Biotechnology Bulletin ›› 2024, Vol. 40 ›› Issue (4): 85-96.doi: 10.13560/j.cnki.biotech.bull.1985.2023-1066
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YANG Qi(
), WEI Zi-di, SONG Juan, TONG Kun, YANG Liu, WANG Jia-han, LIU Hai-yan, LUAN Wei-jiang(), MA Xuan()
Received:2023-11-13
Online:2024-04-26
Published:2024-04-30
Contact:
LUAN Wei-jiang, MA Xuan
E-mail:18722163533@163.com;skylwj@tjnu.edu.cn;skyxma@tjnu.edu.cn
YANG Qi, WEI Zi-di, SONG Juan, TONG Kun, YANG Liu, WANG Jia-han, LIU Hai-yan, LUAN Wei-jiang, MA Xuan. Construction and Transcriptomic Analysis of Rice Histone H1 Triple Mutant[J]. Biotechnology Bulletin, 2024, 40(4): 85-96.
| 引物名称Primer name | 引物序列Primer sequence（5'-3'） | 用途Purpose |
|---|---|---|
| U-F | CTCCGTTTTACCTGTGGAATCG | CRISPR载体构建 CRISPR vector construction |
| gR-R | CGGAGGAAAATTCCATCCAC | |
| OsH1-T1-gRT1 | TACCTCGGCGTACGGCGGGT | |
| OsH1-T1-U6aT1 | ACCCGCCGTACGCCGAGGTAC | |
| OsH1-T2-gRT1 | ATGATCAAGGAGGCGATCA | |
| OsH1-T2-U6aT1 | TGATCGCCTCCTTGATCATC | |
| PpS-GGL | TTCAGAGGTCTCTCTCGACTAGTATGGAATCGGCAGCAAAGG | |
| PgS-GG2 | AGCGTGGGTCTCGTCAGGGTCCATCCACTCCAAGCTC | |
| PpS-GG2 | AGCGTGGGTCTCGTCAGGGTCCATCCACTCCAAGCTC | |
| PgS-GGR | AGCGTGGGTCTCGACCGACGCGTATCCATCCACTCCAAGCTC | |
| OsH1.4F2 | AGCCGGCGAAGGAGAAGAAGAAG | 靶点1检测 Target 1 detection |
| OsH1.4R2 | GGAGAGCTTGAAGGAGTTCTTG | |
| OsH1.3F2 | CTTCGGATAGCGGCAGTAGTCAG | |
| OsH1.3R2 | GCGGCAACTTGTAGGAGTTCTTG | |
| OsH1.2F | AGGCGGAGGGTGAGAAGGAGAAG | 靶点2检测 Target 2 detection |
| OsH1.2R2 | TTCTTCTCCTCGGCAGCCGAC | |
| OsH1.1F2 | TTGGGTTTGGCGGCGTCCTTC | |
| OsH1.1R2 | CAACTTCTCAGCCATGCTCAC | |
| Os04g30420-qRT-F | CAACTTCTCAGCCATGCTCAC | 实时定量PCR RT-qPCR |
| Os04g30420-qRT-R | TCTCAACGACTATCTTGCCGG | |
| Os08g33710-qRT-F | TACACGCTGTCCCAGATCAAG | |
| Os08g33710-qRT-R | TAGAACGCCGGGAACTCGAT | |
| Os03g63950-qRT-F | AGCAGCTAGTGAATGTGGACC | |
| Os03g63950-qRT-R | AGAGTTGGGGATGGTCTCCTT | |
| OsActin-qRT-F | GACTCTGGTGATGGTGTCAGC | |
| OsActin-qRT-R | GGCTGGAAGAGGACCTCAGG | |
| OsH1.1-qRT-F | GTTAAGGCCTCCTACAAGCTCTC | |
| OsH1.1-qRT-R | CTTGGCGACCACCTTCTTCTC | |
| OsH1.2-qRT-F | GAAGGTGAAGGCCTCGTTCAAG | |
| OsH1.2-qRT-R | CTTGTTGGCCTTCTTGGAGATG | |
| OsH1.3-qRT-F | GTGACGAAGACGAAGGCGAC | |
| OsH1.3-qRT-R | CTACTTCTTCGCCTTCCGGG | |
| OsH1.4-qRT-F | CGACCAAGACCAAGATCAAGGT | |
| OsH1.4-qRT-R | CTACTTCATGCTCTTCCTCGCC |
Table 1 Primer information
| 引物名称Primer name | 引物序列Primer sequence（5'-3'） | 用途Purpose |
|---|---|---|
| U-F | CTCCGTTTTACCTGTGGAATCG | CRISPR载体构建 CRISPR vector construction |
| gR-R | CGGAGGAAAATTCCATCCAC | |
| OsH1-T1-gRT1 | TACCTCGGCGTACGGCGGGT | |
| OsH1-T1-U6aT1 | ACCCGCCGTACGCCGAGGTAC | |
