利用RNA-Seq鉴定调控甘蓝型油菜叶片光合特性的低温胁迫应答基因

doi:10.13560/j.cnki.biotech.bull.1985.2021-1048

生物技术通报 ›› 2022, Vol. 38 ›› Issue (4): 126-142.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1048

利用RNA-Seq鉴定调控甘蓝型油菜叶片光合特性的低温胁迫应答基因

金姣姣¹(), 刘自刚², 米文博², 徐明霞², 邹娅², 徐春梅², 赵彩霞³

1.陇东学院农林科技学院陇东旱地作物种质改良及产业化协同创新中心甘肃省旱地冬小麦种质创新与应用工程研究中心,庆阳 745000
2.甘肃省共建干旱生境作物学国家重点实验室甘肃农业大学,兰州 730070
3.西藏自治区农牧科学院农业研究所,拉萨 860000

收稿日期:2021-08-17 出版日期:2022-04-26 发布日期:2022-05-06
作者简介:金姣姣,女,硕士,助教,研究方向：作物遗传育种;E-mail： 631981312@qq.com
基金资助:
甘肃省教育厅产业支撑计划项目(2021CYZC-46);西藏农科院农科所统筹基金项目(2021NKS-TCJJ-001);陇东学院青年科技创新基金项目(XYZK2106);博士基金项目(2021QB-035);中央引导地方科技专项资金(ZCYD2020-2-3)

Identification of Low Temperature Stress-responsive Genes Regulating Photosynthetic Characteristics in the Leaves of Brassica napus by RNA-Seq

JIN Jiao-jiao¹(), LIU Zi-gang², MI Wen-bo², XU Ming-xia², ZOU Ya², XU Chun-mei², ZHAO Cai-xia³

1. College of Agriculture and Forestry,Longdong University/Longdong Co-Innovation Center for Germplasm Improvement and Industrialization of Dryland Crops/ Gansu Province Germplasm Innovation and Application Engineering Research Center of Dryland Winter Wheat,Qingyang 745000
2. Gansu Provincial Key Laboratory of Arid Land Crop Sciences/College of Agronomy,Gansu Agricultural University,Lanzhou 730070
3. Institute of Agriculture,Tibet Academy of Agriculture and Animal Husbandry Sciences,Lhasa 860000

Received:2021-08-17 Published:2022-04-26 Online:2022-05-06

摘要/Abstract

摘要：

明晰甘蓝型油菜光合特性对低温胁迫的响应,鉴定相关应答基因,以耐寒材料17NS和敏感材料NF24为研究对象,通过Illumina HiSeq^TM 2000平台进行RNA-Seq分析,探讨-4℃处理24 h后甘蓝型油菜的叶片形态、光合参数、叶片超微结构、基因调控表达模式等响应低温胁迫的分子机制。结果表明,低温胁迫下甘蓝型油菜响应光合调控的显著富集途径为光合作用途径和光合作用-天线蛋白途径,这两种途径中有64个特异表达基因,其中8个上调表达,56个下调表达,大部分注释到分子组成和生物学过程中,证实了光合作用与低温胁迫导致的幼苗叶片萎蔫、叶绿体数量和形态发生变化、类囊体结构受损、相对电解质渗透率增加等现象有关。通过qRT-PCR鉴定,前17个DEGs的表达模式与RNA-Seq分析结果一致,证实了RNA-Seq结果的可靠性,其中BnFd1（LOC106350097）和BnCP26（LOC106440450）均响应低温胁迫上调表达。BnFd1编码氨基酸产物是叶绿体上的铁氧还蛋白1（BnFd1）,属于不稳定亲水蛋白,定位于叶绿体和细胞质中,存在一个信号肽,属于PLNO3136超家族,存在保守的Fd1结构域;BnCP26编码氨基酸产物是类囊体上叶绿素a-b结合蛋白CP26（BnCP26）,属于稳定亲水蛋白,位于叶绿体中,无信号肽,属于叶绿素a_b结合超家族,存在保守的叶绿素A-B结合蛋白结构域。成功鉴定出2个甘蓝型油菜叶片低温应答基因BnFd1和BnCP26,与光合特性调控关系密切,物种间功能保守。

关键词: 甘蓝型油菜, 低温胁迫, 差异表达基因, BnFd1, BnCP26

Abstract:

