Journal of Agricultural Science and Technology ›› 2022, Vol. 24 ›› Issue (8): 44-54.DOI: 10.13304/j.nykjdb.2022.0393
• BIOTECHNOLOGY & LIFE SCIENCE • Previous Articles Next Articles
Qing LU1(), Ting LIANG1,2, Weiwei WANG2, Dezhou WANG2, Xian WU1,2, Xiaoyan WANG1(
), Yimiao TANG2(
)
Received:
2022-05-11
Accepted:
2022-06-02
Online:
2022-08-15
Published:
2022-08-22
Contact:
Xiaoyan WANG,Yimiao TANG
陆青1(), 梁婷1,2, 王伟伟2, 汪德州2, 吴娴1,2, 王小燕1(
), 唐益苗2(
)
通讯作者:
王小燕,唐益苗
作者简介:
陆青 E-mail:luqing41@163.com
基金资助:
CLC Number:
Qing LU, Ting LIANG, Weiwei WANG, Dezhou WANG, Xian WU, Xiaoyan WANG, Yimiao TANG. Cloning and Expression Analysis of Wheat Heat Shock Protein Gene TaHSP90-1[J]. Journal of Agricultural Science and Technology, 2022, 24(8): 44-54.
陆青, 梁婷, 王伟伟, 汪德州, 吴娴, 王小燕, 唐益苗. 小麦热激蛋白基因TaHSP90-1的克隆与表达分析[J]. 中国农业科技导报, 2022, 24(8): 44-54.
引物名称 Primer name | 引物序列 Primer sequence (5’-3’) | 用途 Purpose | 扩增产物长度PCR product length/bp | 退火温度 Temperature/℃ |
---|---|---|---|---|
TaHSP90-1-A-F | TCGAGAAGTGGCAGCGGAGACGGCA | 克隆基因Gene cloning | 2 318 | 62 |
TaHSP90-1-A-R | AGGAAATGAGACTCTTCTTCAATCT | |||
TaHSP90-1-B-F | CCAATCTTCCGGCGAAAGAGAGGCC | 2 236 | 60 | |
TaHSP90-1-B-R | TGAAATGAGATTCTTCAAGAGAT | |||
TaHSP90-1-D-F | TGACAGCCATGGCGGACGTGCAG | 2 230 | 60 | |
TaHSP90-1-D-R | GAGGTCGACTGAAGAATTTCAGT | |||
TaHSP90-1-A-qPCR-F | GGGGGGAGAGACCACAAG | 荧光定量PCR Real-time PCR | 117 | 60 |
TaHSP90-1-A-qPCR-R | CGGAGATGGGATCGCTAGG | |||
TaHSP90-1-B-qPCR-F | GCAGTATGTGTGGGAGTCGC | 194 | 60 | |
TaHSP90-1-B-qPCR-R | TCCAGAGGTAGATGGGGTAG | |||
TaHSP90-1-D-qPCR-F | AGCAGTACGTGTGGGAGTCGC | 199 | 60 | |
TaHSP90-1-D-qPCR-R | TCGGTCCAGAGGTAGATGGGG | |||
TaHSP90-1-A-promoter-F | CAAGTACATGGACCATCTAGCG | 克隆启动子Cloning promoter | 2 115 | 64 |
TaHSP90-1-A-promoter-R | GACCTTAGCAGAAATGGGGAGGC | |||
TaHSP90-1-B-promoter-F | TAACATTGTGAAACTGATTATTT | 2 234 | 60 | |
TaHSP90-1-B-promoter-R | AATGGACCTCAGAAGAAACGGG | |||
TaHSP90-1-D-promoter-F | GAAAAAGATGTTGAATTTATTGT | 2 178 | 60 | |
TaHSP90-1-D-promoter-R | AGGGAAGCGGTTCGCCTTGGCCT | |||
Actin-F | TACTCCCTCACAACAACCG | 内参 Quantitative reference | 317 | 60 |
Actin-R | AGAACCTCCACTGAGAACAA |
Table 1 Primer name and sequence
引物名称 Primer name | 引物序列 Primer sequence (5’-3’) | 用途 Purpose | 扩增产物长度PCR product length/bp | 退火温度 Temperature/℃ |
---|---|---|---|---|
TaHSP90-1-A-F | TCGAGAAGTGGCAGCGGAGACGGCA | 克隆基因Gene cloning | 2 318 | 62 |
TaHSP90-1-A-R | AGGAAATGAGACTCTTCTTCAATCT | |||
TaHSP90-1-B-F | CCAATCTTCCGGCGAAAGAGAGGCC | 2 236 | 60 | |
TaHSP90-1-B-R | TGAAATGAGATTCTTCAAGAGAT | |||
TaHSP90-1-D-F | TGACAGCCATGGCGGACGTGCAG | 2 230 | 60 | |
TaHSP90-1-D-R | GAGGTCGACTGAAGAATTTCAGT | |||
TaHSP90-1-A-qPCR-F | GGGGGGAGAGACCACAAG | 荧光定量PCR Real-time PCR | 117 | 60 |
TaHSP90-1-A-qPCR-R | CGGAGATGGGATCGCTAGG | |||
TaHSP90-1-B-qPCR-F | GCAGTATGTGTGGGAGTCGC | 194 | 60 | |
TaHSP90-1-B-qPCR-R | TCCAGAGGTAGATGGGGTAG | |||
TaHSP90-1-D-qPCR-F | AGCAGTACGTGTGGGAGTCGC | 199 | 60 | |
TaHSP90-1-D-qPCR-R | TCGGTCCAGAGGTAGATGGGG | |||
TaHSP90-1-A-promoter-F | CAAGTACATGGACCATCTAGCG | 克隆启动子Cloning promoter | 2 115 | 64 |
TaHSP90-1-A-promoter-R | GACCTTAGCAGAAATGGGGAGGC | |||
TaHSP90-1-B-promoter-F | TAACATTGTGAAACTGATTATTT | 2 234 | 60 | |
TaHSP90-1-B-promoter-R | AATGGACCTCAGAAGAAACGGG | |||
TaHSP90-1-D-promoter-F | GAAAAAGATGTTGAATTTATTGT | 2 178 | 60 | |
TaHSP90-1-D-promoter-R | AGGGAAGCGGTTCGCCTTGGCCT | |||
Actin-F | TACTCCCTCACAACAACCG | 内参 Quantitative reference | 317 | 60 |
Actin-R | AGAACCTCCACTGAGAACAA |
Fig. 1 Multi sequence alignment of TaHSP90-1 homologous proteinNote:The amino acid sequence on the dark blue background indicates that the sequence is completely matched; the amino acid sequence on the light blue background indicates that the amino acid sequence matching is different; the blue and red underline indicates HATPase_C domain; the black underline indicates HSP90 domain.