| OsH1-T2-gRT1 | ATGATCAAGGAGGCGATCA | |
| OsH1-T2-U6aT1 | TGATCGCCTCCTTGATCATC | |
| PpS-GGL | TTCAGAGGTCTCTCTCGACTAGTATGGAATCGGCAGCAAAGG | |
| PgS-GG2 | AGCGTGGGTCTCGTCAGGGTCCATCCACTCCAAGCTC | |
| PpS-GG2 | AGCGTGGGTCTCGTCAGGGTCCATCCACTCCAAGCTC | |
| PgS-GGR | AGCGTGGGTCTCGACCGACGCGTATCCATCCACTCCAAGCTC | |
| OsH1.4F2 | AGCCGGCGAAGGAGAAGAAGAAG | 靶点1检测 Target 1 detection |
| OsH1.4R2 | GGAGAGCTTGAAGGAGTTCTTG | |
| OsH1.3F2 | CTTCGGATAGCGGCAGTAGTCAG | |
| OsH1.3R2 | GCGGCAACTTGTAGGAGTTCTTG | |
| OsH1.2F | AGGCGGAGGGTGAGAAGGAGAAG | 靶点2检测 Target 2 detection |
| OsH1.2R2 | TTCTTCTCCTCGGCAGCCGAC | |
| OsH1.1F2 | TTGGGTTTGGCGGCGTCCTTC | |
| OsH1.1R2 | CAACTTCTCAGCCATGCTCAC | |
| Os04g30420-qRT-F | CAACTTCTCAGCCATGCTCAC | 实时定量PCR RT-qPCR |
| Os04g30420-qRT-R | TCTCAACGACTATCTTGCCGG | |
| Os08g33710-qRT-F | TACACGCTGTCCCAGATCAAG | |
| Os08g33710-qRT-R | TAGAACGCCGGGAACTCGAT | |
| Os03g63950-qRT-F | AGCAGCTAGTGAATGTGGACC | |
| Os03g63950-qRT-R | AGAGTTGGGGATGGTCTCCTT | |
| OsActin-qRT-F | GACTCTGGTGATGGTGTCAGC | |
| OsActin-qRT-R | GGCTGGAAGAGGACCTCAGG | |
| OsH1.1-qRT-F | GTTAAGGCCTCCTACAAGCTCTC | |
| OsH1.1-qRT-R | CTTGGCGACCACCTTCTTCTC | |
| OsH1.2-qRT-F | GAAGGTGAAGGCCTCGTTCAAG | |
| OsH1.2-qRT-R | CTTGTTGGCCTTCTTGGAGATG | |
| OsH1.3-qRT-F | GTGACGAAGACGAAGGCGAC | |
| OsH1.3-qRT-R | CTACTTCTTCGCCTTCCGGG | |
| OsH1.4-qRT-F | CGACCAAGACCAAGATCAAGGT | |
| OsH1.4-qRT-R | CTACTTCATGCTCTTCCTCGCC |
Fig. 1 Sequences and expression analysis of rice histone H1 A: Amino acid sequence alignment of four rice histone H1s, red Target 1 and Target 2 are CRISPR mutagenesis sites. B: Phylogenetic tree of histone H1s from different plants（each branch indicates histone H1, and rice and Arabidopsis histone H1s are marked in red and blue, respectively. Species abbreviations: Mpo: Marchantia polymorpha; Smo: Selaginella moellendorffii; Atr: Amborella trichopoda; Ath: Arabidopsis thaliana; Sly: Solanum lycopersicum; Bdi: Brachypodium distachyon）. C: Tissue-specific expressions of rice H1 genes. Expression data is derived from RiceSuperPIRdb database. D: RT-PCR detection of H1 expressions in rice shoot and root. E: RT-qPCR detection of H1 expressions in rice shoot and root
Fig. 2 Construction of CRISPR vector for rice histone H1 A: Map of pYLCRISPR vector containing two target sites. B: Digestion of CRISPR vector by Asc I. M: Molecular marker. C: Sanger sequencing of CRISPR vector
Fig. 3 Screening rice H1 gene mutants from T0 generation
Fig. 4 Sequencing and phenotypic characterization of rice h1 triple mutant A-C: Sequencing chromatograms of the mutated genes OsH1.1, osH1.3 and osH1.4. D-E: Phenotypes of WT and h1 triple mutant
