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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2024, Vol. 44 Issue (2/3): 25-38    DOI: 10.13523/j.cb.2306028
研究报告     
多年宿根甘蔗的转录组分析及差异基因挖掘*
程琴,李佳慧,谭秦亮,宋奇琦,朱鹏锦**(),欧克纬,农泽梅,卢业飞,吕平,周全光,庞新华,彭欣怡**()
广西壮族自治区亚热带作物研究所 南宁 530001
Transcriptome Analysis and Differential Gene Mining of Perennial-root Sugarcane
CHENG Qin,LI Jiahui,TAN Qinliang,SONG Qiqi,ZHU Pengjin**(),OU Kewei,NONG Zemei,LU Yefei,LV Ping,ZHOU Quanguang,PANG Xinhua,PENG Xinyi**()
Guangxi Institute of Subtropical Crops, Nanning 530001, China
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摘要：

甘蔗(Saccharum hybirds)宿根性直接关系到甘蔗生产成本和种植效益,品种、环境和栽培措施均会影响甘蔗宿根能力,但品种的种性是影响宿根性最关键因素。国内外从分子生物学层面解释甘蔗宿根性差异的文献鲜见报道,从转录水平分析不同宿根年限GR2号和ROC22号基因表达的差异。结果表明:转录组测序共得到100 558 条转录本和25 582 条Unigene。获得53 790 条Unigene的注释结果,分别在NR、Swiss-Prot、KEGG、COG、KOG、GO和Pfam数据库进行比对。GO功能注释共分成三大类,51小类,6 年宿根蔗注释到1 029 个差异基因,3 年宿根蔗注释到3 391 个差异基因。主要KEGG代谢通路有8 条,分别涉及植物激素信号转导,淀粉和蔗糖代谢,甘氨酸、丝氨酸和苏氨酸代谢,苯丙素生物合成,同源重组,DNA复制,错配修复及植物病原体相互作用。筛选出脱落酸(ABA)相关差异基因6 个,分别为脱落酸不敏感蛋白2基因(ABI2)、bZIP转录因子超家族蛋白、碱性亮氨酸拉链型转录因子基因(ABI5)、aba响应元件结合因子1基因(ABF1)、G-box结合因子基因(GBFs)、aba响应元件结合因子基因(ABF)。以上差异基因将作为后期进行基因表达和功能分析的参考基因,并联合蛋白质组学深入分析影响甘蔗宿根性的分子机制。
关键词： 甘蔗;  宿根性;  转录组;  脱落酸    
Abstract:

Sugarcane ratooning ability is directly related to its production cost and cultivation efficiency. Sugarcane ratooning productivity is influenced by variety, environment and cultivation measures, but variety is the most critical factor. There are few reports explaining the differences in sugarcane ratooning ability at the molecular biological level both domestically and internationally. In this paper, transcriptome sequencing was used to analyze the differences in gene expression between sugarcane GR2 and ROC22 of different root years. The results showed that 100 558 transcripts and 25 582 unigenes were obtained by transcriptome sequencing. By functional annotation of unigenes, including comparison with NR, Swiss-Prot, KEGG, COG, KOG, GO and Pfam databases, a total of 53 790 unigene annotation results were obtained. The GO functional annotation was divided into three categories and 51 subcategories. A total of 1 029 differential genes were annotated in six-year-old sugarcane, and 3 391 differential genes were annotated in three-year-old sugarcane. There are eight major KEGG metabolic pathways, which are mainly involved in plant hormone signal transduction, starch and sucrose metabolism, glycine, serine and threonine metabolism, phenylpropanoid biosynthesis, homologous recombination, DNA replication, mismatch repair and plant pathogen interaction. Six abscisic acid-related differentially identified genes are: abscisic acid-insensitive 5-like protein 2 (ABI2), putative bZIP transcription factor superfamily protein, protein abscisic acid-insensitive 5 (ABI5), aba responsive element-binding factor 1 (partial ABF1), G-box binding factor 4 (GBFs), and aba responsive element-binding factor 1 (ABF). The above differential genes will serve as reference genes for our later gene expression and function analysis, and further analyze the molecular mechanism affecting sugarcane ratooning ability by combining proteomics.
Key words: Saccharum hybirds    Perennial root    Transcriptome    Abscisic acid
收稿日期: 2023-06-21 出版日期: 2024-04-03
ZTFLH:  Q78  
基金资助: *广西科技计划(桂科AB23026083);广西科技计划(桂科AA22117002-6);广西农业科学院基本科研业务专项(桂农科2021YT151)
通讯作者: **电子信箱:329669037@qq.com; 271147736@qq.com   
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程琴
李佳慧
谭秦亮
宋奇琦
朱鹏锦
欧克纬
农泽梅
卢业飞
吕平
周全光
庞新华
彭欣怡

