Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (3): 218-231.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0802
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WANG Tao1,2(), QI Si-yu1,2, WEI Chao-ling3, WANG Yi-qing1,2, DAI Hao-min1,2, ZHOU Zhe1,2, CAO Shi-xian4, ZENG Wen1,2(), SUN Wei-jiang1,2()
Received:
2022-06-29
Online:
2023-03-26
Published:
2023-04-10
WANG Tao, QI Si-yu, WEI Chao-ling, WANG Yi-qing, DAI Hao-min, ZHOU Zhe, CAO Shi-xian, ZENG Wen, SUN Wei-jiang. Expression Analysis and Interaction Protein Validation of CsPPR and CsCPN60-like in Albino Tea Plant(Camellia sinensis)[J]. Biotechnology Bulletin, 2023, 39(3): 218-231.
基因名称 Gene name | 上游引物 Sense primer(5'-3') | 下游引物 Anti-sense primer(5'-3') | 备注 Note |
---|---|---|---|
CSS0013384 | ATGGCCATCCCCAGTTCTCCAGACTGGTCTGTG | TTACATGGTGCCAAAGCTCTGTTGCAACCATTTTGC | 基因克隆引物 |
CSS0036305 | ATGTACAGAGTTGGTGCAGCCTTCGCTTCATCGAT | TCAGTAACCCATATCATCCAAATTTGGCATTCGAT | 基因克隆引物 |
qCSS0013384 | TGGGGAAAACAGAAGGGCTAA | CAACACCACCACCTGGAACAA | 荧光定量引物 |
qCSS0036305 | CCAAAATGAAGGAAAGAGGGTG | TCAAAGCGGAGCGATAGAGC | 荧光定量引物 |
β-Actin | GCCATCTTTGATTGGAATGG | GGTGCCACAACCTTGATCTT | RNA验证 |
pAbAi- CSS0013384pro | AAATGATGAATTGAAAAGCTTAAGAAATTTTTTGTTGAG | ATACAGAGCACATGCCTCGAGAATTTTGCCTTGCATCAT | 酵母单杂交 |
AD-CSS0002807 | GCCATGGAGGCCAGTGAATTCATGAAAGGAATCATGAA | CTCGATGGATCTCACTTACTTGAGCTAGAA | 酵母单杂交 |
BK-CSS0003881 | ATGGCCATGGAGGCCGAATTCATGGCATCAACTTTGGCT | CCGCTGCAGGTCGACGGATCTTAGTAACCGTATCCTGA | 酵母双杂交 |
BK-CSS0041773 | ATGGCCATGGAGGCCGAATTCATGGCATCAACTTTGGCT | CCGCTGCAGGTCGACGGATCTTAGTAACCGTATCCTGA | 酵母双杂交 |
BK-CSS0043610 | ATGGCCATGGAGGCCGAATTCATGTACCGCTTCGCAG | CCGCTGCAGGTCGACGGATCTCAAAAATCCATACCACC | 酵母双杂交 |
AD-CSS0036305 | GCCATGGAGGCCAGTGAATTCATGTACAGAGTTGGTGC | CAGCTCGAGCTCGATGGATCTCAGTAACCCATATCATCC | 酵母双杂交 |
BK-CSS0036305 | ATGGCCATGGAGGCCGAATTCATGTACAGAGTTGGTGCA | CCGCTGCAGGTCGACGGATCTCAGTAACCCATATCATCC | 酵母双杂交 |
AD-CSS0003881 | GCCATGGAGGCCAGTGAATTCATGGCATCAACTTTGGCT | CAGCTCGAGCTCGATGGATCTTAGTAACCGTATCCTGA | 酵母双杂交 |
AD-CSS0041773 | GCCATGGAGGCCAGTGAATTCATGGCATCAACTTTGGCT | CAGCTCGAGCTCGATGGATCTTAGTAACCGTATCCTGA | 酵母双杂交 |
AD-CSS0043610 | GCCATGGAGGCCAGTGAATTCATGTACCGCTTCGCAG | CAGCTCGAGCTCGATGGATCTCAAAAATCCATACCAC | 酵母双杂交 |
Table 1 Primers required for the experiment
基因名称 Gene name | 上游引物 Sense primer(5'-3') | 下游引物 Anti-sense primer(5'-3') | 备注 Note |
---|---|---|---|
CSS0013384 | ATGGCCATCCCCAGTTCTCCAGACTGGTCTGTG | TTACATGGTGCCAAAGCTCTGTTGCAACCATTTTGC | 基因克隆引物 |
CSS0036305 | ATGTACAGAGTTGGTGCAGCCTTCGCTTCATCGAT | TCAGTAACCCATATCATCCAAATTTGGCATTCGAT | 基因克隆引物 |
qCSS0013384 | TGGGGAAAACAGAAGGGCTAA | CAACACCACCACCTGGAACAA | 荧光定量引物 |
qCSS0036305 | CCAAAATGAAGGAAAGAGGGTG | TCAAAGCGGAGCGATAGAGC | 荧光定量引物 |
β-Actin | GCCATCTTTGATTGGAATGG | GGTGCCACAACCTTGATCTT | RNA验证 |
pAbAi- CSS0013384pro | AAATGATGAATTGAAAAGCTTAAGAAATTTTTTGTTGAG | ATACAGAGCACATGCCTCGAGAATTTTGCCTTGCATCAT | 酵母单杂交 |
AD-CSS0002807 | GCCATGGAGGCCAGTGAATTCATGAAAGGAATCATGAA | CTCGATGGATCTCACTTACTTGAGCTAGAA | 酵母单杂交 |
BK-CSS0003881 | ATGGCCATGGAGGCCGAATTCATGGCATCAACTTTGGCT | CCGCTGCAGGTCGACGGATCTTAGTAACCGTATCCTGA | 酵母双杂交 |
