广丰千金薯和铁棍山药脱毒微型块茎的转录组分析

doi:10.3969/j.issn.1004-1524.2022.10.15

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (10): 2209-2219.DOI: 10.3969/j.issn.1004-1524.2022.10.15

广丰千金薯和铁棍山药脱毒微型块茎的转录组分析

尹明华¹^,²^,³^,⁴(), 白丽¹, 陈舒敏¹, 程佳慧¹, 冯丽文¹

1.上饶师范学院生命科学学院,江西上饶 334001
2.上饶农业技术创新研究院,江西上饶 334001
3.上饶市药食同源植物资源保护与利用重点实验室,江西上饶 334001
4.上饶市薯芋类作物种质保存与利用重点实验室,江西上饶 334001

收稿日期:2022-01-18 出版日期:2022-10-25 发布日期:2022-10-26
作者简介:尹明华(1973—),女,江西永新人,硕士,教授,主要从事植物生物技术研究。E-mail: yinminghua04@163.com
基金资助:
2021年度江西省教育厅科学技术研究项目(GJJ211729);上饶市科技局平台载体建设项目(2020I001)

Transcriptome analysis of virus-free microtubers of Dioscorea polystachya Turczaninow. cv. Guangfeng Qianjin and Dioscorea polystachya Turczaninow. cv. Tiegun

YIN Minghua¹^,²^,³^,⁴(), BAI Li¹, CHEN Shumin¹, CHENG Jiahui¹, FENG Liwen¹

1. College of Life Sciences, Shangrao Normal University, Shangrao 334001, Jiangxi, China
2. Shangrao Agricultural Technology Innovation Research Institute, Shangrao 334001, Jiangxi, China
3. Key Laboratory of Protection and Utilization of Medicinal and Edible Plant Resources in Shangrao City, Shangrao 334001, Jiangxi, China
4. Shangrao Key Laboratory of Germplasm Conservation and Utilization of Potato and Taro Crops, Shangrao 334001, Jiangxi, China

Received:2022-01-18 Online:2022-10-25 Published:2022-10-26

摘要/Abstract

摘要：

为探究广丰千金薯和铁棍山药在分子水平的差异,以广丰千金薯(QJS)和铁棍山药(TGSY)试管苗的微型块茎为试验材料进行转录组分析。结果表明:QJS组和TGSY组样本间相关系数为0.42。QJS组和TGSY组表达量FPKM的对数值在0~1.5,表达量密度在0~1.0。与TGSY组相比,QJS组有4 765个基因下调,有5 112个基因上调。QJS组和TGSY组表达的共有基因有25 207个,QJS组单独表达的基因有5 261个,TGSY组单独表达的基因有3 571个。QJS组和TGSY组共发现611 498个单核苷酸多态性(single nucleotide polymorphism,SNP)位点和12 056个简单重复序列(simple sequence repeats,SSR)。与TGSY组相比,QJS组的淀粉合成酶3、α-淀粉酶3、β-淀粉酶、类黄酮3'-单加氧酶、类黄酮3'-羟化酶、花青素合酶等基因上调,而颗粒结合淀粉合酶2、蔗糖合酶1、扩展蛋白2和苯丙氨酸解氨酶等基因下调。这可能是广丰千金薯微型块茎肉质绵密粉嫩、软糯爽口、胁迫防御、食后易胀的内在原因。结果可以为广丰千金薯的品种鉴定和分子育种提供参考。

关键词: 广丰千金薯, 铁棍山药, 脱毒, 微型块茎, 转录组分析

Abstract:

