 1.The plasmid of expression vector pBI121-pAtTUF (Positive control);2—9.Transgenic Arabidopsis;10.Wild type Arabidopsis(Negative control);M.Marker.

Fig.5 The GUS staining results of pBI121-pAtTUF transgenic Arabidopsis plants A—Q.Homozygous seedlings and plant tissues of T₃ generation;R—S.Germinated seedlings of T₃ generation seeds.A.Cotyledon stage;B.Four-leaf stage;C.Six-leaf stage;D.Seven-leaf stage;E.Rosette leaf;F.Main stem,cauline leaf and lateral branch of the cauline leaf;G.The top leaves and inflorescence of cauline leaf's lateral branch(enlarged red box in F);H.Inflorescences of the main stem;I.Sepal;J.A single flower;K.Lateral branches of rosette leaves;L.Young silique;M.Siliques of different developmental stages;N.Pericarp,pseudoseptum and immature seed;O.A seed at developing stage(enlarged view of the seed circled in red in N);P.Siliques at maturation stage;Q.Seeds at maturation stage;R.Germinated 48 h seedling;S.Germinated 60 h seedling.

Fig.6 Response patterns of AtTUF promoter to different hormones and stress treatments A.Staining results of seedlings cultured for 9 days under light,and then cultured for 3 days under light or dark after transferred to different MS plates harboring hormones or NaCl;B.Staining results of seedlings cultured for 10 days under light,and then cultured for 2 days under light after transferred to MS plate harboring PEG.

Fig.7 AtTUF gene expression in different tissues of Arabidopsis The different lowercase letters indicate significant differences in the expression of AtTUF gene in different tissues at 0.05 level.

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