In order to clarify the response of photosynthetic characteristics to low temperature stress in Brassica napus L.,using cold-resistant material 17NS and cold-sensitive material NF24 as study object,Illumina HiSeq^TM 2000 platform was applied to have RNA-Seq analysis,the molecular mechanism of B. napus responding to low temperature,such as leaf morphology,photosynthetic parameters,leaf ultrastructure and gene regulation expression pattern treated at -4℃ for 24 h was investigated. The results showed that the photosynthetic pathway and the photosynthesis-antenna protein pathway were significantly enriched in B. napus in response to low temperature stress. In these two pathways,64 genes were specifically expressed,including 8 up-regulated genes and 56 down-regulated genes,most of which were noted in molecular composition and biological process. It was confirmed that photosynthesis was related to leaf wilt,chloroplast number and morphology change,thylakoid structure damage and relative electrolyte permeability increase. qRT-PCR analysis showed that the expression patterns of the first 17 DEGs were consistent with RNA-Seq analysis results,confirming the reliability of RNA-Seq results. BnFd1（LOC106350097）and BnCP26（LOC106440450）were up-regulated in response to cold stress. The amino acid product encoded by BnFd1was the chloroplast ferridoretin 1（BnFd1）,an unstable hydrophilic protein,which were located in the chloroplast and cytoplasm,there was a signal peptide,belonging to the PLNO3136 superfamily and had a conserved Fd1 domain. The amino acid product encoded by BnCP26 was the chlorophyll a-b binding protein CP26（BnCP26）on thylakoid,it was a stable hydrophilic protein located in chloroplast without signal peptide,and belonged to the chlorophyll a_b binding superfamily and had a conserved chlorophyll a-b binding protein domain. Two cold response genes BnFd1 and BnCP26 are successfully identified in B. napus L.,which are closely related to the regulation of photosynthetic characteristics and had conserved interspecies functions.

Key words: Brassica napus L., low temperature stress, differential expressed genes, BnFd1, BnCP26

金姣姣, 刘自刚, 米文博, 徐明霞, 邹娅, 徐春梅, 赵彩霞. 利用RNA-Seq鉴定调控甘蓝型油菜叶片光合特性的低温胁迫应答基因[J]. 生物技术通报, 2022, 38(4): 126-142.

JIN Jiao-jiao, LIU Zi-gang, MI Wen-bo, XU Ming-xia, ZOU Ya, XU Chun-mei, ZHAO Cai-xia. Identification of Low Temperature Stress-responsive Genes Regulating Photosynthetic Characteristics in the Leaves of Brassica napus by RNA-Seq[J]. Biotechnology Bulletin, 2022, 38(4): 126-142.

图/表 20

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KEGG通路 KEGG pathway	基因号 Gene ID	引物序列 Sequence of primer（5'-3'）	扩增长度 Amplification length/bp	扩增效率 Amplification efficiency/%
P	ncbi_106366341	AGATCCTAATGCCACCATCATC（F）	100	101.80
	ncbi_106366341	GTAGTCCTCATGCTCCTCAAAG（R）	100	101.80
	ncbi_106360470	CTCCTTACAACCCACTTCAGAG（F）	100	96.80
	ncbi_106360470	GAGCCACCTCCAAGAATCAA（R）	100	96.80
	ncbi_106400646	CTACCGACAAGAAGTCCATCAC（F）	102	88.28
	ncbi_106400646	AGGCAGTCTCACCGAAGTA（R）	102	88.28
	ncbi_106430073	GCAGAGGCAAGGATCAAAGA（F）	99	93.16
	ncbi_106430073	GTAAGGACGACGGAGGAAATAAG（R）	99	93.16
	ncbi_106381576	GCAGAGGCAAGGATCAAAGA（F）	99	100.09
	ncbi_106381576	GTAAGGACGACGGAGGAAATAAG（R）	99	100.09
	ncbi_106401683	CCGAGATTTGCTCTCCTTATCC（F）	119	103.61
	ncbi_106401683	CCGACGTATCTTTCACGTACTC（R）	119	103.61
	ncbi_106405529	CTCCTTACAACCCACTTCAGAG（F）	100	95.07
	ncbi_106405529	GAGCCTCCTCCAAGAATCAAA（R）	100	95.07
	ncbi_106350097	CATAACACCAGATGGAGAGAAGG（F）	149	102.17
	ncbi_106350097	CAACCGAGCCAGAGACTATTT（R）	149	102.17
PAP	ncbi_106371874	TAGCTGGAGATTACGGGTTTG（F）	143	122.90
	ncbi_106371874	GGTACGAAGTAGGTCGGTTAAG（R）	143	122.90
	ncbi_106382137	CTCGGAAACCCTAACTTGATCC（F）	100	101.97
	ncbi_106382137	CACCTCCAATTCTGTACCCTTC（R）	100	101.97
	ncbi_106439795	AACCGTGAGCTCGAAGTAATC（F）	100	98.06
	ncbi_106439795	CTCCGAATTTGACTCCGTTCT（R	100	98.06
	ncbi_106349237	AACCGTGAGCTCGAAGTAATC（F）	131	102.84
	ncbi_106349237	ATCTGAGAACCTGCCTTGAAC（R）	131	102.84
	ncbi_106396209	GAAGGTGGGCTCGACTATTT（F）	112	94.26
	ncbi_106396209	CGACTCTGTAACCCTCAACAG（R）	112	94.26
	ncbi_106447496	GAAGGAGGGCTTGACTACTTG（F）	99	103.51
	ncbi_106447496	CTCAACAGCTCCCATGAGAAT（R）	99	103.51
	ncbi_106447497	AAACGGAAGGTTGGCTATGT（F）	96	100.88
	ncbi_106447497	GCCAAATGGTCAGCAAGATTC（R）	96	100.88
	ncbi_106421061	CTGTCAAGCAAGGAGCAAAC（F）	102	97.35
	ncbi_106421061	CAAGAGGGTCGAATCCATAGTC（R）	102	97.35
	ncbi_106440450	AACCTTGTTCTGGCTGTAGTT（F）	98	93.57
	ncbi_106440450	GGGTGTAGCTTGTCCTCAAA（R）	98	93.57
RG	Bnactin-F	TCCATCCATCGTCCACAG		106.80
RG	Bnactin-R	GCATCATCACAAGCATCCTT		106.80