BK-CSS0041773 | ATGGCCATGGAGGCCGAATTCATGGCATCAACTTTGGCT | CCGCTGCAGGTCGACGGATCTTAGTAACCGTATCCTGA | 酵母双杂交 |
BK-CSS0043610 | ATGGCCATGGAGGCCGAATTCATGTACCGCTTCGCAG | CCGCTGCAGGTCGACGGATCTCAAAAATCCATACCACC | 酵母双杂交 |
AD-CSS0036305 | GCCATGGAGGCCAGTGAATTCATGTACAGAGTTGGTGC | CAGCTCGAGCTCGATGGATCTCAGTAACCCATATCATCC | 酵母双杂交 |
BK-CSS0036305 | ATGGCCATGGAGGCCGAATTCATGTACAGAGTTGGTGCA | CCGCTGCAGGTCGACGGATCTCAGTAACCCATATCATCC | 酵母双杂交 |
AD-CSS0003881 | GCCATGGAGGCCAGTGAATTCATGGCATCAACTTTGGCT | CAGCTCGAGCTCGATGGATCTTAGTAACCGTATCCTGA | 酵母双杂交 |
AD-CSS0041773 | GCCATGGAGGCCAGTGAATTCATGGCATCAACTTTGGCT | CAGCTCGAGCTCGATGGATCTTAGTAACCGTATCCTGA | 酵母双杂交 |
AD-CSS0043610 | GCCATGGAGGCCAGTGAATTCATGTACCGCTTCGCAG | CAGCTCGAGCTCGATGGATCTCAAAAATCCATACCAC | 酵母双杂交 |
Fig. 1 Gene amplification electropherogram and protein structure prediction a: Electrophoresis of PCR amplification of CSS0013384 and CSS0036305 in C. sinensis. b: Conserved domain prediction of CSS0013384 in C. sinensis. c: Motif distribution map of CSS0013384. d: Conserved domain prediction of CSS0036305 in C. sinensis
基因 Gene | 分子量 Mw/kD | 等电点 pI | 不稳定系数 Instability index | 脂肪族氨基酸指数Aliphatic index | 平均疏水性 GRAVY | 信号肽 Signal peptide | 跨膜结构 Transmembrane domain | 亚细胞定位预测 Subcellular localization |
---|---|---|---|---|---|---|---|---|
CSS0013384 | 71.87 | 8.93 | 40.73 | 85.90 | -0.167 | 无 | 无 | 叶绿体 |
CSS0036305 | 60.79 | 6.21 | 21.85 | 103.99 | 0.002 | 无 | 无 | 线粒体 |
Table 2 Basic physicochemical properties of CSS0013384 and CSS0036305 protein
基因 Gene | 分子量 Mw/kD | 等电点 pI | 不稳定系数 Instability index | 脂肪族氨基酸指数Aliphatic index | 平均疏水性 GRAVY | 信号肽 Signal peptide | 跨膜结构 Transmembrane domain | 亚细胞定位预测 Subcellular localization |
---|---|---|---|---|---|---|---|---|
CSS0013384 | 71.87 | 8.93 | 40.73 | 85.90 | -0.167 | 无 | 无 | 叶绿体 |
CSS0036305 | 60.79 | 6.21 | 21.85 | 103.99 | 0.002 | 无 | 无 | 线粒体 |
Fig. 2 Gene chromosomal location, gene structure and protein tertiary structure a: Chromosomal localization of CSS0013384 and CSS0036305 in tea plant(C. sinensis). b: Gene structure of CSS0013384 and CSS0036305 in tea plant. c: The tertiary structure prediction of CSS0013384 in tea plant. d: The tertiary structure prediction of CSS0036305 in tea plant
Fig. 3 Genetic tree a: PPR neighbor-joining tree of CSS0036305 in tea pant and other plants. b: CPN60-like neighbor-joining tree of CSS0036305 in tea plant and other plants
类型Type | 基因名称Gene name | 注释Function | |
---|---|---|---|
CSS0013384 | CSS0036305 | ||
光响应 Light response | A-box(2)、ATCT-motif(1)、Box 4(6)、Box Ⅱ(1)、GA-motif(1)、GATA-motif(1)、G-box(5)、I-box(1) | Box 4(1)、Box Ⅱ(1)、GATA-motif(3)、G-box(2)、GT1-motif(2)、TCCC-motif(1)、TCT-motif(2)、 | 参与光反应的顺式作用元件cis-acting element involved in light responsiveness |
转录因子结合位点 Transcription factor binding site | MRE(1)、MBS(1)、MBSI(1)、Myb-binding site(1) | HD-Zip3(1)、MRE(1)、MBS(2) | 转录因子结合位点 Transcription factor binding site |
激素响应 Hormone response | AAGAA-motif(1)、ABRE(2)、ABRE2(1) | — | 参与脱落酸反应的顺式作用元件cis-acting element involved in the abscisic acid responsiveness |
