山葡萄‘双优’组织培养生根期愈伤组织的转录组分析

doi:10.6048/j.issn.1001-4330.2023.06.018

摘要/Abstract

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

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

中图分类号:

S663.1

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.

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.

图/表 14

表1 正交试验设计

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

表2 qRT-PCR引物序列

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

图1 各组‘双优’葡萄组织培养主根数

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

图2 S1和S6‘双优’葡萄生根及组培苗

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

表3 样品总RNA 质检

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

图3 总RNA 琼脂糖凝胶电泳检测注:M. Trans 2KPlus

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

表4 样品测序数据统计

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

图4 差异表达基因的火山图

Fig.4 Volcanic map analysis of differentially expressed genes

图5 差异表达基因的COG功能分类注:A. RNA加工与修饰; B. 染色质结构与动力学; C. 能源生产和转换;D. 细胞周期控制,细胞分裂,染色体分配; E. 氨基酸转运与代谢; F. 核苷酸转运与代谢; G. 碳水化合物运输和代谢; H. 辅酶转运与代谢; I. 脂质转运与代谢; J. 翻译、核糖体结构与生物发生; K. 转录; L. 复制、重组和修复; M. 细胞壁/膜/包膜生物发生; O. 翻译后修饰,蛋白质周转,伴侣; P. 无机离子转运与代谢; Q. 次生代谢物生物合成、运输和分解代谢; S. 功能未知; T. 信号转导机制; U. 细胞内运输、分泌和囊泡转运; V. 防御机制; Z. 细胞骨架