In order to explore the differences between Dioscorea polystachya Turczaninow. cv. Guangfeng Qianjin (QJS) and D. polystachya Turczaninow. cv. Tiegun (TGSY) at molecular level, the transcriptome analysis was carried out on the microtubers of plantlets of QJS and TGSY. The results showed that correlation coefficient between QJS group and TGSY group was 0.42. The logarithm of expression FPKM in QJS group and TGSY group was between 0-1.5 and the expression density was between 0-1.0. Compared with TGSY group, 4 765 genes were down regulated and 5 112 genes were up regulated in QJS group. The number of common genes expressed in QJS group and TGSY group was 25 207, the number of genes expressed alone in QJS group was 5 261, and the number of genes expressed alone in TGSY group was 3 571. A total of 611 498 single nucleotide polymorphism (SNP) loci and 12 056 simple sequence repeats (SSRs) were found in QJS group and TGSY group. Compared with TGSY group, genes of starch synthase 3, alpha-amylase 3, beta-amylase, flavonoid 3'-monooxygenas, flavonoid 3'-hydroxylas and anthocyanin synthase were up-regulated, while genes of granule-bound starch synthase, sucrose synthase 1, expansin 2 and phenylalanine ammonia lyase were down regulated. This might be the internal reason why D. polystachya Turczaninow. cv. Guangfeng Qianjin microtuber was tender, soft, waxy and refreshing, stress defense and easy to swell after eating. The results of this study would provide reference for variety identification and molecular breeding of D. polystachya Turczaninow. cv. Guangfeng Qianjin.

Key words: D. polystachya Turczaninow. cv. Guangfeng Qianjin, D. polystachya Turczaninow. cv. Tiegun, virus-free, microtuber, transcriptome analysis

中图分类号:

S632.1

尹明华, 白丽, 陈舒敏, 程佳慧, 冯丽文. 广丰千金薯和铁棍山药脱毒微型块茎的转录组分析[J]. 浙江农业学报, 2022, 34(10): 2209-2219.

YIN Minghua, BAI Li, CHEN Shumin, CHENG Jiahui, FENG Liwen. Transcriptome analysis of virus-free microtubers of Dioscorea polystachya Turczaninow. cv. Guangfeng Qianjin and Dioscorea polystachya Turczaninow. cv. Tiegun[J]. Acta Agriculturae Zhejiangensis, 2022, 34(10): 2209-2219.

图/表 11

参考文献 36

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目的基因 Target gene	上游引物 Forward primer(5'→3')	下游引物 Reverse primer(5'→3')	退火温度 Annealing temperature/℃	长度 Length/bp
淀粉合成酶3 Starch synthase 3(SS3)	CTGTGGAAGGTGGAATGGATG	TCTGCTTCTTTCCGACGCTGT	52.3	136
α-淀粉酶3 Alpha-amylase 3(α-AMY3)	GCCCTGCGTAGAACCAGTATCAT	TGCGAGTATTGGCGGCTATCT	53.6	114
β-淀粉酶 Beta-amylase(β-AMY)	TTATGCTCCCTCTGGGTGTTA	GCTCCTGGGCTTGGACTCTAT	52.8	149
颗粒结合淀粉合酶2 Granule-bound starch synthase 2(GBSS2)	ATTAAAGTGCTCACCTCCTACT	ATACGTGCCAGAAATCCAAGA	49.8	164
蔗糖合酶1 Sucrose synthase 1(SUS1)	CTCAAGAAGGTCTAGGAGCAG	ATCCCAAATGATACACCATACTC	51.3	126
扩展蛋白2 Expansin 2(EXP2)	CGCTGTGCTTAGTGCTGCTGT	ATTCTACGGTGGTGGTGATGC	52.3	106
类黄酮3'-单加氧酶 Flavonoid 3'-monooxygenase(F3'MO)	TCTGACATTGCCCATTCTACT	TTCTTCAACTTGCTGCTGATC	51.8	130
类黄酮3'-羟化酶 Flavonoid 3'-hydroxylase(F3'H)	CATTCACCGCCTGTCCTACAT	CTGCACCTGTTCTCAGTTAAAGC	53.4	127
苯丙氨酸解氨酶 Phenylalanine ammonia lyase(PAL)	AATCCTAACTAACATTGCTGCTC	GGCTTTGGTGCTACTTCTCAT	51.2	156
花青素合酶 Anthocyanin synthase(CHS)	TGAAGGATAGGTGGCTGACGT	GAAGCAGGTGGAATGGAGAAC	52.4	121
甘油醛磷酸脱氢酶 Glyceraldehyde phosphate dehydrogenase (GAPD)	TCCTAGCAAGGATGCTCCGATGT	AAACCCTCCACGATGCCAAAG	53.3	153