ERE(3) | — | 参与乙烯反应的顺式作用元件 cis-acting element involved in ethylene responsiveness | |
TCA-element(1) | TCA-element(2) | 与水杨酸反应有关的顺式作用元件 cis-acting element involved in salicylic acid responsiveness | |
— | as-1(3)、CGTCA-motif(3)、TGACG-motif(3) | 参与MeJA反应的顺式作用元件 cis-acting element involved in MeJA responsiveness | |
组织特异性 Tissue specificity | CAT-box(2)、CCGTCC motif(4)、GCN4 motif(1) | — | 组织特异性元件 Tissue specific element |
胁迫响应 Stress response | ARE(1)、STRE(1)、WUN-motif(4) | ACE(1)、ARE(4)、STRE(2)、WRE3(2)、WUN-motif(2) | 胁迫响应元件Stress response element |
Table 3
类型Type | 基因名称Gene name | 注释Function | |
---|---|---|---|
CSS0013384 | CSS0036305 | ||
光响应 Light response | A-box(2)、ATCT-motif(1)、Box 4(6)、Box Ⅱ(1)、GA-motif(1)、GATA-motif(1)、G-box(5)、I-box(1) | Box 4(1)、Box Ⅱ(1)、GATA-motif(3)、G-box(2)、GT1-motif(2)、TCCC-motif(1)、TCT-motif(2)、 | 参与光反应的顺式作用元件cis-acting element involved in light responsiveness |
转录因子结合位点 Transcription factor binding site | MRE(1)、MBS(1)、MBSI(1)、Myb-binding site(1) | HD-Zip3(1)、MRE(1)、MBS(2) | 转录因子结合位点 Transcription factor binding site |
激素响应 Hormone response | AAGAA-motif(1)、ABRE(2)、ABRE2(1) | — | 参与脱落酸反应的顺式作用元件cis-acting element involved in the abscisic acid responsiveness |
ERE(3) | — | 参与乙烯反应的顺式作用元件 cis-acting element involved in ethylene responsiveness | |
TCA-element(1) | TCA-element(2) | 与水杨酸反应有关的顺式作用元件 cis-acting element involved in salicylic acid responsiveness | |
— | as-1(3)、CGTCA-motif(3)、TGACG-motif(3) | 参与MeJA反应的顺式作用元件 cis-acting element involved in MeJA responsiveness | |
组织特异性 Tissue specificity | CAT-box(2)、CCGTCC motif(4)、GCN4 motif(1) | — | 组织特异性元件 Tissue specific element |
胁迫响应 Stress response | ARE(1)、STRE(1)、WUN-motif(4) | ACE(1)、ARE(4)、STRE(2)、WRE3(2)、WUN-motif(2) | 胁迫响应元件Stress response element |
Fig. 4 Expression patterns of genes in different parts of tea plant a: Expression pattern of CSS0013384 in different tissues of tea plant. b: Expression pattern of CSS0036305 in different tissues of tea plant. Different lower letters mean the significant difference in P<0.05
Fig. 5 Gene expression patterns of genes from different tea cultivars under shading and restored light a: Expression pattern analysis of tea plant CSS0013384 under shading and restored light. b: Expression pattern analysis of tea plant CSS0036305 under shading and restored light. c: Expression analysis of CSS0013384 in different tea plant varieties. d: Expression analysis of CSS0036305 in different tea plant varieties. N0: No shading. S1: Shading 1 d. S3: Shading 3 d. RL1: Restored light for 1 d. RL2: Restored light for 2 d. MD: Jinmudan. AJBC: Anjibaiha. BJG: Baijiguan. JG: Jinguang. QNX: Qiannianxue. ZH1: Zhonghuang 1. HYZ: Huangyazao. HJC: Huangjincha. HH: Albino tea plant germplasm. HJY: Huangjinya. YJX: Yujinxiang. Different letters indicate significant differences between samples(P<0.05). Relative expressions of genes under shading and restored light were compared with N0, and the gene expression patterns among different tea varieties were compared with Jinmudan(MD)
