The Grapevine Calmodulin-Like Protein Gene CML21 Is Regulated by Alternative Splicing and Involved in Abiotic Stress Response
Abstract
:1. Introduction
2. Results
2.1. VaCML21 Is Regulated by Alternative Splicing
2.2. CML21 Expression Profile under Different Abiotic Stresses
2.3. Genetic Plant Transformation with the VaCML21v2 and VaCML21v4 Splice Variants and Selection of the Transformants
2.4. Stress Tolerance of VaCML21-Overexpressing V. amurensis Cell Cultures and A. thaliana Plants
2.5. Transformation with VaCML21 Enhanced Transcription of Cold-Inducible Regulatory Genes in Stressed Arabidopsis
3. Discussion
4. Materials and Methods
4.1. Plant Materials and Growth Conditions
4.2. Isolation and Sequencing of VaCML21 Spliced Transcript Variants
4.3. Generation of Transgenic Grapevine Cell Cultures
4.4. Generation of Transgenic Arabidopsis Plants
4.5. RNA Isolation and cDNA Synthesis
4.6. Gene Expression Analysis by qRT-PCR
4.7. V. vinifera RNAseq Library Analsis
4.8. Abiotic Stress Treatments of Transgenic Cell Cultures and Plants
4.9. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Vitis amurensis (cDNA Clone) | No. of EF Hands | Predicted Lipid Modification Sites (Position) | MW (kDA) | Vitis vinifera (mRNA Prediction) | No. of EF Hands | Predicted Lipid Modification Sites (Position) | MW (kDA) | ||||
---|---|---|---|---|---|---|---|---|---|---|---|
Transcript Variant | ID (NCBI GenBank) | CDS (Amino Acids) | Transcript Variant | ID | CDS (Amino Acids) | ||||||
nd | nd | nd | nd | nd | nd | VviCML21v.1 | VIT_219s0015g01200.1 | 708 (235) | 4 | N-Myrist(2,6,8) | 26.9 |
VaCML21v.2 | MN540599 | 699 (232) | 4 | N-Myrist(2,6,8) N-Palmit(120) | 26.4 | VviCML21v.2 | VIT_219s0015g01200.2 | 699 (232) | 4 | N-Myrist(2,6,8) N-Palmit(120) | 26.5 |
VaCML21v.3 | MW033207 | 594 (197) | 4 | N-Palmit(3) | 22.7 | VviCML21v.3 | VIT_219s0015g01200.7 | 594 (197) | 4 | N-Palmit(3) | 22.7 |
VaCML21v.4 | MW033208 | 585 (194) | 4 | N-Palmit(3,82) | 22.4 | VviCML21v.4 | VIT_219s0015g01200.8 | 585 (194) | 4 | N-Palmit(3,82) | 22.4 |
Transcript Name (GenBankID) | Primers, 5′–3′ |
---|---|
Cloning and sequencing full-length cDNA coding sequences of VaCML21, 5′–3′ | |
CML21v1/v2 (no sequence/MN540599) | F1:ATGGGAGGCGTGGTGGG R:TCAAACTTTCTCTTCACCTTC |
CML21v3/v4 (MW033207/ MW033208) | F2:ATGCTGTGTATCATCCTTCATG R:TCAAACTTTCTCTTCACCTTC |
Cloning VaCML21v2 and VaCML21v4 for plant transformation by the BglII and SalI sites (underlined), 5’–3’ | |
VaCML21v2 (MN540599) | F:GCTCAGATCTATGGGAGGCGTGGTGGGAAAA R:TCGAGTCGACTTAAACTTTCTCTTCACCTTC |
VaCML21v4 (MW033208) | F:GCTCAGATCTATGCTGTGTATCATCCTTCAT R:TCGAGTCGACTCAAACTTTCTCTTCACCTT |
Primers for real-time PCR, 5′–3′ | |