引用本文:

程琴, 李佳慧, 谭秦亮, 宋奇琦, 朱鹏锦, 欧克纬, 农泽梅, 卢业飞, 吕平, 周全光, 庞新华, 彭欣怡. 多年宿根甘蔗的转录组分析及差异基因挖掘*[J]. 中国生物工程杂志, 2024, 44(2/3): 25-38.

CHENG Qin, LI Jiahui, TAN Qinliang, SONG Qiqi, ZHU Pengjin, OU Kewei, NONG Zemei, LU Yefei, LV Ping, ZHOU Quanguang, PANG Xinhua, PENG Xinyi. Transcriptome Analysis and Differential Gene Mining of Perennial-root Sugarcane. China Biotechnology, 2024, 44(2/3): 25-38.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2306028    或    https://manu60.magtech.com.cn/biotech/CN/Y2024/V44/I2/3/25

样品 Clean Reads数 比对上的读长 碱基和 GC含量/% Q20含量/% Q30含量/%
S-GR-T-1 24 679 239 (100%) 18 270 708 (74.03%) 7 382 461 956 55.17 97.74 93.83
S-GR-T-2 24 356 811 (100%) 18 081 214 (74.23%) 7 289 302 138 54.83 97.74 93.81
S-GR-T-3 32 105 941 (100%) 23 800 101 (74.13%) 9 597 818 186 55.30 97.75 93.92
S-ROC-T-1 26 523 983 (100%) 19 655 684 (74.11%) 7 932 528 412 55.34 97.96 94.27
S-ROC-T-2 26 636 115 (100%) 19 895 642 (74.69%) 7 972 651 934 55.57 97.81 93.94
S-ROC-T-3 28 279 985 (100%) 21 002 557 (74.27%) 8 456 504 582 55.47 97.78 93.92
T-GR-T-1 21 281 893 (100%) 15 965 989 (75.02%) 6 368 694 450 54.20 98.27 95.31
T-GR-T-2 23 747 346 (100%) 17 465 407 (73.55%) 7 099 029 056 54.60 98.00 94.84
T-GR-T-3 24 833 434 (100%) 18 411 800 (74.14%) 7 424 914 922 55.17 98.19 95.04
T-ROC-T-1 25 923 473 (100%) 19 361 506 (74.69%) 7 750 471 616 54.36 98.32 95.42
T-ROC-T-2 22 033 766 (100%) 16 049 898 (72.84%) 6 574 468 010 55.26 98.09 95.07
T-ROC-T-3 27 024 567 (100%) 20 102 036 (74.38%) 8 078 974 654 54.66 98.22 95.25
表1  甘蔗幼根转录组分析结果
长度范围/bp 转录本 独立基因
数量/个 百分比/% 数量/个 百分比/%
200~300 40 151 15.94 32 617 32.44
300~500 42 015 16.68 23 909 23.78
500~1 000 58 540 23.23 18 450 18.35
1 000~2 000 65 068 25.83 13 774 13.70
2 000+ 46 177 18.33 11 808 11.74
总数/个 251 951 100 558
总长度/bp 301 843 833 86 622 242
N50长度/bp 1 871 1 663
平均长度/bp 1 198.03 861.42
表2  组装结果统计表
注释数据库 注释数量 片段长度/bp
300~1 000 ≥1 000
COG 14 604 4 157 7 272
