Transcriptome analysis of callus at rooting stage in tissue culture of vitis amurensis 'shuangyou'

doi:10.6048/j.issn.1001-4330.2023.06.018

Abstract

Abstract:

【Objective】 In order to explore the molecular mechanism of rooting of Vitis amurensis and provide theoretical data and reference basis for rooting in production and reproduction of Vitis amurensis.【Methods】 From the tissue culture to the rooting stage of Vitis amurensis ‘Shuangyou’, the two groups with large differences in the number of roots were treated with different concentrations of IBA (0.1, 0.3 and 0.5 mg/L), NAA (0.1, 0.3 and 0.5 mg/L) and sucrose (15, 25 and 35 g/L),and the transcriptome of the callus rooting at the base was sequenced.【Results】 A total of 284.40 Gb high-quality sequences were obtained from 6 samples, and 89.15%-90.3% of Clean Reads were compared to the reference genome. Compared with S1, there were 537 differentially expressed genes significantly up-regulated in S6 and 277 differentially expressed genes significantly down regulated in S1, a total of 814 differentially expressed genes. A total of 805 differentially expressed genes were annotated into 21 categories of COG, mainly including unknown function, intracellular transport and signal transduction mechanism. GO enrichment analysis showed that the differentially expressed genes were mainly enriched in polysaccharide binding, kinesin complex and auxin activated signal pathway. KEGG enrichment analysis showed that the differentially expressed genes were enriched in phenylpropane biosynthesis, flavonoid biosynthesis and plant hormone signal transduction. The expression levels of 16 differentially expressed genes in plant hormone signal transduction pathway were analyzed. The 11 differentially expressed genes in auxin pathway were up-regulated, and the 5 genes with large differences were IAA4, IAA22, IAA27, LAX3 and A-ARR genes. The transcriptome data were verified by qRT-PCR. 【Conclusion】 By sequencing the callus of 'Shuangyou' grape tissue culture plantlets induced by different concentrations of IBA, NAA and sucrose, a relatively complete transcriptome data is obtained, and its function annotation, enrichment analysis and hormone related gene expression analysis are carried out.

Key words: Vitis amurensis; ‘Shuangyou’; rooting; callus; transcriptome sequencing

摘要：

【目的】研究山葡萄生根的分子机理,为山葡萄生产繁殖中生根提供理论数据和参考依据。【方法】对山葡萄‘双优’进行组织培养至生根阶段,设置不同浓度IBA(0.1、0.3和0.5 mg/L)、NAA(0.1、0.3和0.5 mg/L)和蔗糖(15、25和35 g/L)处理,选择根系数目差别较大的两组,对其基部生根的愈伤组织进行转录组测序分析。【结果】6个样本共获得的高质量序列共284.40 Gb,89.15%~90.3%的Clean Reads 比对到参考基因组上, S6 较S1中显著上调的差异表达基因 537个,显著下调的差异表达基因 277个,共814个差异表达基因。共805个差异表达基因注释到COG的21个分类中,主要包括未知功能、细胞内运输和信号转导机制等条目。差异表达基因主要富集于多糖结合、驱动蛋白复合体和生长素激活信号通路等条目。差异表达基因富集在苯丙烷生物合成、类黄酮生物合成和植物激素信号转导等通路。生长素途径中的11个差异表达基因均为上调表达,表达量差异较大的5个基因为IAA14、IAA22、IAA27、LAX3和A-ARR基因。转录组数据可信。【结论】通过对不同浓度的IBA、NAA和蔗糖诱导‘双优’葡萄组培苗生根的愈伤组织进行测序,获得了较为完整的转录组数据,并对其进行功能注释、富集分析和激素相关基因表达量分析。

关键词: 山葡萄, ‘双优’, 生根, 愈伤组织, 转录组测序

CLC Number:

S663.1

WEI Wei, SHAN Shouming, XU Wendi, LI Guangzong. Transcriptome analysis of callus at rooting stage in tissue culture of vitis amurensis 'shuangyou'[J]. Xinjiang Agricultural Sciences, 2023, 60(6): 1451-1459.

韦伟, 单守明, 徐文娣, 李光宗. 山葡萄‘双优’组织培养生根期愈伤组织的转录组分析[J]. 新疆农业科学, 2023, 60(6): 1451-1459.