Fig. 6 Gene interaction network prediction map a: Protein interaction network of CSS0013384 in tea plant. b: Protein interaction network of CSS0036305 in tea plant. Diamond represents transcription factor. Circle represents functional gene
靶蛋白 Target protein | 互作蛋白 Interacting protein | 基因注释 Gene annotation |
---|---|---|
CSS0036305 | CSS0029920 | TCP-1/cpn60 伴侣蛋白家族蛋白 TCP-1/cpn60 chaperonin family protein |
CSS0043610 | TCP-1/cpn60 伴侣蛋白家族蛋白 TCP-1/cpn60 chaperonin family protein | |
CSS0003881 | β Rubisco大亚基结合蛋白亚基 Rubisco large subunit-binding protein subunit beta | |
CSS0041773 | β Rubisco大亚基结合蛋白亚基 Rubisco large subunit-binding protein subunit beta | |
CSS0036200 | Rubisco亚基结合蛋白β亚基 Rubisco subunit binding-protein beta subunit | |
CSS0044373 | GAGA结合转录激活因子 GAGA-binding transcriptional activator | |
CSS0007067 | GAGA结合转录激活因子 GAGA-binding transcriptional activator | |
CSS0008658 | GAGA结合转录激活因子 GAGA-binding transcriptional activator | |
CSS0046266 | MYB_related蛋白 MYB_related protein | |
CSS0035646 | MYB_related蛋白 MYB_related protein | |
CSS0028905 | MADS-box蛋白 MADS-box protein | |
CSS0013384 | CSS0002939 | 鸟嘌呤核苷酸结合蛋白亚基β-蛋白异构体X2 Guanine nucleotide-binding protein subunit beta-like protein isoform X2 |
CSS0002807 | PIF转录因子 Transcription factor PIF7 | |
CSS0028905 | MADS家族蛋白 MADS-box protein |
Table 4 Functional annotations of interacting proteins of CSS0013384 and CSS0036305
靶蛋白 Target protein | 互作蛋白 Interacting protein | 基因注释 Gene annotation |
---|---|---|
CSS0036305 | CSS0029920 | TCP-1/cpn60 伴侣蛋白家族蛋白 TCP-1/cpn60 chaperonin family protein |
CSS0043610 | TCP-1/cpn60 伴侣蛋白家族蛋白 TCP-1/cpn60 chaperonin family protein | |
CSS0003881 | β Rubisco大亚基结合蛋白亚基 Rubisco large subunit-binding protein subunit beta | |
CSS0041773 | β Rubisco大亚基结合蛋白亚基 Rubisco large subunit-binding protein subunit beta | |
CSS0036200 | Rubisco亚基结合蛋白β亚基 Rubisco subunit binding-protein beta subunit | |
CSS0044373 | GAGA结合转录激活因子 GAGA-binding transcriptional activator | |
CSS0007067 | GAGA结合转录激活因子 GAGA-binding transcriptional activator | |
CSS0008658 | GAGA结合转录激活因子 GAGA-binding transcriptional activator | |
CSS0046266 | MYB_related蛋白 MYB_related protein | |
CSS0035646 | MYB_related蛋白 MYB_related protein | |
CSS0028905 | MADS-box蛋白 MADS-box protein | |
CSS0013384 | CSS0002939 | 鸟嘌呤核苷酸结合蛋白亚基β-蛋白异构体X2 Guanine nucleotide-binding protein subunit beta-like protein isoform X2 |
CSS0002807 | PIF转录因子 Transcription factor PIF7 | |
CSS0028905 | MADS家族蛋白 MADS-box protein |
Fig. 7 Yeast one-hybrid and yeast two-hybrid results a: CSS0013384 promoter self-activation detection. b: Results of yeast one-hybrid for CSS0013384. c: Results of yeast two-hybrid for CSS0036305. SD/-Leu+AbA: SD medium lacking leucine supplemented with aureobasidin A. SD/-Trp/-Leu: SD medium lacking tryptophan and leucine. SD/-Trp/-Leu/- Ade/-Leu: SD medium lacking tryptophan, histidine, adenine and leucine. SD/-Trp/-Leu/-Ade/-Leu/X-α-gal: SD medium lacking tryptophan, histidine, adenine and leucine supplemented with X-α-Gal
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