目的基因 Target gene	上游引物 Forward primer(5'→3')	下游引物 Reverse primer(5'→3')	退火温度 Annealing temperature/℃	长度 Length/bp
淀粉合成酶3 Starch synthase 3(SS3)	CTGTGGAAGGTGGAATGGATG	TCTGCTTCTTTCCGACGCTGT	52.3	136
α-淀粉酶3 Alpha-amylase 3(α-AMY3)	GCCCTGCGTAGAACCAGTATCAT	TGCGAGTATTGGCGGCTATCT	53.6	114
β-淀粉酶 Beta-amylase(β-AMY)	TTATGCTCCCTCTGGGTGTTA	GCTCCTGGGCTTGGACTCTAT	52.8	149
颗粒结合淀粉合酶2 Granule-bound starch synthase 2(GBSS2)	ATTAAAGTGCTCACCTCCTACT	ATACGTGCCAGAAATCCAAGA	49.8	164
蔗糖合酶1 Sucrose synthase 1(SUS1)	CTCAAGAAGGTCTAGGAGCAG	ATCCCAAATGATACACCATACTC	51.3	126
扩展蛋白2 Expansin 2(EXP2)	CGCTGTGCTTAGTGCTGCTGT	ATTCTACGGTGGTGGTGATGC	52.3	106
类黄酮3'-单加氧酶 Flavonoid 3'-monooxygenase(F3'MO)	TCTGACATTGCCCATTCTACT	TTCTTCAACTTGCTGCTGATC	51.8	130
类黄酮3'-羟化酶 Flavonoid 3'-hydroxylase(F3'H)	CATTCACCGCCTGTCCTACAT	CTGCACCTGTTCTCAGTTAAAGC	53.4	127
苯丙氨酸解氨酶 Phenylalanine ammonia lyase(PAL)	AATCCTAACTAACATTGCTGCTC	GGCTTTGGTGCTACTTCTCAT	51.2	156
花青素合酶 Anthocyanin synthase(CHS)	TGAAGGATAGGTGGCTGACGT	GAAGCAGGTGGAATGGAGAAC	52.4	121
甘油醛磷酸脱氢酶 Glyceraldehyde phosphate dehydrogenase (GAPD)	TCCTAGCAAGGATGCTCCGATGT	AAACCCTCCACGATGCCAAAG	53.3	153

样品 Sample	原始数据中的read数 Raw read	原始数据过滤后的read数 Clean read	原始数据过滤后的碱基数 Clean base	数据整体测序错误率 Error rate/%	Phred数值大于 20的碱基占总碱基的百分比 Q20/%	Phred数值大于 30的碱基占总碱基的百分比 Q30/%	Clean read中G与 C占4种碱基的百分比 GC content/%
广丰千金薯QJS	22 686 888	21 821 043	6.55 G	0.03	98.00	94.1	46.00
铁棍山药TGSY	22 152 964	21 470 765	6.44 G	0.02	98.08	94.3	45.59

样品 Sample	原始数据中的read数 Raw read	原始数据过滤后的read数 Clean read	原始数据过滤后的碱基数 Clean base	数据整体测序错误率 Error rate/%	Phred数值大于 20的碱基占总碱基的百分比 Q20/%	Phred数值大于 30的碱基占总碱基的百分比 Q30/%	Clean read中G与 C占4种碱基的百分比 GC content/%
广丰千金薯QJS	22 686 888	21 821 043	6.55 G	0.03	98.00	94.1	46.00
铁棍山药TGSY	22 152 964	21 470 765	6.44 G	0.02	98.08	94.3	45.59

