植物学报

Primer name	Sequence (5′-3′)	Purpose
BnaA02.CPSF6-FP	GGAGCTTTAAGATGGATGAAGGAG	Gene cloning
BnaA02.CPSF6-RP	CCTAGTCATTATTCAGTTGTAAGCCG
35S-GFP-BnaA02.CPSF6-FP	GACAGCCCAGATCAACTAGTATGGATGAAGGAGATGGGAGAGATG	Subcellular localization
35S-GFP-BnaA02.CPSF6-RP	CTTGCTCACCATGGATCCTTCAGTTGTAAGCCGCCTTCGTTTC
BnaA02.CPSF6-qRT-FP	GGAACAGAAGTCGATCGTCCAGAG	qRT-PCR
BnaA02.CPSF6-qRT-RP	GAGAGCCTTCTTTCTGTTACAAGGC
BnaUBC9-FP	GCATCTGCCTCGACATCTTGA	Reference gene
BnaUBC9-RP	CGATAGCAGCACCTTGGAGATA
35S-BnaA02.CPSF6-NOS-FP	GGGACTCTTGACCATGGATGGATGAAGGAGATGGGAGAGATG	Vector construction
35S-BnaA02.CPSF6-NOS-RP	CAATTCACACGTGACGCGTTTATTCAGTTGTAAGCCGCCTTCG

Primer name	Sequence (5′-3′)	Purpose
BnaA02.CPSF6-FP	GGAGCTTTAAGATGGATGAAGGAG	Gene cloning
BnaA02.CPSF6-RP	CCTAGTCATTATTCAGTTGTAAGCCG
35S-GFP-BnaA02.CPSF6-FP	GACAGCCCAGATCAACTAGTATGGATGAAGGAGATGGGAGAGATG	Subcellular localization
35S-GFP-BnaA02.CPSF6-RP	CTTGCTCACCATGGATCCTTCAGTTGTAAGCCGCCTTCGTTTC
BnaA02.CPSF6-qRT-FP	GGAACAGAAGTCGATCGTCCAGAG	qRT-PCR
BnaA02.CPSF6-qRT-RP	GAGAGCCTTCTTTCTGTTACAAGGC
BnaUBC9-FP	GCATCTGCCTCGACATCTTGA	Reference gene
BnaUBC9-RP	CGATAGCAGCACCTTGGAGATA
35S-BnaA02.CPSF6-NOS-FP	GGGACTCTTGACCATGGATGGATGAAGGAGATGGGAGAGATG	Vector construction
35S-BnaA02.CPSF6-NOS-RP	CAATTCACACGTGACGCGTTTATTCAGTTGTAAGCCGCCTTCG

Element name	Sequence	Quantity	Function	Position (bp)
ATC-motif	AGTAATCT	1	Light responsive element	-160 (-)
GT1-motif	GGTTAA	6	Light responsive element	-368 (-); -705 (-); -889 (-); -1225 (-); -1803 (-)
Box 4	ATTAAT	9	Light responsive element	-407 (+/-); -566 (+/-); -928 (+/-); -1087 (+/-); -1292 (+/-); -1443 (+/-); -1689 (+/-); -1842 (+/-); -1992 (+/-)
I-box	AAGATAAGGCT	1	Light responsive element	-473 (-)
TCT-motif	TCTTAC	3	Light responsive element	-1274 (+); -1372 (+); -1770 (+)
G-box	CACGTT	3	Light responsive element	-687 (-); -1207 (-); -1603 (-)
MYB-like sequence	TAACCA	4	MYB-like binding site	-367 (+); -888 (+); -1404 (+); -1802 (+)
CCAAT-box	CAACGG	1	MYBHv1 binding site	-1591 (-)
ABRE	ACGTG	3	Abscisic acid responsiveness	-687 (+)

