月季插穗不定根起始的转录组分析和关键基因筛选

陈静; 陈芸; 热依麦阿依·阿布都艾尼; 方志刚; 凯迪日耶·玉苏普; 马刘峰

doi:10.6054/j.jscnun.2021044

月季插穗不定根起始的转录组分析和关键基因筛选

doi: 10.6054/j.jscnun.2021044

喀什大学生命与地理科学学院//新疆帕米尔高原生物资源与生态重点实验室，喀什 844000

基金项目:

新疆维吾尔自治区青年科技创新人才培养工程资助项目 QN2016YX0611

详细信息

通讯作者:
马刘峰, Email：maliufeng@126.com

中图分类号: Q943.2;Q786
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- 文章访问数: 229
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- PDF下载量: 40
- 被引次数: 0
出版历程
- 收稿日期: 2020-12-19
- 网络出版日期: 2021-07-06
- 刊出日期: 2021-06-25

A Transcriptomic Analysis of Early Adventitious Roots of Rosa chinensis Cuttings and Key Genes Screening

College of Life and Geography Science, Kashi University//The Key Laboratory of Biological Resources and Ecology of Plateau in Xinjiang Uygur Autonomous Region, Kashi 844000, China

摘要

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.
- 月季 /
- 扦插 /
- 不定根 /
- 转录组
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.
- Rosa chinensis /
- cuttings /
- adventitious roots /
- transcriptome

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图 1 月季扦插生根的3个时期

Figure 1. The 3 periods of rose cutting rooting

下载: 全尺寸图片幻灯片

图 2 差异表达基因火山图

Figure 2. The differentially expressed gene volcano plot

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图 3 不定根未启动期与愈伤组织阶段GO富集分析柱状图

注：横坐标为Go level2等级的term，纵坐标为每个term富集的-log10(p值).

Figure 3. The histogram of the GO enrichment analysis at the cutting and callus stages

下载: 全尺寸图片幻灯片

图 4 KEGG富集分析图

Figure 4. The diagram of the KEGG enrichment analysis

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图 5 扦插生根不同时期的差异表达基因维恩图

注：A-B: 不定根未启动期与愈伤组织期；A-C: 不定根未启动期与不定根伸长期；B-C: 愈伤组织期与不定根伸长期.

Figure 5. The Venn diagram of differentially expressed genes at different stages of rooting

下载: 全尺寸图片幻灯片

图 6 qRT-PCR检测月季扦插生根过程中2个阶段差异基因的表达

Figure 6. The differential gene expression at 2 rooting stages detected with qRT-PCR

下载: 全尺寸图片幻灯片

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.

下载: 导出CSV

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

下载: 导出CSV

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.

下载: 导出CSV

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

下载: 导出CSV

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