Transcript/unigene	不同长度(bp)区间Different length interval				总数 Total
Transcript/unigene	300~<500	500~<1 000	1 000~<2 000	≥2 000	总数 Total
Transcript数量 Number of transcript	21 413	25 033	23 152	11 940	81 538
Unigene数量 Number of unigene	11 661	9 924	8 117	4 365	34 067

广丰千金薯和铁棍山药脱毒微型块茎的转录组分析

Transcriptome analysis of virus-free microtubers of Dioscorea polystachya Turczaninow. cv. Guangfeng Qianjin and Dioscorea polystachya Turczaninow. cv. Tiegun

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GO标号 GO accession	描述 Description	项目类别 Term type	P值 P value	校正后的P值 Corrected P value	差异表达基因数 Number of differentially expressed genes
GO:0003735	核糖体的结构成分Structural constituent of ribosome	MF	0.0 000 001	0.0 005 988	215
GO:0005198	结构分子活性Structural molecule activity	MF	0.0 000 014	0.0 033 944	301
GO:0071840	细胞成分组织或生物发生	BP	0.0 000 275	0.0 404 870	537
	Cellular component organization or biogenesis
GO:0005840	核糖体Ribosome	CC	0.0 000 413	0.0 404 870	240
GO:0044085	细胞成分生物发生Cellular component biogenesis	BP	0.0 000 415	0.0 404 870	394
GO:0042254	核糖体生物发生Ribosome biogenesis	BP	0.0 000 722	0.0 586 230	240
GO:0004523	RNA-DNA杂交核糖核酸酶活性	MF	0.0 000 841	0.0 586 230	53
	RNA-DNA hybrid ribonuclease activity
GO:0004521	内核糖核酸酶活性Endoribonuclease activity	MF	0.0 001 260	0.0 768 270	68
GO:0022613	核糖核蛋白复合物生物发生	BP	0.0 001 420	0.0 769 580	242
	Ribonucleoprotein complex biogenesis
GO:0016891	产生5'-磷酸单酯的内核糖核酸酶活性	MF	0.0 004 694	0.2 289 300	58
	Endoribonuclease activity, producing 5'-phosphomonoesters
GO:0006412	翻译Translation	BP	0.0 010 554	0.4 679 300	268
GO:0006979	氧化应激反应Response to oxidative stress	BP	0.0 011 666	0.4 741 100	34
GO:0004298	苏氨酸型内肽酶活性	MF	0.0 015 389	0.5 361 000	12
	Threonine-type endopeptidase activity
GO:0070003	苏氨酸型肽酶活性Threonine-type peptidase activity	MF	0.0 015 389	0.5 361 000	12
GO:0043043	肽生物合成过程Peptide biosynthetic process	BP	0.0 016 825	0.5 470 400	269
GO:0071554	细胞壁组织或生物发生	BP	0.0 021 782	0.6 639 400	43
	Cell wall organization or biogenesis
GO:0043232	细胞内非膜结合细胞器	CC	0.0 025 134	0.6 982 100	487
	Intracellular non-membrane-bounded organelle
GO:0043228	非膜结合细胞器Non-membrane-bounded organelle	CC	0.0 025 769	0.6 982 100	491
GO:0016209	抗氧化活性Antioxidant activity	MF	0.0 028 077	0.7 206 800	46
GO:0006518	肽代谢过程Peptide metabolic process	BP	0.0 0384 40	0.8 927 200	280