Fig. 5 Rice h1 mutant genetics and phenotypic analysis of T2 generation A: Construction of rice h1 mutant. The T1 triple plant #1 used for RNA-seq analysis, triple plant #2 used for maintaining mutant alleles. B: Drought stress and GA phenotype identification of rice h1 mutant T2. C: Statistics of shoot lengths of different treatments. D: Statistics of root lengths of different treatments. * P<0.05
Fig. 6 Gene expression and GO analysis of rice h1 triple mutant A: Comparison of the gene expressions between WT and h1 mutant. B: Gene expression analysis of h1 mutant. FC: Fold-change. C: GO analysis of up-regulated genes in h1 mutant. FDR: False discovery rate. D: GO analysis of down-regulated genes in h1 mutant. E: Network analysis of up-regulated genes. Red circles indicate ribosomal and chloroplast pathway genes. F: Network analysis of down-regulated genes. Red circles indicate stress-related hydrogenase genes
| 基因ID Gene ID | 差异倍数log2值 log2 of fold-change | P值 P value | 功能注释 Functional annotation |
|---|---|---|---|
| LOC_Os03g63950 | 2.701 833 350 | 5.00E-05 | 质体特异性30S核糖体蛋白Plastid-specific 30S ribosomal protein |
| LOC_Os01g14070 | 1.560 583 912 | 0.005 65 | 60S核糖体蛋白L18a-1 60S ribosomal protein L18a-1 |
| LOC_Os03g37970 | 1.212 029 246 | 0.010 8 | 核糖体蛋白L13 Ribosomal protein L13 |
| LOC_Os02g01332 | 1.268 533 286 | 0.011 6 | 核糖体蛋白L6 Ribosomal protein L6 |
| LOC_Os01g01060 | 1.178 811 181 | 0.011 7 | 40S核糖体蛋白S5 40S ribosomal protein S5 |
| LOC_Os07g10720 | 1.276 663 714 | 0.011 7 | 40核糖体蛋白S15a 40S ribosomal protein S15a |
| LOC_Os02g18090 | 1.730 630 752 | 0.013 35 | 线粒体核糖体蛋白L53 Mitochondrial ribosomal protein L53 |
| LOC_Os07g08660 | 1.408 912 387 | 0.014 7 | 40S核糖体蛋白S15 40S ribosomal protein S15 |
| LOC_Os01g10820 | 1.170 916 250 | 0.015 45 | 核糖体蛋白L5 Ribosomal protein L5 |
| LOC_Os04g52361 | 1.177 325 645 | 0.016 1 | 核糖体蛋白S17 Ribosomal protein S17 |
| LOC_Os07g42450 | 1.199 616 805 | 0.016 9 | 核糖体蛋白S2 Ribosomal protein S2 |
| LOC_Os08g13690 | 1.113 868 750 | 0.017 | 60S核糖体蛋白L7 60S ribosomal protein L7 |
| LOC_Os03g60400 | 1.217 299 391 | 0.0175 5 | 40S核糖体蛋白S23 40S ribosomal protein S23 |
| LOC_Os07g10300 | 1.591 065 282 | 0.0188 5 | 线粒体28S核糖体蛋白S29相关 Mitochondrial 28S ribosomal protein S29-related |
| LOC_Os06g19640 | 1.383 070 486 | 0.0202 50 | 线粒体39S核糖体蛋白L46 Mitochondrial 39S ribosomal protein L46 |
| LOC_Os04g28180 | 1.154 726 863 | 0.025 60 | 核糖体蛋白 Ribosomal protein |
| LOC_Os02g30050 | 1.052 697 490 | 0.028 70 | 核糖体蛋白L29 Ribosomal protein L29 |
| LOC_Os07g10660 | 1.148 487 268 | 0.035 30 | 核糖体蛋白Ribosomal protein |
