Chinese Bulletin of Botany ›› 2024, Vol. 59 ›› Issue (4): 558-573.DOI: 10.11983/CBB23129 cstr: 32102.14.CBB23129
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Laipeng Zhao1,2, Baike Wang2, Tao Yang2, Ning Li2, Haitao Yang2, Juan Wang2,*(), Huizhuan Yan1,*()
Received:
2023-09-15
Accepted:
2023-12-19
Online:
2024-07-10
Published:
2024-07-10
Contact:
*E-mail: drjuanwang@126.com; hzhyan1118@163.com
Laipeng Zhao, Baike Wang, Tao Yang, Ning Li, Haitao Yang, Juan Wang, Huizhuan Yan. Investigation of the Regulation of Drought Tolerance by the SlHVA22l Gene in Tomato[J]. Chinese Bulletin of Botany, 2024, 59(4): 558-573.
Primer name | Forward primer (5'-3') | Reverse primer (5'-3') |
---|---|---|
SlHVA22l | GGCATGGCTAGTTTTTCCACATT | TGGGAGTGATGAAGTCCACAAA |
SlPDS | GTGAACCCTGTCGGCCTTTA | AACTACGCTTGCTTCCGACA |
Slactin | AGCAGGAACTTGAAACCGCT | CTCATGGATACCCGCAGCTT |
SlDREB1A | GGTTTGTGTGCCGTTGGATT | AGGAACTTTACGCACTGGCT |
SlHK1 | GCTCTGAGTGGGTTTGCTCT | GGCAAAGACTCTCTGGCCTT |
SlAREB1 | AATGAGGGCAGGGATGGTTG | TCCATTGCTCTTCCCAAGTCC |
SlPYL9 | CGGTTCATCCCGAGGTCATT | GTACCGCCAAACGCTCTGAT |
SlAVP1 | CAACACCGCAAATATCGCCC | CGTCCATACCAGCCATCTCC |
SlABI1 | AGTCAGCCGCAGTGTTTTTG | AGACAGTCCATCAAGCACGC |
SlAAPK | TCTGGAGGGGAGCTGTTTGA | GTGCAGGACTTCCATCCAGT |
SlMPK8 | CCAAGGTACCCAAGGCAACA | GCCTCGGACAAACAGGTTCT |
Table 1 Primers for qRT-PCR
Primer name | Forward primer (5'-3') | Reverse primer (5'-3') |
---|---|---|
SlHVA22l | GGCATGGCTAGTTTTTCCACATT | TGGGAGTGATGAAGTCCACAAA |
SlPDS | GTGAACCCTGTCGGCCTTTA | AACTACGCTTGCTTCCGACA |
Slactin | AGCAGGAACTTGAAACCGCT | CTCATGGATACCCGCAGCTT |
SlDREB1A | GGTTTGTGTGCCGTTGGATT | AGGAACTTTACGCACTGGCT |
SlHK1 | GCTCTGAGTGGGTTTGCTCT | GGCAAAGACTCTCTGGCCTT |
SlAREB1 | AATGAGGGCAGGGATGGTTG | TCCATTGCTCTTCCCAAGTCC |
SlPYL9 | CGGTTCATCCCGAGGTCATT | GTACCGCCAAACGCTCTGAT |
SlAVP1 | CAACACCGCAAATATCGCCC | CGTCCATACCAGCCATCTCC |
SlABI1 | AGTCAGCCGCAGTGTTTTTG | AGACAGTCCATCAAGCACGC |
SlAAPK | TCTGGAGGGGAGCTGTTTGA | GTGCAGGACTTCCATCCAGT |
SlMPK8 | CCAAGGTACCCAAGGCAACA | GCCTCGGACAAACAGGTTCT |
Figure 1 Amino acid sequence analysis of SlHVA22l (A) Phylogenetic tree of the HVA22l protein in eukaryotic organisms; (B) Phylogenetic tree of HVA22l proteins in dicotyledonous and monocotyledonous plants; (C) Comparison of homologous proteins between SlHVA22l and other plant species through multiple sequence alignment; (D) The TB2/DP1 domain. Cs: Citrus sinensis; Gh: Gossypium hirsutum; Tc: Theobroma cacao; Vv: Vitis vinifera; Cas: Camellia sinensis; Ah: Arachis hypogaea; Coa: Coffea arabica; Nt: Nicotiana tomentosiformis; Ca: Capsicum annuum; Sl: Solanum lycopersicum; St: So. tuberosum; Mb: Musa balbisiana; Zo: Zingiber officinale; Os: Oryza sativa; Bd: Brachypodium distachyon; Ta: Triticum aestivum; Hv: Hordeum vulgare; Sb: Sorghum bicolor; Zm: Zea mays; Si: Setaria italica; Sv: Se. viridis; Pv: Panicum virgatum; Pm: P. miliaceum; Ph: P. hallii
Figure 2 Cis-acting elements and expression pattern analysis of the SlHVA22l gene (A) Cis-acting elements within the 3 000 bp upstream promoter region of the SlHVA22l gene; (B) Tissue-specific expression of the SlHVA22l gene; (C)-(I) SlHVA22l expression pattern under PEG6000, ABA (100, 200, and 300 µmol∙L-1), and MeJA (100, 200, and 300 µmol∙L-1) treatments at 0-24 h. Slactin was used as a reference gene. Different lowercase letters indicate significant differences among different treatments (P<0.05).
Figure 3 Subcellular localization of SlHVA22l (A) Schematic representation of SlHVA22l in the pCAMBIA1300-GFP vector; (B) Localization of the SlHVA22l protein in Ben’s tobacco cells. HDEL is an endoplasmic reticulum localization marker. Bars=50 μm
Figure 4 Expression analysis of SlHVA22l in Saccharomyces cerevisiae (A) Responses of recombinant pYES2-SlHVA22l and control pYES2 under simulated drought conditions; (B) Growth kinetics of recombinant pYES2-SlHVA22l and control pYES2 under drought stress; (C) Drip plate experiment of recombinant pYES2- SlHVA22l and control pYES2 under drought stress. * P<0.05; ** P<0.01; *** P<0.001
Figure 5 The effect of SlHVA22l gene silencing on drought tolerance in tomato (A), (B) TRV::SlPDS-carrying Agrobacterium was introduced into 2-week-old tomato plants, after two weeks, phenotypic changes (A) and the efficiency of SlPDS silencing was quantified using qRT-PCR (B); (C) The phenotypes of the control and silenced plants were visually documented and recorded 48 hours after treatment with a 20% PEG6000 solution; (D) The silencing efficiency of SlHVA22l was assessed via qRT-PCR analysis; (E) DAB and NBT staining of leaves from drought-stressed control and silenced plants, respectively, were performed before and after the stress treatment; (F) H2O2 contents; (G) O2-. clearance rate; (H) Malondialdehyde (MDA) contents; (I) Identification of silenced plants; (J) Water loss rate; (K) Dehydration phenotype of detached leaves. CK: TRV::00; SG: TRV::SlHVA22l. * P<0.05; ** P<0.01; *** P<0.001. (A), (C) Bars=3 cm; (E), (K) Bars=1 cm
Figure 6 The antioxidant enzyme activities in SlHVA22l silenced tomato leaf under drought stress (A) Peroxidase (POD) activity; (B) Superoxide dismutase (SOD) activity; (C) Catalase (CAT) activity; (D) Ascorbate peroxidase (APX) activity. ** P<0.01; *** P<0.001
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