SNP位点所在unigene ID Gene ID	SNP位点 SNP site	变异位点在参考序列中的碱基 Base of variant site in reference sequence	变异位点在样本中的实际碱基 The actual base of the variant site in the sample
Cluster-10348.0	213	A	G
Cluster-10348.0	264	T	A
Cluster-10348.0	312	C	T
Cluster-10348.0	339	A	G
Cluster-10348.0	378	T	C
Cluster-10348.0	419	A	T
Cluster-10348.0	420	T	A
Cluster-10348.0	480	C	T
Cluster-10348.0	933	T	A
Cluster-10348.0	975	T	C
Cluster-10348.0	1 122	G	A
Cluster-10348.0	1 335	A	G
Cluster-10348.0	1 438	T	C
Cluster-10348.0	1 443	G	A
Cluster-10348.0	1 519	G	C
Cluster-10348.0	1 749	A	C
Cluster-10348.0	1 884	A	G
Cluster-10348.0	1 993	G	A
Cluster-10348.0	2 016	G	A
Cluster-10348.0	2 029	A	G

基因ID Gene ID	SSR类型 SSR type	重复序列的核苷酸构成以及重复数 Nucleotide composition and number of repeats	大小 Size/bp	开始位置 Start	结尾位置 End	SSR位置区域 SSR position
Cluster-8671.0	p1	(A)12	12	243	254	undetermined
Cluster-8532.707	p2	(TC)7	14	46	59	undetermined
Cluster-4711.0	p1	(A)11	11	349	359	undetermined
Cluster-15386.0	p2	(AT)8	16	477	492	undetermined
Cluster-12681.1	p1	(A)15	15	1	15	undetermined
Cluster-9382.0	p1	(T)10	10	705	714	undetermined
Cluster-9382.0	p1	(A)10	10	1 203	1 212	undetermined
Cluster-9382.0	p1	(T)10	10	1 499	1 508	undetermined
Cluster-4739.0	c	(A)10(AAAT)11	108	9	116	undetermined
Cluster-8532.12872	p4	(ATTT)5	20	5 026	5 045	undetermined
Cluster-8532.12872	p2	(TA)6	12	5 178	5 189	undetermined
Cluster-8532.12872	p4	(TTTA)5	20	5 291	5 310	undetermined
Cluster-8532.12872	p1	(T)11	11	5 504	5 514	undetermined
Cluster-8532.12870	p1	(A)13	13	45	57	undetermined
Cluster-8532.12877	p1	(T)10	10	1 056	1 065	undetermined
Cluster-8532.12875	c	(AAAT)5(AAG)6	38	353	390	undetermined
Cluster-8532.13795	p1	(A)12	12	303	314	utr5
Cluster-8532.12878	p2	(CT)7	14	1 047	1 060	utr3
Cluster-8532.12878	p1	(A)18	18	1 402	1 419	utr3
Cluster-10579.0	p2	(AT)10	20	916	935	undetermined

目的基因 Target gene	RNA-Seq		qRT-PCR
目的基因 Target gene	QJS	TGSY	QJS	TGSY
淀粉合成酶3	29.61	5.93	1 a	0.584 b
Starch synthase 3
α-淀粉酶3	41.41	16	1 a	0.558 b
Alpha-amylase 3
β-淀粉酶 Beta-amylase	41.42	9.61	1 a	0.413 b
颗粒结合淀粉合酶2	32.23	106.97	1 b	1.815 a
Granule-bound starch synthase 2
蔗糖合酶1	223.73	840.19	1 b	2.733 a
Sucrose synthase 1
扩展蛋白2 Expansin 2	0.63	62.31	1 b	5.487 a
类黄酮3'-单加氧酶	12.72	0.72	1 a	0.097 b
Flavonoid 3'-monooxygenase
类黄酮3'-羟化酶	24.89	2.66	1 a	0.320 b
Flavonoid 3'-hydroxylase
苯丙氨酸解氨酶	0	10.04	1 b	1.610 a
Phenylalanine ammonia lyase
花青素合酶	25.18	4.05	1 a	0.596 b
Anthocyanin synthase

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