KEGG通路 KEGG pathway	基因号 Gene ID	引物序列 Sequence of primer（5'-3'）	扩增长度 Amplification length/bp	扩增效率 Amplification efficiency/%
P	ncbi_106366341	AGATCCTAATGCCACCATCATC（F）	100	101.80
	ncbi_106366341	GTAGTCCTCATGCTCCTCAAAG（R）	100	101.80
	ncbi_106360470	CTCCTTACAACCCACTTCAGAG（F）	100	96.80
	ncbi_106360470	GAGCCACCTCCAAGAATCAA（R）	100	96.80
	ncbi_106400646	CTACCGACAAGAAGTCCATCAC（F）	102	88.28
	ncbi_106400646	AGGCAGTCTCACCGAAGTA（R）	102	88.28
	ncbi_106430073	GCAGAGGCAAGGATCAAAGA（F）	99	93.16
	ncbi_106430073	GTAAGGACGACGGAGGAAATAAG（R）	99	93.16
	ncbi_106381576	GCAGAGGCAAGGATCAAAGA（F）	99	100.09
	ncbi_106381576	GTAAGGACGACGGAGGAAATAAG（R）	99	100.09
	ncbi_106401683	CCGAGATTTGCTCTCCTTATCC（F）	119	103.61
	ncbi_106401683	CCGACGTATCTTTCACGTACTC（R）	119	103.61
	ncbi_106405529	CTCCTTACAACCCACTTCAGAG（F）	100	95.07
	ncbi_106405529	GAGCCTCCTCCAAGAATCAAA（R）	100	95.07
	ncbi_106350097	CATAACACCAGATGGAGAGAAGG（F）	149	102.17
	ncbi_106350097	CAACCGAGCCAGAGACTATTT（R）	149	102.17
PAP	ncbi_106371874	TAGCTGGAGATTACGGGTTTG（F）	143	122.90
	ncbi_106371874	GGTACGAAGTAGGTCGGTTAAG（R）	143	122.90
	ncbi_106382137	CTCGGAAACCCTAACTTGATCC（F）	100	101.97
	ncbi_106382137	CACCTCCAATTCTGTACCCTTC（R）	100	101.97
	ncbi_106439795	AACCGTGAGCTCGAAGTAATC（F）	100	98.06
	ncbi_106439795	CTCCGAATTTGACTCCGTTCT（R	100	98.06
	ncbi_106349237	AACCGTGAGCTCGAAGTAATC（F）	131	102.84
	ncbi_106349237	ATCTGAGAACCTGCCTTGAAC（R）	131	102.84
	ncbi_106396209	GAAGGTGGGCTCGACTATTT（F）	112	94.26
	ncbi_106396209	CGACTCTGTAACCCTCAACAG（R）	112	94.26
	ncbi_106447496	GAAGGAGGGCTTGACTACTTG（F）	99	103.51
	ncbi_106447496	CTCAACAGCTCCCATGAGAAT（R）	99	103.51
	ncbi_106447497	AAACGGAAGGTTGGCTATGT（F）	96	100.88
	ncbi_106447497	GCCAAATGGTCAGCAAGATTC（R）	96	100.88
	ncbi_106421061	CTGTCAAGCAAGGAGCAAAC（F）	102	97.35
	ncbi_106421061	CAAGAGGGTCGAATCCATAGTC（R）	102	97.35
	ncbi_106440450	AACCTTGTTCTGGCTGTAGTT（F）	98	93.57
	ncbi_106440450	GGGTGTAGCTTGTCCTCAAA（R）	98	93.57
RG	Bnactin-F	TCCATCCATCGTCCACAG		106.80
RG	Bnactin-R	GCATCATCACAAGCATCCTT		106.80

