不同硬度西瓜果皮的转录组测序及相关基因表达分析

doi:10.7668/hbnxb.20192957

摘要/Abstract

摘要：

为解析西瓜果皮硬度不同的相关分子机制,挖掘西瓜果皮硬度相关的关键基因提供理论基础,以生育期相似但果皮硬度差异显著的高硬度(901)4-1-1-M和低硬度BSH为试验材料,选取果皮硬度差异最大时期即西瓜授粉30 d后的果皮,利用Illumina HiSeq^TM测序平台进行转录组测序分析,采用实时荧光定量的PCR技术对测序结果进行验证。结果显示,转录组测序共筛选获得1 085个差异表达基因,其中,上调表达基因555个,下调表达基因530个。GO富集分析表明,1 085个差异表达基因显著富集在细胞组分、分子功能和生物过程中的细胞壁、细胞外围、外部封装结构、胞外区、四吡咯骨架、氧化还原酶活性、转移酶活性、果胶酯酶活性等功能类别。KEGG富集分析表明,Cla97C04G075800、Cla97C02G044950、Cla97C09G165820、Cla97C10G195660、Cla97C01G025380等19个差异基因被注释富集程度最高的苯丙烷代谢通路,该路径最终形成木质素、5羟基愈创木酚木质素、愈创木酚木质素和对羟基苯酚木质素代谢物,qRT-PCR和转录组测序数据相关系数R²为0.791,证明转录组试验数据可靠,从转录水平解释了(901)4-1-1-M和BSH西瓜果皮硬度存在差异的原因。

关键词: 西瓜, 果皮硬度, 转录组, 差异表达基因, 苯丙烷, 木质素

Abstract:

In order to analyze the molecular mechanism of different watermelon peel firmness,and provided a theoretical basis for discovering key genes related to watermelon peel firmness,the high firmness(901)4-1-1-M and low firmness BSH with similar growth period but significant difference in peel hardness were used as experimental materials.The peel with the maximum difference 30 days after pollination was selected for transcriptome sequencing analysis and the Illumina HiSeq^TM sequencing platform was used to analyze the molecular mechanism of different watermelon peel firmness,Real-time fluorescence quantitative PCR (qRT-PCR) was used to verify the sequencing results.A total of 1 085 differentially expressed genes (DEGs) were screened by transcriptome sequencing,including 555 up-regulated genes and 530 down-regulated genes.Gene Ontology(GO)analysis showed that 1 085 DEGs were significantly enriched in cell components,molecular functions and biological processes,including cell wall,cell periphery,external encapsulation structure,extracellular region,tetrapyrrole skeleton,redox enzyme activity,transferase activity and pectin esterase activity.Kyoto Encyclopedia of Genes and Genomes (KEGG)analysis indicated that 19 DEGs,including Cla97C04G075800,Cla97C02G044950,Cla97C09G165820,Cla97C10G195660,Cla97C01G025380,etc.,were enriched in the most significant enrichment biosynthesis of phenylpropanoid,which eventually lead to the metabolites of Syringyl lignin,5-Hydroxy-guaiacy lifnin,Guaiacy lifnin and P-Hydroxy-phenyl lifnin.The correlation coefficient of DEGs expression levels by qRT-PCR and RNA-seq data was 0.791,which indicated that the transcriptome test data were reliable.This study explained the reason of watermelon peel firmness difference between(901)4-1-1-M and BSH from the transcriptional level.

Key words: Watermelon, Peel firmness, Transcriptomic, Differentially expressed genes, Styrene acrylic pigment, Lignin

张敬敬, 李冰, 史宇凡, 高秀瑞, 潘秀清, 宋雪, 武彦荣. 不同硬度西瓜果皮的转录组测序及相关基因表达分析[J]. 华北农学报, 2022, 37(3): 44-52. doi: 10.7668/hbnxb.20192957.

ZHANG Jingjing, LI Bing, SHI Yufan, GAO Xiurui, PAN Xiuqing, SONG Xue, WU Yanrong. Transcriptome Sequencing and Gene Expression Analysis of Watermelon Peel with Different Firmness[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(3): 44-52. doi: 10.7668/hbnxb.20192957.