GO 36 135 12 135 16 204
KEGG 16 679 5 665 7 179
KOG 25 724 8 157 11 550
Pfam 31 365 9 527 15 847
Swiss-Prot 27 617 8 722 14 455
eggNOG 47 362 16 338 20 075
NR 51 138 18 418 21 317
合计 53 790 19 261 21 445
表3  Unigene 注释统计
图1  与NR数据库比对上的物种分布
差异表达基因集
名称		 差异表达
基因数目		 上调基因
数目		 下调基因
数目
S-ROC_vs_S-GR 2 484 1 206 1 278
T-ROC_vs_T-GR 6 224 2 334 3 890
表4  差异表达基因数目统计表
图2  不同宿根年限甘蔗品种间差异表达基因火山图
差异表达基因集名称 注释到的差异
表达基因数目		 各功能数据库注释到的差异表达基因数目
COG GO KEGG KOG Pfam Swiss-Prot eggNOG NR
S-ROC_vs_S-GR 1 728 402 1 029 302 681 1 046 968 1 470 1 714
T-ROC_vs_T-GR 4 831 1 347 3 391 1 190 2 092 3 225 3 080 4 385 4 813
表5  注释的差异表达基因数量统计表
图3  6年宿根蔗差异表达基因GO功能富集
图4  3年宿根蔗差异表达基因GO功能富集
图5  6年宿根蔗差异表达基因COG功能分类
图6  3年宿根蔗差异表达基因COG功能分类 A:RNA加工和修饰;B:染色质结构和动力学;C:能源生产与转换;D:细胞周期控制,细胞分裂,染色体分裂;E:氨基酸运输和代谢;F:核苷酸迁移和代谢;G:碳水化合物运输和代谢;H:辅酶运输和代谢;I:脂质运输与代谢;J:翻译、核糖体结构和生物发生;K:转录;L:复制、重组和修复;M:细胞壁/膜/信封生源论;N:细胞运动性;O:翻译后修饰,蛋白质转换,分子伴侣;P:无机离子的转运和代谢;Q:次级代谢产物生物合成、运输和分解代谢;R:通用功能预测;S:未知功能;T:信号转导机制;U:细胞内运输、分泌和囊泡运输;V:防御机制;W:细胞外结构;X:噬菌体原,转座子;Z:细胞骨架
图7  6年宿根蔗差异表达基因KEGG注释
图8  3年宿根蔗差异表达基因KEGG注释
图9  T-ROC_vs_T-GR.KEGG富集因子
图10  S-ROC_vs_S-GR.KEGG富集因子
ID 条目 数量
S-ROC_vs_S-GR T-ROC_vs_T-GR
ko04075 植物激素信号转导 6 74
ko00500 淀粉和蔗糖代谢 7 33
ko00260 甘氨酸、丝氨酸和苏氨酸代谢 5 20
ko00940 苯丙素生物合成 7 48
ko03440 同源重组 11 13
ko03030 DNA复制 11 12
ko03430 错配修复 10 12
ko04626 植物病原体相互作用 9 49
表6  主要 KEGG 代谢通路注释
基因ID 脱落酸相关差异基因表达量FPKM 差异倍数 上下调 基因名称
S-GR1 S-GR2 S-GR3 S-ROC1 S-ROC2 S-ROC3 S-GR-vs-
S-ROC
c83449 9.99 9.42 12.21 4.26 4.16 4.33 7.40* 1.065 上调 脱落酸不敏感5-like蛋白2
c73830 0.00 0.00 0.00 3.75 6.31 6.64 5.57* -5.669 下调 碱性亮氨酸拉链型转录因子(ABI5)
c87455 13.75 13.75 14.68 36.57 36.08 20.25 22.52 -1.448 下调 aba响应元件结合因子1(ABF1)
c99528 9.91 10.61 12.13 15.55 21.68 15.00 14.18* -1.165 下调 G-box结合因子(GBFs)
c77791 3.54 3.73 3.90 0.69 1.07 1.22 2.36* 1.509 上调 bZIP转录因子超家族蛋白
c87455 6.23 16.08 9.26 37.83 41.91 26.21 22.92* -1.981 下调 aba响应元件结合因子(ABF)
表7  脱落酸差异基因表达分析
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