反应物 Reagent	体积Volume/μL
SYBR^R Premix Ex Taq ^TM II	10
Forward primer	0.8
Reverse primer	0.8
ROX Reference Dye II	0.4
Template cDNA	2
ddH₂O	6

反应物 Reagent	体积Volume/μL
SYBR^R Premix Ex Taq ^TM II	10
Forward primer	0.8
Reverse primer	0.8
ROX Reference Dye II	0.4
Template cDNA	2
ddH₂O	6

组合 Paired comparison	显著上调差异基因数 Significantly up-regulated differential genes	显著下调差异基因数 Significantly down-regulated differential genes	总差异基因数 Significantly different number of genes
NF24 t0 - NF24 t	12 583	24 414	36 997
17NS t0 - 17NS t	11 710	23 141	34 851
NF24 t0-17NS t0	10 708	8 270	18 978
NF24 t - 17NS t	11 447	10 572	22 019

利用RNA-Seq鉴定调控甘蓝型油菜叶片光合特性的低温胁迫应答基因

Identification of Low Temperature Stress-responsive Genes Regulating Photosynthetic Characteristics in the Leaves of Brassica napus by RNA-Seq

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基因号 Gene ID	基因表达特性 Gene expression characteristics	基因描述 Description
ncbi_106366341	下调 Down	铁氧还蛋白—NADP还原酶,叶同工酶1,类叶绿体; Ferredoxin-NADP reductase,leaf isozyme 1,and chloroplastic-like
ncbi_106360470		光系统I反应中心亚基VI,类叶绿体; Photosystem I reaction center subunit VI,and chloroplastic-like
ncbi_106400646		进化氧增强蛋白2,叶绿体; Oxygen-evolving enhancer protein 2,and chloroplastic
ncbi_106430073		ATP合酶链1,类叶绿体; ATP synthase gamma chain 1,and chloroplastic-like
ncbi_106381576		ATP合酶链1,类叶绿体; ATP synthase gamma chain 1,and chloroplastic-like
ncbi_106405529		光系统I反应中心亚基VI,类叶绿体; Photosystem I reaction center subunit VI,and chloroplastic-like
ncbi_106401683		光系统II修复蛋白PSB27-H1,叶绿体; Photosystem II repair protein PSB27-H1,and chloroplastic
ncbi_106350097	上调 Up	铁氧还蛋白-1,叶绿体; Ferredoxin-1,and chloroplastic
ncbi_106371874	下调 Down	光系统I叶绿素a/b结合蛋白5,类叶绿体 Photosystem I chlorophyll a/b-binding protein 5,and chloroplastic-like
ncbi_106382137		叶绿素a-b结合蛋白2.4,类叶绿体; Chlorophyll a-b binding protein 2.4,and chloroplastic-like
ncbi_106439795		叶绿素a-b结合蛋白2.1,叶绿体; Chlorophyll a-b binding protein 2.1,and chloroplastic
ncbi_106349237		叶绿素a-b结合蛋白2.4,类叶绿体; Chlorophyll a-b binding protein 2.4,and chloroplastic-like
ncbi_106396209		叶绿素a-b结合蛋白1,叶绿体; Chlorophyll a-b binding protein 1,and chloroplastic
ncbi_106447496		叶绿素a-b结合蛋白1,叶绿体; Chlorophyll a-b binding protein 1,and chloroplastic
ncbi_106447497		叶绿素a-b结合蛋白1,叶绿体; Chlorophyll a-b binding protein 1,and chloroplastic-like
ncbi_106421061		光系统I叶绿素a/b结合蛋白3-1,叶绿体; Photosystem I chlorophyll a/b-binding protein 3-1,and chloroplastic
ncbi_106440450	上调 Up	叶绿素a-b结合蛋白CP26,类叶绿体; Chlorophyll a-b binding protein CP26,and chloroplastic-like

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