VaCML21v1 (no sequence) | GAACTGCAAAGCTATTTTTCAGCA CCCATCCGTGATTTTTTGGATAC |
VaCML21v2 (MN540599) | GAACTGCAAAGCTATTTTTCAGCA ATCCGTGATTTGGCCTGAAGG |
VaCML21v3 (MW033207) | TGCCAGAGGTTGAAATTCGTTGTT CCCATCCGTGATTTTTTGGATAC |
VaCML21v4 (MW033208) | TGCCAGAGGTTGAAATTCGTTGTT ATCCGTGATTTGGCCTGAAGG |
VaActin1 (DQ517935) | GTATTGTGCTGGATTCTGGTGAT AGCAAGGTCAAGACGAAGGATAG |
VaGAPDH (XM_002263109) | CACTGAAGATGATGTTGTTTCC GCTATTCCAGCCTTGGCAT |
AtGAPDH (NM_111283.4) | TTGGTGACAACAGGTCAAGCA AAACTTGTCGCTCAATGCAAT |
AtEF1a (XM_002864638) | TGAGCACGCTCTTCTTGCTTTCA GGTGGTGGCATCCATCTTGTTACA |
Primers for cDNA check-up on DNA contamination, 5′–3′ | |
VaActin1 (DQ517935) | TTGCCATTCAGGCTGTTCTTTCT AGGAGCTGCTCTTTGCAGTTTCC |
AtActin1 (NM_112764) | GATTCAGATGCCCAGAAGTC TCTGTGAACGATTCCTGGA |
Primers for qRT-PCR quantification of the VaCML21v2 and VaCML21v4 transgene and endogene mRNAs in transformed plant cells, 5′–3′ | |
VaCML21v2 transgene (MN540599) | CAAGCATTCTACTTCTATTG ACCATTTTGGCCTCAAGC |
VaCML21v1/v2 endogene (no sequence/MN540599) | CATACTTAGCACTTGTCCCTTTTCC ACCATTTTGGCCTCAAGC |
VaCML21v4 transgene (MW033208) | CAAGCATTCTACTTCTATTG TCTTCATAGATGAGGATTCAAATG |
VaCML21v3/v4 endogene (MW033207/ MW033208) | TGAACCAGTTTTCATTTTAATTCAACGTG TCTTCATAGATGAGGATTCAAATG |
Sangiovese | Cabernet Franc | Cultivation Conditions a | ||
---|---|---|---|---|
SRR6026721 | SRR6026711 | SRR6026737 | SRR6026699 | Control |
SRR6026722 | SRR6026712 | SRR6026736 | SRR6026700 | Freezing (−3) for 45 min |
SRR6026716 | SRR6026735 | SRR6026738 | SRR6026698 | Chilling stress (+4) for 48 h |
SRR6026715 | SRR6026734 | SRR6026739 | SRR6026697 | Acclimated freezing (+4 → −3) |
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Aleynova, O.A.; Kiselev, K.V.; Ogneva, Z.V.; Dubrovina, A.S. The Grapevine Calmodulin-Like Protein Gene CML21 Is Regulated by Alternative Splicing and Involved in Abiotic Stress Response. Int. J. Mol. Sci. 2020, 21, 7939. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21217939
Aleynova OA, Kiselev KV, Ogneva ZV, Dubrovina AS. The Grapevine Calmodulin-Like Protein Gene CML21 Is Regulated by Alternative Splicing and Involved in Abiotic Stress Response. International Journal of Molecular Sciences. 2020; 21(21):7939. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21217939
Chicago/Turabian StyleAleynova, Olga A., Konstantin V. Kiselev, Zlata V. Ogneva, and Alexandra S. Dubrovina. 2020. "The Grapevine Calmodulin-Like Protein Gene CML21 Is Regulated by Alternative Splicing and Involved in Abiotic Stress Response" International Journal of Molecular Sciences 21, no. 21: 7939. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21217939