VaCBL6 from Vitis amurensis Involved in Abiotic Stress Response and ABA-mediated Pathway

doi:10.16420/j.issn.0513-353x.2020-0902

Acta Horticulturae Sinica ›› 2021, Vol. 48 ›› Issue (6): 1079-1093.doi: 10.16420/j.issn.0513-353x.2020-0902

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VaCBL6 from Vitis amurensis Involved in Abiotic Stress Response and ABA-mediated Pathway

MA Junjie¹, SONG Lina¹, LI Le¹, MA Xiaochun¹, JIN Lei¹, XU Weirong²^,³^,⁴^,^*()

¹College of Agronomy,Ningxia University,Yinchuan 750021,China
²School of Food & Wine,Ningxia University,Yinchuan 750021,China
³China Wine Industry Technology Institute,Yinchuan 750021,China
⁴Key Laboratory of Modern Molecular Breeding of Dominant and Special Crops in Ningxia,Yinchuan 750021,China

Received:2021-03-16 Revised:2021-05-11 Online:2021-06-25 Published:2021-07-07
Contact: XU Weirong E-mail:xuwr@nxu.edu.cn

Abstract

Abstract:

A calcineurin B-like phosphatase subunit protein gene VaCBL6 from Vitis amurensis ‘Zuoshan-1'was cloned and functionally analyzed. VaCBL6is located on chromosome 4 and has an open reading frame of 777 bp,encoding 258 aa;protein domain analysis shows that the protein contains four EF-hands and one transmembrane domain. Phylogenetic analysis using amino acid sequence showed that high similarities between VaCBL6 and VvCBL9,VaCBL6 and VvCBL6 are 98.84% and 94.96%,respectively. VaCBL6 is located in the nucleus and cell membrane,and is highly expressed in roots and tendrils,and its expression is induced by low temperature,drought,NaCl and ABA. Analysis of transcriptional activation based on the yeast system displayed that VaCBL6 had no transcriptional activation activity. Experiments on the growth stage ofArabidopsis seedlings overexpressingVaCBL6with different treatments found that 100-125 μmol · L ^-1 NaCl and 0.25-1.25 μmol · L ^-1 ABA treatments inhibited the germination of VaCBL6 transgenic Arabidopsis seeds,and the transgenic Arabidopsis seedlings increased the sensitivity to NaCl and ABA. The results indicated that VaCBL6 plays an important role in plant stress response.

Key words: Vitis amurensis, calcineurin B-like phosphatase subunit protein, gene cloning, abiotic stress, functional analysis

CLC Number:

S633.1

MA Junjie, SONG Lina, LI Le, MA Xiaochun, JIN Lei, XU Weirong. VaCBL6 from Vitis amurensis Involved in Abiotic Stress Response and ABA-mediated Pathway[J]. Acta Horticulturae Sinica, 2021, 48(6): 1079-1093.

Figures/Tables 15

Table 1 Primers of vector generation and the expression of VaCBL6

引物 Primer	引物序列（5′-3′） Primer sequence
qPCR-CBL6-F	TGCTGCGACTTCCACAAA
qPCR-CBL6-R	CTCATACAGTGCCTCCACTTC
RT-VvActin-F	CTATCCTTCGTCTTGACCTTGCTG
RT-VvActin-R	AGTGGTGAACATGTAACCCCTCTC
Semi-CBL6-F	GGAAGAGCTTCAATTGGCGT
Semi-CBL6-R	ACTGTGGTGATGTCCTTCAAAT
AtActin2-RT-F	TGAGCAAAGAAATCACAGCACT
AtActin2-RT-R	CCTGGACCTGCCTCATCATAC
VaCBL6-ORF-F	ATGAGTTCTTGGCAGGGAACGGCG
VaCBL6-ORF-R	TCATTCTTCAACCTCAGTATTGAAAACAAAGC
221-VaCBL6-F	GAGAGAACACGGGGGACTCTAGAATGAGTTCTTGGCAGGGAACGGCG
221-VaCBL6-R	TTACCCATGGTACCCCGCTCGAGTTCTTCAACCTCAGTATTGAAAACAAAGC
BD-CBL6-F	atggccatggaggccgaattcATGAGTTCTTGGCAGGGAACG
BD-CBL6-R	ccgctgcaggtcgacggatccTCATTCTTCAACCTCAGTATTGAAAAC
Entry-VaCBL6-F	AAAAAAgCAggCTTTgACTTTATgAgTTCTTggCAgggAACg
Entry-VaCBL6-R	AAAgCTgggTCTAgAgACTTTCCTTCTTCAACCTCAgTATTgA

Fig. 1 Cloning（A）and chromosome location（B）of VaCBL6 gene from Vitis amurensis

Fig. 2 Phylogenetic relationships of Vitis amurensis VaCBL6 with CBLs from other plant species Protein sequences were aligned to construct a phylogenetic tree using the Neighbor-Joining（NJ）method by MEGA 7.0 software. The percentage of trees with related clusters were displayed next to the branches,and the phylogenetic tree was drawn proportionally with branch lengths measured in the number of substitutions per site. Bootstrap value was 1 000.

Fig. 3 Amino acid sequence alignment of VaCBL6 from Vitis amurensis

Fig. 4 Expression pattern of VaCBL6 in various tissues of Vitis amurensis *,** indicated significant differences compared with the expression level of root（expression level is 1）（* α = 0.05,** α = 0.01).

Fig. 5 Expression pattern of VaCBL6 in grape leaves under abiotic stress and ABA treatment * and ** indicated significant differences compared with control groups（* α = 0.05,** α = 0.01）.

Fig. 6 Subcellular localization of VaCBL6 protein in Arabidopsis thaliana protoplasts

Fig. 7 Analysis of self activation of VaCBL6 from Vitis amurensis

Fig. 8 Identification of VaCBL6-overexpressing lines in Arabidopsis thaliana A：Agarose gel electrophoresis of RNA;B：Expression of VaCBL6 analyzed by semi quantitative RT-PCR;C：Expression level of VaCBL6 analyzed by qRT-PCR. WT：Wild type. ** α = 0.01.

Fig. 9 Phenotypes of Arabidopsis thaliana overexpressing VaCBL6 lines（#1,#2 and #6）on 1/2 MS medium supplemented with different concentrations of NaCl WT：Wild type;#1,#2 and #6：35S::VaCBL6 transgenic lines. The same below.

Fig. 10 Germination rates of Arabidopsis thaliana overexpressing VaCBL6 lines（#1,#2 and #6）under different concentrations of NaCl at 0-7 d

Fig. 11 Germination rates of green cotyledon of seedlings of Arabidopsis thaliana overexpressing VaCBL6 lines （#1,#2 and #6）grown for 10 days under different concentrations of NaCl

Fig. 12 Phenotypes of Arabidopsis thaliana overexpressing VaCBL6 lines（#1,#2 and #6) on 1/2 MS medium supplemented with different concentrations of ABA

Fig. 13 Germination rates of Arabidopsis thaliana overexpressing VaCBL6 lines（#1,#2 and #6） under different concentrations of ABA at 0-7 d

Fig. 14 Germination rates of green cotyledon of seedlings of Arabidopsis thaliana overexpressing VaCBL6 lines （#1,#2 and #6）grown for 10 d under different concentrations of ABA

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VaCBL6 from Vitis amurensis Involved in Abiotic Stress Response and ABA-mediated Pathway

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