Element name	Sequence	Quantity	Function	Position (bp)
ATC-motif	AGTAATCT	1	Light responsive element	-160 (-)
GT1-motif	GGTTAA	6	Light responsive element	-368 (-); -705 (-); -889 (-); -1225 (-); -1803 (-)
Box 4	ATTAAT	9	Light responsive element	-407 (+/-); -566 (+/-); -928 (+/-); -1087 (+/-); -1292 (+/-); -1443 (+/-); -1689 (+/-); -1842 (+/-); -1992 (+/-)
I-box	AAGATAAGGCT	1	Light responsive element	-473 (-)
TCT-motif	TCTTAC	3	Light responsive element	-1274 (+); -1372 (+); -1770 (+)
G-box	CACGTT	3	Light responsive element	-687 (-); -1207 (-); -1603 (-)
MYB-like sequence	TAACCA	4	MYB-like binding site	-367 (+); -888 (+); -1404 (+); -1802 (+)
CCAAT-box	CAACGG	1	MYBHv1 binding site	-1591 (-)
ABRE	ACGTG	3	Abscisic acid responsiveness	-687 (+)

甘蓝型油菜BnaA02.CPSF6基因的克隆及功能分析

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李青洋 , 刘翠 , 何李 , 彭姗 , 马嘉吟 , 胡子祎 , 刘宏波 ^*

植物学报 | 研究报告 2025,60(1): 62-73

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植物学报 |研究报告 2025 , 60 (1) : 62 -73

甘蓝型油菜BnaA02.CPSF6基因的克隆及功能分析

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李青洋, 刘翠, 何李, 彭姗, 马嘉吟, 胡子祎, 刘宏波^*

作者信息

浙江农林大学现代农学院, 杭州 311300

通讯作者:

* E-mail: hbliu@zafu.edu.cn

Cloning and Functional Analysis of the BnaA02.CPSF6 Gene from Brassica napus

Qingyang Li, Cui Liu, Li He, Shan Peng, Jiayin Ma, Ziyi Hu, Hongbo Liu^*

Affiliations

College of Advanced Agricultural Sciences, Zhejiang A&F University, Hangzhou 311300, China

Corresponding Author:

* E-mail: hbliu@zafu.edu.cn

出版时间: 2025-01-10 doi: 10.11983/CBB24068

文章导航

摘要

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CPSF家族蛋白是植物体内mRNA前体中多聚腺苷酸化信号识别、剪切和添加poly(A)的重要因子, 对开花时间调控、环境响应和种子发育等具有重要作用。目前, 甘蓝型油菜(Brassica napus) CPSF家族基因的功能尚不明确。为探究甘蓝型油菜CPSF家族基因的功能和表达模式, 从甘蓝型油菜品种中双11号中克隆得到BnaA02.CPSF6, 并对其进行生物信息学、编码蛋白质亚细胞定位、表达模式和基因功能分析。结果表明, BnaA02.CPSF6基因编码区全长1 938 bp, 编码646个氨基酸残基, 无内含子结构, 其在甘蓝型油菜中有6个同源基因; BnaA02.CPSF6启动子区存在多个参与光反应的顺式作用元件和MYB结合位点; BnaA02.CPSF6在根、茎、叶、花和不同发育时期种子中均有表达, 特别是在发育15-35天的种子中显著高表达, 其编码的蛋白定位于细胞核; BnaA02.CPSF6受盐和干旱胁迫诱导上调表达; 在ABA、IAA、GA₃、SA和MeJA激素处理下, BnaA02.CPSF6基因表达先受到抑制再逐渐恢复至正常水平; 在正常条件下, 在拟南芥(Arabidopsis thaliana)中过表达BnaA02.CPSF6会出现提前抽薹开花的表型, 且莲座叶数量显著减少。综上所述, BnaA02.CPSF6参与非生物胁迫响应并受植物激素调控, 可能在开花调控中起促进作用。