Fig. 2 Amplification results and analysis of the full-length sequence and promoter of wheat TaHSP90-1 geneA:Full length amplification of wheat TaHSP90-1 gene; B:Amplification results of wheat TaHSP90-1 promoter; C:Sketch map of structure and sequence variation of gene TaHSP90-1 in Xiaobaimai,Wanmai 33 and Aikang 58; D:Sketch map of structure and sequence variation of gene TaHSP90-1 promoter in Xiaobaimai,Wanmai 33 and Aikang 58. M—DL 2 000 marker; UTR—Untranslated region; CDS—Coding sequence
Fig.3 Phylogenetic analysis of wheat TaHSP90-1 protein and other plant homologous protein sequencesNote:At—Arabidopsis thaliana; Os—Oryza sativa; Tu—Triticum urartu; Hv—Hordeum vulgare; Td—Triticum dicoccoide; Sl—Solanum lycopersicum; St—Solanumtuberosum; Ta—Triticum aestivum.
基因 Gene | 顺式作用元件序列 Cis-acting element sequence (5’-3’) | 顺式作用元件 Cis-acting element | 长度 Length/bp |
---|---|---|---|
TaHSP90-1-A | CAACTG | MBS | 6 |
TaHSP90-1-D | CAACTG | MBS | 6 |
TaHSP90-1-D | CAACTG | MBS | 6 |
TaHSP90-1-A | ACATCCTCCG | HSE | 10 |
TaHSP90-1-A | AGATCCTCTAG | HSE | 11 |
TaHSP90-1-A | GAATCCTTGTG | HSE | 11 |
TaHSP90-1-A | TTTTTCCTTT | HSE | 10 |
TaHSP90-1-A | GGCATCCTGGG | HSE | 11 |
TaHSP90-1-B | AGATTGGAACTCCC | HSE | 14 |
TaHSP90-1-B | CTCACGAATCGCA | HSE | 13 |
TaHSP90-1-B | CTCACGAATCGCA | HSE | 13 |
TaHSP90-1-D | ACGAGAAAATGCA | HSE | 13 |
TaHSP90-1-D | ACAACTCCCCAGT | HSE | 13 |
TaHSP90-1-D | ACAAATCCATGAAC | HSE | 14 |
TaHSP90-1-D | TTTTGAACCGGT | HSE | 12 |
TaHSP90-1-D | ATGGTCCGTATGT | HSE | 13 |
TaHSP90-1-D | CAAGTAGAAGCTTCG | HSE | 15 |
Table 2 Cis-acting element statistics of TaHSP90-1
基因 Gene | 顺式作用元件序列 Cis-acting element sequence (5’-3’) | 顺式作用元件 Cis-acting element | 长度 Length/bp |
---|---|---|---|
TaHSP90-1-A | CAACTG | MBS | 6 |
TaHSP90-1-D | CAACTG | MBS | 6 |
TaHSP90-1-D | CAACTG | MBS | 6 |
TaHSP90-1-A | ACATCCTCCG | HSE | 10 |
TaHSP90-1-A | AGATCCTCTAG | HSE | 11 |
TaHSP90-1-A | GAATCCTTGTG | HSE | 11 |
TaHSP90-1-A | TTTTTCCTTT | HSE | 10 |
TaHSP90-1-A | GGCATCCTGGG | HSE | 11 |
TaHSP90-1-B | AGATTGGAACTCCC | HSE | 14 |
TaHSP90-1-B | CTCACGAATCGCA | HSE | 13 |
TaHSP90-1-B | CTCACGAATCGCA | HSE | 13 |
TaHSP90-1-D | ACGAGAAAATGCA | HSE | 13 |
TaHSP90-1-D | ACAACTCCCCAGT | HSE | 13 |
TaHSP90-1-D | ACAAATCCATGAAC | HSE | 14 |
TaHSP90-1-D | TTTTGAACCGGT | HSE | 12 |
TaHSP90-1-D | ATGGTCCGTATGT | HSE | 13 |
TaHSP90-1-D | CAAGTAGAAGCTTCG | HSE | 15 |
Fig. 5 Expression analysis of wheat TaHSP90-1 in different varieties under drought stressA: 12 h growth of Xiaobaimai and Wanmai 33 under drought stress; B: Expression of TaHSP90-1 gene in Xiaobaimai and Wanmai 33 after drought stress
Fig. 6 Expression analysis of wheat TaHSP90-1 in different varieties under heat stressA: Phenotype of Xiaobaimai and Aikang 58 after heat stress 12 h; B: Expression of TaHSP90-1 gene in Xiaobaimai and Aikang 58 after heat stress
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