A Transcriptomic Analysis of Early Adventitious Roots of Rosa chinensis Cuttings and Key Genes Screening
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摘要: 为研究月季插穗在不定根发生过程中的关键基因调控机理,利用Illumina平台测序技术对切花月季品种‘卡罗拉’插穗的3个发育阶段(不定根未启动期、愈伤组织形成期和不定根伸长期)插穗基部1 cm皮层进行转录组测序分析,结果表明:月季插穗不定根发生的3个阶段中,在不定根未启动期与愈伤组织形成期之间共筛选出差异表达基因5 033个,其中2 313个基因上调,2 720个基因下调;在愈伤组织形成期与不定根伸长期之间共筛选出差异表达基因1 865个,其中1 332个基因上调,533个基因下调;GO功能分析表明,差异表达基因主要参与生物过程、分子功能和细胞组分3大功能;KEGG富集分析结果表明,差异表达基因主要参与植物激素信号转导、次生代谢产物的合成以及碳水化合物的合成等代谢通路;将月季插穗生根过程中差异性最为显著的8个基因通过实时荧光定量PCR检测其转录水平变化,结果表明: 实时荧光定量PCR的验证结果与转录组测序结果基本一致.Abstract: In order to study the key gene regulation mechanism of Rosa chinensis cuttings in the process of adventitious root formation, the Illumina platform sequencing technology was used to analyze the three developmental stages of cutting rose cultivar 'Carola', i.e., adventitious root initiation period, callus formation period and adventitious root elongation period. A transcriptome sequencing analysis was performed on the 1 cm cortex at the base of cuttings. The results showed that at the three stages of adventitious root formation of rose cuttings, a total of 5 033 differentially expressed genes were screened between the adventitious root initiation stage and the callus formation stage, of which 2 313 genes were up-regulated and 2 720 genes were down-regulated; a total of 1 865 differentially expressed genes were screened between the callus formation period and adventitious root elongation period, of which 1 332 genes were up-regulated and 533 genes were down-regulated. The GO functional analysis showed that differentially expressed genes were mainly involved in the 3 major functions of biological process, molecule function and cell composition. The KEGG enrichment analysis showed that the differentially expressed genes were mainly involved in the metabolic pathways of plant hormone signal transduction, the synthesis of secondary metabolites and the synthesis of carbohydrates. The changes in the transcription levels of the 8 genes that exhibited the most significant differences in the rooting process were detected with real-time fluorescent quantitative PCR. The results showed that the verification results of real-time fluorescent quantitative PCR were basically consistent with the results of transcriptome sequencing.
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Key words:
- Rosa chinensis /
- cuttings /
- adventitious roots /
- transcriptome
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表 1 文库基本情况
Table 1. The basic information of the library
样品 样品名称 文库名 不定根未启动期 A1 LRA13418 A2 LRA13419 愈伤组织期 B1 LRA13420 B2 LRA13421 不定根伸长期 C1 LRA13422 C2 LRA13423 注:A1、A2为不定根未启动期的2个平行样本;B1、B2为愈伤组织期的2个平行样本;C1、C2为不定根伸长期的2个平行样本. 表 2 检测基因表达的引物序列
Table 2. The primer sequence of gene expression detected
基因编号 正向序列(5'-3') 反向序列(5'-3') PCR产物/bp GAPDH GGAAAGGTTCTGCCTGCTC CTGGTCATAGGTTGCCTTCTTC 139 LOC112189488 CTCTACCCTGCTGCCCCACT GCGACTCATCACCACCGTTT 187 LOC112186298 GGAGCATTTGTCCAGTTCGT GCCATTAGTAGCCGCCTTT 180 LOC112165178 GCATGGGAGTTGTGGAAAGA AGGTGCAGGTAATCGTGAGC 198 LOC112172220 CCACCCTCTTGAGCCTTACC AGCATTTCCTCCATCTCCTTC 165 LOC112179697 TCTATCCTCGCCAGCCACT ACCGTCCTCGAATACTCCTTC 187 LOC112164736 TCCTAAGCAATGGTCTAAAGCC TCCGCATAGTCCCGAAAA 124 LOC112193119 TGAAAACCCGATTCCCATC GGTCAAGAACCCACCCAATG 115 LOC112194361 TGTTGTCATTCCTGCGTTTG TGGTGCTGCTGAGGTTGC 127 表 3 下机数据统计
Table 3. The statistics of offline data
样品 Reads/条 碱基总数/bp Q30/bp N/% Q20占碱基数的百分数/% Q30占碱基数的百分数/% Clean Reads占Reads的百分数/% A1 41 432 804 6 214 920 600 5 608 791 753 0.003 266 95.89 90.24 92.26 A2 44 353 390 6 653 008 500 6 057 901 035 0.003 008 96.30 91.05 92.80 B1 39 577 678 5 936 651 700 5 384 791 893 0.003 314 96.09 90.70 92.83 B2 43 275 996 6 491 399 400 5 900 420 774 0.003 278 96.19 90.89 92.73 C1 45 240 742 6 786 111 300 6 172 944 331 0.003 262 96.26 90.96 92.52 C2 48 695 120 7 304 268 000 6 625 080 997 0.003 226 96.11 90.70 92.91 注:Q20(bp):碱基识别准确率在99%以上的碱基总数;Q30 (bp):碱基识别准确率在99.9%以上的碱基总数;N(%):模糊碱基所占百分比;Clean Reads占Reads的百分数(%):高质量序列碱基占测序碱基的百分比. 表 4 参考基因组信息
Table 4. The information of reference genome
数据库 基因注释数 基因注释率/% NCBI_GeneID 30 216 100 UniProt 25 170 83.30 GO 19 242 63.68 KEGG 8 754 28.97 -
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