关键词

甘蓝型油菜 / BnaA02.CPSF6 / 基因功能 / 表达模式 / 生长发育

Abstract

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INTRODUCTION: The CPSF family (cleavage and polyadenylation specificity factor) is a crucial protein family that is responsible for polyadenylation signal recognition in mRNA precursors, cleavage and the addition of poly(A) tails to mRNAs in plants. This family plays crucial roles in the regulation of flowering time, the environmental response, and seed development. Currently, the function of the CPSF family genes in Brassica napus is unclear.
RATIONALE: To explore the function and expression patterns of the CPSF gene family, this study cloned BnaA02.CPSF6 from B. napus variety Zhongshuang No.11 and conducted bioinformatics analysis, subcellular localization, expression pattern, and functional characterization of the gene.
RESULTS: These results indicate that the coding region of the BnaA02.CPSF6 gene is 1 938 bp in length and encodes 646 amino acids without intron structures. Its promoter region contains multiple cis-acting elements involved in light responses and MYB binding sites. Additionally, there are six genes homologous to BnaA02.CPSF6 in B. napus. The BnaA02.CPSF6 gene expressed in the roots, stems, leaves, flowers and different developmental seeds of B. napus, especially significantly higher in 15-35 d developmental seeds, and its encoded protein was localized in the nucleus. The BnaA02.CPSF6 gene expression is upregulated under salt and drought stress. Under treatment with hormones such as ABA, IAA, GA₃, SA, and MeJA, the expression of BnaA02.CPSF6 gene is initially inhibited and then gradually recovers to normal levels. Under normal conditions, the overexpression of the BnaA02.CPSF6 gene in Arabidopsis thaliana results in an early bolting phenotype, along with a reduced number of rosette leaves.
CONCLUSION: In summary, the above results indicate that the BnaA02.CPSF6 is involved in abiotic stress responses, is regulated by phytohormones, and may also play a promoting role in flowering regulation.

Key words

Brassica napus / BnaA02.CPSF6 / gene function / expression patterns / growth and development

引用本文

李青洋, 刘翠, 何李, 彭姗, 马嘉吟, 胡子祎, 刘宏波. 甘蓝型油菜BnaA02.CPSF6基因的克隆及功能分析. 植物学报, 2025 , 60 (1) : 62 -73 . DOI: 10.11983/CBB24068

Qingyang Li, Cui Liu, Li He, Shan Peng, Jiayin Ma, Ziyi Hu, Hongbo Liu. Cloning and Functional Analysis of the BnaA02.CPSF6 Gene from Brassica napus[J]. Chinese Bulletin of Botany, 2025 , 60 (1) : 62 -73 . DOI: 10.11983/CBB24068

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在真核生物中, mRNA的3'末端加工对于基因表达调控至关重要, 这一过程包括前体mRNA的剪切和多聚腺苷酸化(cleavage and polyadenylation), 主要由CPSF (cleavage and polyadenylation specificity factor)家族蛋白和CFI_m(Cleavage Factor I_m)复合体等共同完成(Hardy and Norbury, 2016; Kumar et al., 2019; Boreikaitė and Passmore, 2023)。CFI_m复合体是细胞内mRNA前体3'端加工的重要因子之一, 主要参与多聚腺苷酸化信号识别和切割反应, 其在基因表达调控中发挥关键作用(Brown and Gilmartin, 2003; Venkataraman et al., 2005)。CFI_m由多个亚基组成, 包括CFI_m25、CFI_m59和CFI_m68 (Yang et al., 2011)。研究发现, 拟南芥(Arabidopsis thaliana) AtCFI_m25a基因功能缺失导致莲座叶和花药变小, 以及育性降低等表型(Zhang et al., 2022)。多聚腺苷酸化特异因子CPSF家族作为mRNA 3′末端加工的核心复合物之一, 决定着mRNA的成熟、运输、稳定性及翻译效率(Edwalds-Gilbert and Milcarek, 1995)。