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

图6 差异表达基因的GO富集

Fig.6 GO function analysis of differentially expressed genes

图7 KEGG富集散点图

Fig.7 Scatter plot of KEGG enrichment

图8 KEGG富集分析显著富集通路基因数

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

图9 激素相关基因热图注:使用log10(FPKM)值进行聚类分析。红色代表高表达基因,蓝色代表低表达基因,从蓝色到红色为log10(FPKM)逐渐上升

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

图10 部分基因的qRT-PCR 验证

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

参考文献 23

[1]	张宇, 徐智慧, 任邵琦, 等. 山葡萄F3'H基因及其启动子的克隆与表达分析[J]. 农业生物技术学报, 2021, 29(11): 2099-2108.
	ZHANG Yu, XU Zhihui, REN Shaoqi, et al. Cloning and Expression Analysis of F3'H Gene and Promoter from Vitis amurensis[J]. Journal of Agricultural Biotechnology, 2021, 29(11): 2099-2108.
[2]	Xin S, Lan D, Bao G, et al. A stress associated protein from Chinese wild Vitis amurensis, VaSAP 15, enhances the cold tolerance of transgenic grapes[J]. Scientia Horticulturae, 2021: 285.
[3]	沈育杰, 赵淑兰, 杨义明, 等. 我国山葡萄种质资源研究与利用现状[J]. 特产研究, 2006,(3): 53-57.
	SHEN Yujie, ZHAO Shulan, YANG Yiming, et al. The research and utilization on Amur grape (Vitis. amurensis Rupr) germplasm resources in China[J]. Special Wild Economic Animal and Plant Research, 2006,(3): 53-57.
[4]	Oh K E, Shin H, Lee M K, et al. Characterization and optimization of the tyrosinase inhibitory Activity of Vitis amurensis root using LC-Q-TOF-MS coupled with a bioassay and response surface methodology[J]. Molecules, 2021, 26(2): 446. DOI URL
[5]	戴彩虹, 马绍英, 李胜, 等. 山葡萄‘双红’和‘双优’的试管快繁研究[J]. 甘肃农业大学学报, 2014, 49(4): 63-68, 72.
	DAI Caihong, MA Shaoying, LI Sheng, et al. Rapid proliferation of Vitis amurensis ‘Shuanghong’ and ‘Shuangyou’[J]. Journal of Gansu Agricultural University, 2014, 49(4): 63-68, 72.
[6]	莫银屏. 刺葡萄离体快繁体系建立及扦插生根机理研究[D]. 长沙: 湖南农业大学, 2015.
	MO Yinping. Establishment of in vitro rapid propagation system and rooting mechanism of cutting of Vitis spinosa[D]. Changsha: Hunan Agricultural University, 2015.
[7]	崔凯, 吴伟伟, 刁其玉. 转录组测序技术的研究和应用进展[J]. 生物技术通报, 2019, 35(7): 1-9. DOI
	CUI Kai, WU Weiwei, DIAO Qiyu. Application and research progress on transcriptomics[J]. Biotechnology Bulletin, 2019, 35(7): 1-9. DOI
[8]	石田培, 张莉. 全转录组学在畜牧业中的应用[J]. 遗传, 2019, 41(3):193-205.
	SHI Tianpei, ZHANG Li. Application of whole transcriptomics in animal husbandry[J]. Hereditas(Beijing), 2019, 35(7): 1-9. DOI URL
[9]	林茜, 高营营, 覃换玲, 等. ‘阳光玫瑰’葡萄组培脱毒快繁技术研究[J]. 果树学报, 2021, 38(3): 435-443.
	LIN Xi, GAO Yingying, QIN Huanling, et al. Study on the technology of virus-free and rapid propagation of ‘Shine Muscat’ grape in tissue culture[J]. Journal of Fruit Science, 2021, 38(3): 435-443.
[10]	齐向丽, 师校欣, 杜国强. ‘红国王’葡萄组织培养快速繁殖[J]. 分子植物育种, 2020, 18(3): 982-987.
	QI Xiangli, SHI Xiaoxin, DU Guoqiang. Rapid propagation of ‘Hongguowang’ grape in Vitro[J]. Molecular Plant Breeding, 2020, 18(3): 982-987.
[11]	Saini S, Sharma I, Kaur N, et al. Auxin: a master regulator in plant root development[J]. Pant Cell Rep, 2013, 32(6): 741-757.
[12]	Ljung K, Bhalerao RP, Sandberg G. Sites and homeostatic control of auxin biosynthesis in Arabidopsis during vegetative growth.[J]. The Plant Journal, 2001, 28(4): 465-474. DOI URL
[13]	Ikeda Y, Men S Z, Fischer U, et al. Local auxin biosynthesis modulates gradient-directed planar polarity in Arabidopsis[J]. Nature Cell Biology, 2009, 11(6):731-738. DOI PMID
[14]	Robert H S, Friml J. Auxin and other signals on the move in plants[J]. Nature Chemical Ciology, 2009, 5(5): 325-332.
[15]	林雨晴, 齐艳华. 生长素输出载体PIN家族研究进展[J]. 植物学报, 2021, 56(2):151-165.
	LIN Yuqing, QI Yanhua. Advances in auxin efflux carrier PIN proteins[J]. Chinese Bulletin of Botany, 2021, 56(2): 151-165.
[16]	孙雪丽, 刘范, 田娜, 等. 香蕉Aux/IAA基因家族的全基因组鉴定及表达分析[J]. 园艺学报, 2019, 46(10): 1919-1935. DOI
	SUN Xueli, LIU Fan, TIAN Na, et al. Genome-wide identification and expression analysis of Aux/IAA gene family in banana[J]. Acta Horticulturae Sinica, 2019, 46(10): 1919-1935. DOI
[17]	李俊男, 燕晓杰, 李枢航, 等. 植物AUX/IAA基因家族研究进展[J]. 中国农学通报, 2018, 34(15): 89-92. DOI
	LI Junnan, YAN Xiaojie, LI Shuhang, et al. Plants AUX/IAA gene family: research progress[J]. Chinese Agricultural Science Bulletin, 2018, 34(15): 89-92.
[18]	曾文芳, 王小贝, 潘磊, 等. 桃Aux/IAA家族基因鉴定及在果实成熟过程中的表达分析[J]. 园艺学报, 2017, 44(2): 233-244. DOI
	ZENG Wenfang, WANG Xiaobei, PAN Lei, et al. Identification and expression profiling of Aux/IAA family gene during peach fruit ripening[J]. Acta Horticulturae Sinica, 2017, 44(2): 233-244. DOI
[19]	黎颖, 左开井, 唐克轩. 植物GH3基因家族的功能研究概况[J]. 植物学通报, 2008,(5): 507-515.
	LI Ying, ZUO Kaijing, TANG Kexuan. A survey of functional studies of the GH3 gene family in plants[J]. Chinese Bulletin of Botany, 2008,(5): 507-515.
[20]	曾文芳, 潘磊, 牛良, 等. 桃GH3基因家族的生物信息学分析及其在果实发育中的表达[J]. 园艺学报, 2015, 42(5): 833-842. DOI
	ZENG Wenfang, PAN Lei, NIU Liang, et al. Bioinformatics analysis and expression of the nectarine indole-3-aceticacid-amido synthase(GH 3)gene family during fruit development[J]. Acta Horticulturae Sinica, 2015, 42(5): 833-842. DOI
[21]	刘晓东, 王若仲, 焦彬彬, 等. 拟南芥IAA酰胺合成酶GH3-6负调控干旱和盐胁迫的反应[J]. 植物学报, 2016, 51(5): 586-593. DOI
	LIU Xiaodong, WANG Ruozhong, JIAO Binbin, et al. Indole acetic acid-amido synthetase GH3-6 negatively regulates response to drought and salt in Arabidopsis[J]. Chinese Bulletin of Botany, 2016, 51(5): 586-593.
[22]	刘帅, 徐伟荣, 张亚红, 等. 基于转录组研究补光对设施‘红地球’葡萄萌芽的影响[J]. 果树学报, 2021, 38(3): 305-317.
	LIU Shuai, XU Weirong, ZHANG Yahong, et al. Effects of supplementary light on the bud burst of ‘Red Globe’ grape under protected cultivation based on transcriptome sequencing[J]. Journal of Fruit Science, 2021, 38(3): 305-317.
[23]	尚骁尧, 周玲芳, 石欣玥, 等. 蒺藜苜蓿细胞分裂素响应调节因子ARR9自激活检测及表达分析[J]. 中国草地学报, 2021, 43(12): 1-10.
	SHANG Xiaorao, ZHOU Linfang, SHI Xinyue, et al. Self-activation detection and expression analysis of cytokinin response regulator ARR9 in Medicago truncatula[J]. Chinese Journal of Grassland, 2021, 43(12): 1-10.