| LOC_Os04g39700 | 1.055 227 902 | 0.040 20 | 60S核糖体蛋白L6 60S ribosomal protein L6 |
| LOC_Os02g32760 | 1.570 591 039 | 0.042 45 | 60S酸性核糖体蛋白 60S acidic ribosomal protein |
| LOC_Os03g48840 | 3.563 924 435 | 0.042 95 | 核糖体蛋白L18p/L5e家族蛋白 Ribosomal L18p/L5e family protein |
| LOC_Os12g38000 | 1.022 593 563 | 0.047 05 | 60S核糖体蛋白L8 60S ribosomal protein L8 |
| LOC_Os08g33820 | 2.001 613 891 | 5.00E-05 | 叶绿素A-B结合蛋白 Chlorophyll A-B binding protein |
| LOC_Os04g38410 | 2.743 324 248 | 5.00E-05 | 叶绿素A-B结合蛋白 Chlorophyll A-B binding protein |
| LOC_Os02g36850 | 1.942 328 010 | 0.000 60 | 放氧增强蛋白3 Oxygen-evolving enhancer protein 3 |
| LOC_Os07g37240 | 1.907 040 368 | 0.000 75 | 叶绿素A-B结合蛋白 Chlorophyll A-B binding protein |
| LOC_Os09g26810 | 1.457 308 201 | 0.001 75 | 叶绿素A-B结合蛋白 Chlorophyll A-B binding protein |
| LOC_Os07g36080 | 1.656 294 981 | 0.002 45 | 放氧增强蛋白3结构域蛋白 Oxygen-evolving enhancer protein 3 domain protein |
| LOC_Os07g30670 | 1.267 700 635 | 0.014 15 | 2Fe-2S铁硫簇结合域蛋白 2Fe-2S iron-sulfur cluster binding domain protein |
| LOC_Os11g13890 | 1.502 035 534 | 0.014 35 | 叶绿素A-B结合蛋白 Chlorophyll A-B binding protein |
| LOC_Os12g08770 | 1.118 250 231 | 0.021 50 | 光系统I反应中心亚基 Photosystem I reaction center subunit |
| LOC_Os08g25720 | 1.467 877 984 | 0.023 85 | 焦磷酸-果糖6-磷酸1-磷酸转移酶亚基 Pyrophosphate-fructose 6-phosphate 1-phosphotransferase subunit |
| LOC_Os02g10390 | 1.148 377 518 | 0.029 20 | 叶绿素A-B结合蛋白 Chlorophyll A-B binding protein |
| LOC_Os06g22060 | 1.526 688 176 | 0.033 45 | 焦磷酸-果糖6-磷酸1-磷酸转移酶亚基 Pyrophosphate-fructose 6-phosphate 1-phosphotransferase subunit |
| LOC_Os06g21590 | 1.221 345 436 | 0.043 55 | 叶绿素A-B结合蛋白 Chlorophyll A-B binding protein |
Table 2 Significantly up-regulated ribosomal and photosynthesis genes
| 基因ID Gene ID | 差异倍数log2值 log2 of fold-change | P值 P value | 功能注释 Functional annotation |
|---|---|---|---|
| LOC_Os03g63950 | 2.701 833 350 | 5.00E-05 | 质体特异性30S核糖体蛋白Plastid-specific 30S ribosomal protein |
| LOC_Os01g14070 | 1.560 583 912 | 0.005 65 | 60S核糖体蛋白L18a-1 60S ribosomal protein L18a-1 |
| LOC_Os03g37970 | 1.212 029 246 | 0.010 8 | 核糖体蛋白L13 Ribosomal protein L13 |
| LOC_Os02g01332 | 1.268 533 286 | 0.011 6 | 核糖体蛋白L6 Ribosomal protein L6 |
| LOC_Os01g01060 | 1.178 811 181 | 0.011 7 | 40S核糖体蛋白S5 40S ribosomal protein S5 |
| LOC_Os07g10720 | 1.276 663 714 | 0.011 7 | 40核糖体蛋白S15a 40S ribosomal protein S15a |
| LOC_Os02g18090 | 1.730 630 752 | 0.013 35 | 线粒体核糖体蛋白L53 Mitochondrial ribosomal protein L53 |
| LOC_Os07g08660 | 1.408 912 387 | 0.014 7 | 40S核糖体蛋白S15 40S ribosomal protein S15 |
| LOC_Os01g10820 | 1.170 916 250 | 0.015 45 | 核糖体蛋白L5 Ribosomal protein L5 |
| LOC_Os04g52361 | 1.177 325 645 | 0.016 1 | 核糖体蛋白S17 Ribosomal protein S17 |