植物CPSF家族在调控开花和响应外界环境变化等方面发挥重要作用。目前已报道的CPSF家族成员有12个, 分别为CPSF_A、CPSF1、CPSF2、CPSF3、CPSF4、CPSF5、CPSF6、CPSF7、CPSF30、CPSF73、CPSF100和CPSF160。它们协同识别mRNA的3′末端多聚腺苷酸化信号序列(polyadenylation signal sequence), 影响mRNA的3′末端加工, 产生多种不同的mRNA 3′末端形式, 从而决定mRNA的成熟、运输、稳定性及翻译效率(Schul et al., 1996; Proudfoot, 2004; Mandel et al., 2008; Thomas et al., 2012)。植物CPSF蛋白主要通过可变多聚腺苷酸化(alternative polyadenylation, APA)过程来调控开花时间(Henderson et al., 2005; Feng et al., 2011; 蔡芳芳等, 2022)、表观遗传(Ma et al., 2014)以及氮吸收和同化(Hou et al., 2021)。研究表明, CPSF100参与mRNA的正常加工, 从而影响植物的开花时间以及生殖器官发育; CPSF100功能缺失导致开花时间延迟, 胚胎发育异常(Tzafrir et al., 2004; Herr et al., 2006; Lin et al., 2017)。CPSF30-L蛋白在拟南芥中不仅具有识别mRNA上N6-甲基腺嘌呤(m6A)的功能, 还参与mRNA的3'末端加工; CPSF30-L能通过影响mRNA的poly(A)位点选择调控氮信号相关基因的表达, 从而影响植物对氮的吸收和同化(Hou et al., 2021), 且cpsf30-l突变体呈现出开花延迟以及ABA响应敏感的表型(Song et al., 2021)。同时, Hao等(2023)发现抗疟疾药物苯并硼唑化合物AN3661与CPSF73蛋白结合能够抑制其功能, 从而影响拟南芥的主根、叶片以及根毛的生长(Hao et al., 2023)。此外, 植物FCA (Flowering Control Locus A)蛋白和FPA (Flowering Locus PA)蛋白也利用APA途径负调控开花抑制基因FLC (Flowering Locus C)的转录(Eckardt, 2002; Mouradov et al., 2002; Quesada et al., 2003; Hornyik et al., 2010; 张长生等, 2021)。开花关键基因FCA有2个APA位点, 若近端的APA位点被使用, 转录本编码的蛋白就会失去活性, 导致开花时间发生变化(Macknight et al., 2002; Quesada et al., 2003)。

甘蓝型油菜(Brassica napus)是一种全球范围内广泛栽培的油料作物, 既是食用植物油的主要来源, 也是重要的饲用蛋白来源(黄郢等, 2024; 李谷成等, 2024)。近年来, 为了保障我国油料作物供给, 已明确提出利用冬闲田扩大甘蓝型油菜的种植面积。然而, 受限于长江流域甘蓝型油菜主产区的作物生产茬口矛盾, 早熟甘蓝型油菜新品种选育已逐渐受到育种家的重视。拟南芥CPSF家族基因多次被报道与开花时间调控相关(Herr et al., 2006; Yu et al., 2019; Song et al., 2021), 而目前甘蓝型油菜CPSF家族调控开花的功能研究还未见报道。本研究克隆了甘蓝型油菜BnaA02.CPSF6基因, 分析了其表达模式和生物学功能, 旨在为甘蓝型油菜早熟育种奠定理论基础。

1 材料与方法

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1.1 实验材料

本研究所用材料为甘蓝型油菜品种中双11号(Brassica napus L. cv. ‘Zhongshuang No.11’)和本氏烟草(Nicotiana benthamiana Domin.), 均由本实验室保存。挑选饱满无破皮的中双11号种子和本氏烟草种子, 春化后置于生长室中培养。培养条件: 光照强度为100 μmol·m^-2·s^-1, 光周期为16小时光照/8小时黑暗, 温度23°C, 相对湿度60%。所用载体为本实验室保存的载体pCAMBIA1305.1-35S-NOS和pCAMBIA1305.1- 35S-sGFP (经本实验室改造, 融合GFP报告基因序列的pCAMBIA1305.1载体)。引物合成及基因测序均由杭州擎科生物技术公司完成。