Figures/Tables 14

Tab.1 Orthogonal experimental design table

试验组 Test group	蔗糖浓度 Sucrose concentration (g/L)	NAA浓度 NAA concentration (mg/L)	IBA浓度 IBA concentration (mg/L)
S₁	15	0.1	0.1
S₂	15	0.5	0.3
S₃	15	0.3	0.5
S₄	25	0.3	0.1
S₅	25	0.1	0.3
S₆	25	0.5	0.5
S₇	35	0.5	0.1
S₈	35	0.3	0.3
S₉	35	0.1	0.5

Tab.2 Primer sequences of qRT-PCR

基因ID Gene ID	注释 Annotated	序列 Sequence (5'-3')
VIT_07s0289g00080	糖基转移酶	F: GCTACACGCTTCATCTCCAAGTCC R: GAGCCGCCTTCATCATAGCCATC
VIT_13s0067g02360	过氧化物酶	F: GGTGTGGTCTCGTGTGCTGATG R: TTGGAGGAGGGATGCTGTTGTTTG
VIT_18s0041g00710	糖基转移酶	F: AAAGGATTGGAGAACAGCGGACAG R: ATCGGCTTCTTCACTCTGCTTGC
VIT_00s0615g00020	含PKS_ER结构域的蛋白质	F: GAGATTGTAGGCATCGTGACAGAGG R: GCTCCAACCAGACACCCAACAC
VIT_14s0128g00660	胚蛋白样蛋白	F: TCATGGCATTGGCTTCCTCTCTTG R: TCTTGGGTTCAGGAATGGCAACAC
VIT_07s0005g06450	含蛋白激酶结构域的蛋白质	F: GTTTCCTTTCCGTGGCTCCAGAG R: GGCTTCCGCTACTTCTCGTTCAC

Fig.1 Number of taproots in tissue culture of ‘Shuangyou’ grapes in each group

Fig.2 Rooting and tissue culture seedlings of ‘Shuangyou’grape in S1 and S6 groups

Tab.3 The sample total RNA quality test

样品名称 Sample name	RNA 浓度 RNA concentration (ng/μL)	OD_260/280	OD_260/230	RIN值 RIN value
S₁-1	454.50	2.07	2.19	10
S₁-2	387.20	2.10	1.77	10
S₁-3	437.10	2.07	1.87	10
S₆-1	199.90	2.15	1.09	9.9
S₆-2	316.50	2.09	1.75	10
S₆-3	188.90	2.07	1.78	10

Fig.3 Total RNA agarose gel electrophoresis Note: M. Trans 2KPlus

Tab.4 Statistical of sample sequencing data

样品名称 Sample name	原始数据 Raw reads	过滤数据 Clean reads	错误率 Error rate (%)	Q20 (%)	Q30 (%)	GC含量 Content (%)
S₁-1	48.77	48. 50	0.02	98.16	94.36	46.66
S₁-2	47.93	47.69	0.02	98.22	94.5	46.67
S₁-3	47.61	47.36	0.02	98.24	94.56	46.5
S₆-1	44.48	44.19	0.03	98.04	94.07	46.18
S₆-2	47.89	47.65	0.02	98.19	94.38	45.95
S₆-3	49.24	49.02	0.02	98.23	94.47	46.21

Fig.4 Volcanic map analysis of differentially expressed genes

Fig.5 COG function classification of differentially expressed genes Note: A. RNA processing and modification; B. Chromatin structure and dynamics; C. Energy production and conversion; D. Cell cycle control, cell division, chromosome partitioning; E. Amino acid transport and metabolism; F. Nucleotide transport and metabolism; G. Carbohydrate transport and metabolism; H. Coenzyme transport and metabolism; I. Lipid transport and metabolism; J. Translation, ribosomal structure and biogenesis; K. Transcription; L. Replication, recombination and repair; M. Cell wall/membrane/envelope biogenesis; O. Posttranslational modification, protein turnover, chaperones; P. Inorganic ion transport and metabolism; Q. Secondary metabolites biosynthesis, transport and catabolism; S. Function unknown; T. Signal transduction mechanisms; U. Intracellular trafficking, secretion, and vesicular transport; V. Defense mechanisms; Z. Cytoskeleton

Fig.6 GO function analysis of differentially expressed genes

Fig.7 Scatter plot of KEGG enrichment

Fig.8 Display of gene number of significant enrichment pathway in KEGG enrichment analysis

Fig.9 Heat map of genes related to hormone Note: Using log10(FPKM) value for cluster analysis, red represents high expression genes, blue represents low expression genes, log10(FPKM) gradually increases from blue to red

Fig.10 qRT-PCR validation of partially selected gene expression

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