| LOC_Os07g42450 | 1.199 616 805 | 0.016 9 | 核糖体蛋白S2 Ribosomal protein S2 |
| LOC_Os08g13690 | 1.113 868 750 | 0.017 | 60S核糖体蛋白L7 60S ribosomal protein L7 |
| LOC_Os03g60400 | 1.217 299 391 | 0.0175 5 | 40S核糖体蛋白S23 40S ribosomal protein S23 |
| LOC_Os07g10300 | 1.591 065 282 | 0.0188 5 | 线粒体28S核糖体蛋白S29相关 Mitochondrial 28S ribosomal protein S29-related |
| LOC_Os06g19640 | 1.383 070 486 | 0.0202 50 | 线粒体39S核糖体蛋白L46 Mitochondrial 39S ribosomal protein L46 |
| LOC_Os04g28180 | 1.154 726 863 | 0.025 60 | 核糖体蛋白 Ribosomal protein |
| LOC_Os02g30050 | 1.052 697 490 | 0.028 70 | 核糖体蛋白L29 Ribosomal protein L29 |
| LOC_Os07g10660 | 1.148 487 268 | 0.035 30 | 核糖体蛋白Ribosomal protein |
| LOC_Os04g39700 | 1.055 227 902 | 0.040 20 | 60S核糖体蛋白L6 60S ribosomal protein L6 |
| LOC_Os02g32760 | 1.570 591 039 | 0.042 45 | 60S酸性核糖体蛋白 60S acidic ribosomal protein |
| LOC_Os03g48840 | 3.563 924 435 | 0.042 95 | 核糖体蛋白L18p/L5e家族蛋白 Ribosomal L18p/L5e family protein |
| LOC_Os12g38000 | 1.022 593 563 | 0.047 05 | 60S核糖体蛋白L8 60S ribosomal protein L8 |
| LOC_Os08g33820 | 2.001 613 891 | 5.00E-05 | 叶绿素A-B结合蛋白 Chlorophyll A-B binding protein |
| LOC_Os04g38410 | 2.743 324 248 | 5.00E-05 | 叶绿素A-B结合蛋白 Chlorophyll A-B binding protein |
| LOC_Os02g36850 | 1.942 328 010 | 0.000 60 | 放氧增强蛋白3 Oxygen-evolving enhancer protein 3 |
| LOC_Os07g37240 | 1.907 040 368 | 0.000 75 | 叶绿素A-B结合蛋白 Chlorophyll A-B binding protein |
| LOC_Os09g26810 | 1.457 308 201 | 0.001 75 | 叶绿素A-B结合蛋白 Chlorophyll A-B binding protein |
| LOC_Os07g36080 | 1.656 294 981 | 0.002 45 | 放氧增强蛋白3结构域蛋白 Oxygen-evolving enhancer protein 3 domain protein |
| LOC_Os07g30670 | 1.267 700 635 | 0.014 15 | 2Fe-2S铁硫簇结合域蛋白 2Fe-2S iron-sulfur cluster binding domain protein |
| LOC_Os11g13890 | 1.502 035 534 | 0.014 35 | 叶绿素A-B结合蛋白 Chlorophyll A-B binding protein |
| LOC_Os12g08770 | 1.118 250 231 | 0.021 50 | 光系统I反应中心亚基 Photosystem I reaction center subunit |
| LOC_Os08g25720 | 1.467 877 984 | 0.023 85 | 焦磷酸-果糖6-磷酸1-磷酸转移酶亚基 Pyrophosphate-fructose 6-phosphate 1-phosphotransferase subunit |
| LOC_Os02g10390 | 1.148 377 518 | 0.029 20 | 叶绿素A-B结合蛋白 Chlorophyll A-B binding protein |
| LOC_Os06g22060 | 1.526 688 176 | 0.033 45 | 焦磷酸-果糖6-磷酸1-磷酸转移酶亚基 Pyrophosphate-fructose 6-phosphate 1-phosphotransferase subunit |
| LOC_Os06g21590 | 1.221 345 436 | 0.043 55 | 叶绿素A-B结合蛋白 Chlorophyll A-B binding protein |
Fig. 7 Expressions of three genes are disrupted in rice h1 triple mutant A-C: Transcriptomic sequencing peaks, there are 2 replicates for each sample. D-F: RT-qPCR validation. ** P < 0.01
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