http://www.hbnxb.net/CN/Y2022/V37/I3/44

图/表 11

参考文献 23

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基因 Gene ID	正向引物(5'-3') Forward primer sequence(5'-3')	反向引物(5'-3') Reverse primer sequence(5'-3')	扩增片段长度/bp Product size
Actin	TCAACCCAAAGGCAAACAGG	AAAACGGCCTGGATTGCAAC	91
Cla97C04G075800	TGGCTTCGAGTTTTGCATCG	AATGGCTTCCCTTCAACGAC	92
Cla97C02G044950	ATCGCCATTTCCAAGCTTCG	TTGCGGTGGTTCAAGTCATC	110
Cla97C10G195660	TTGCAAAGGAGCCATTTCCG	TGTTTCGAGGCAATGAGCTG	108
Cla97C09G165820	AAGCGTGGCTCAACAAATGG	ATGTGGAAAAACGGCAGCAC	90
Cla97C09G165630	AAAACGGCAACACCACCAAC	CGTCGGTGGCAATGTAAAGC	123
Cla97C02G032910	ACGGACAAGAAGACCAGAACC	ACAGAGCATCGCTTTGCAAC	78
Cla97C07G144540	TTTGACAGCGTTCCAAAGGG	TGCATTCCGCCACAATTACC	139
Cla97C02G045070	AGACGCTTCATTCGCAAAGC	AAAGCTTTGGGGTAGCAACG	95
Cla97C07G139710	ACATGAAACCGAGCAAGCTC	TCGCCGCTATGATCTTCAAC	138

基因 Gene ID	正向引物(5'-3') Forward primer sequence(5'-3')	反向引物(5'-3') Reverse primer sequence(5'-3')	扩增片段长度/bp Product size
Actin	TCAACCCAAAGGCAAACAGG	AAAACGGCCTGGATTGCAAC	91
Cla97C04G075800	TGGCTTCGAGTTTTGCATCG	AATGGCTTCCCTTCAACGAC	92
Cla97C02G044950	ATCGCCATTTCCAAGCTTCG	TTGCGGTGGTTCAAGTCATC	110
Cla97C10G195660	TTGCAAAGGAGCCATTTCCG	TGTTTCGAGGCAATGAGCTG	108
Cla97C09G165820	AAGCGTGGCTCAACAAATGG	ATGTGGAAAAACGGCAGCAC	90
Cla97C09G165630	AAAACGGCAACACCACCAAC	CGTCGGTGGCAATGTAAAGC	123
Cla97C02G032910	ACGGACAAGAAGACCAGAACC	ACAGAGCATCGCTTTGCAAC	78
Cla97C07G144540	TTTGACAGCGTTCCAAAGGG	TGCATTCCGCCACAATTACC	139
Cla97C02G045070	AGACGCTTCATTCGCAAAGC	AAAGCTTTGGGGTAGCAACG	95
Cla97C07G139710	ACATGAAACCGAGCAAGCTC	TCGCCGCTATGATCTTCAAC	138

样品 Sample	原始reads/条 Raw reads	滤后reads/条 Clean reads	错误率/% Error rate	Q20百分率/% Q20 percentage	Q30百分率/% Q30 percentage	GC 含量/% GC percentage
A1	41 758 886	41 376 900	0.02	98.13	94.40	43.83
A2	43 554 750	43 143 786	0.02	98.18	94.47	43.80
A3	50 305 470	49 846 930	0.02	98.08	94.24	43.67
B1	45 484 004	44 895 382	0.03	97.35	92.63	43.72
B2	47 849 050	47 228 278	0.02	98.25	94.61	43.67
B3	46 091 088	45 586 596	0.02	98.22	94.55	43.48

样品 Sample	原始reads/条 Raw reads	滤后reads/条 Clean reads	错误率/% Error rate	Q20百分率/% Q20 percentage	Q30百分率/% Q30 percentage	GC 含量/% GC percentage
A1	41 758 886	41 376 900	0.02	98.13	94.40	43.83
A2	43 554 750	43 143 786	0.02	98.18	94.47	43.80
A3	50 305 470	49 846 930	0.02	98.08	94.24	43.67
B1	45 484 004	44 895 382	0.03	97.35	92.63	43.72
B2	47 849 050	47 228 278	0.02	98.25	94.61	43.67
B3	46 091 088	45 586 596	0.02	98.22	94.55	43.48

样品 Sample	总数/条 Total reads	数量/条(比对率/%) Quantity (ratio)			外显子比对率/% Exon mapped ratio
样品 Sample	总数/条 Total reads	定位到基因组上序列 Total mapped	唯一比对位置序列 Uniquely mapped	多个比对位置序列 Multiple mapped	外显子比对率/% Exon mapped ratio
A1	41 376 900	33 568 170(81.13)	32 939 299(79.61)	628 871(1.52)	76.50
A2	43 143 786	35 596 732(82.51)	34 956 941(81.02)	639 791(1.48)	75.60
A3	49 846 930	40 630 024(81.51)	39 734 776(79.71)	895 248(1.80)	76.03
B1	44 895 382	35 359 625(78.76)	34 779 983(77.47)	579 642(1.29)	75.23
B2	47 228 278	35 818 334(75.84)	35 071 296(74.26)	747 038(1.58)	76.03
B3	45 586 596	32 768 449(71.88)	32 165 238(70.56)	603 211(1.32)	76.31

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