1.2 实验方法

1.2.1 BnaA02.CPSF6基因的克隆与生物信息学分析

从BnTIR数据库中检索甘蓝型油菜BnaA02.CPSF6基因的cDNA序列(No.BnaA02T0126500ZS), 利用SnapGene软件设计引物, 扩增BnaA02.CPSF6的CDS全长。PCR反应体系: 8 µL 5× buffer, 3.2 µL 10 mmol∙L^-1 dNTPs, 0.4 µL 5 U∙µL^-1 PrimeSTAR HS DNA聚合酶(Takara, Cat No.R045Q), 10 μmol∙L^-1正反向引物各1 µL, 2 µL 50 ng∙µL^-1 cDNA模板, 用ddH₂O补足至40 µL。PCR反应程序: 98°C预变性3分钟; 98°C变性10秒, 55°C退火15秒, 72°C延伸2分钟, 35个循环; 72°C终延伸10分钟。4°C保存。引物序列见表1。

通过BnTIR和TAIR数据库下载BnaA02.CPSF6基因在不同物种中的同源氨基酸序列, 采用MEGA-X软件中的Neighbor-joining方法构建系统进化树, 检验方式为Bootstrap method并重复1 000次。利用PlantCare网站对BnaA02.CPSF6基因上游2 000 bp的启动子区域进行调控元件预测。

1.2.2 BnaA02.CPSF6亚细胞定位

采用同源重组法构建pCAMBIA1305.1-35S-BnaA02. CPSF6-sGFP融合表达载体, 将扩增到的BnaA02. CPSF6基因片段和线性化载体进行同源重组。利用根癌农杆菌GV3101将阴性对照空载体和融合表达载体注射到本氏烟草叶片中, 置于生长室培养48小时。通过激光共聚焦扫描显微镜观察荧光信号。

1.2.3 BnaA02.CPSF6组织特异性表达分析

取幼苗时期的根、茎、叶和花, 授粉后第15、20、25、30、35、40、45和50天的发育种子, 在液氮中磨样,使用AG RNAex Pro RNA提取试剂(艾科瑞生物,Cat No.AG21101)、SteadyPure RNA提取试剂盒(不含裂解液) (艾科瑞生物, Cat No.AG21024)和Hifair^® III 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus)试剂盒(YEASEN, Cat No.11141ES60)进行油菜不同组织RNA的提取及反转录。以甘蓝型油菜SUMO E2结合酶编码基因(BnaUBC9)作为内参基因, 通过qRT-PCR分析BnaA02. CPSF6基因的表达水平。

1.2.4 外源激素及非生物胁迫下BnaA02.CPSF6基因的表达模式分析

室温条件下甘蓝型油菜中双11号种子在水中浸泡12小时, 以去除壳层和促进发芽, 再将其移至铺有湿润滤纸的培养皿上。待2片子叶完全展开时, 将幼苗移至Hoagland水培营养液中, 每隔7天更换1次营养液。待幼苗长至2片真叶时, 分别换用200 mmol∙L^-1 NaCl、15% PEG6000、0 μmol∙L^-1 P水培液作为盐、干旱和低磷胁迫处理。外源激素处理采用喷施法, 包括生长素(100 µmol∙L^-1 IAA)、脱落酸(50 µmol∙L^-1 ABA)、茉莉酸甲酯(100 µmol∙L^-1 MeJA)、水杨酸(100 µmol∙L^-1 SA)和赤霉素(20 mg∙L^-1 GA₃)。在处理后12和24小时取样, 每次进行混合取样(至少取5-8株混样)并将样品置于-80°C冰箱保存。参照1.2.3节所述方法进行RNA提取及反转录, 利用qRT-PCR检测BnaA02.CPSF6基因的表达量。

1.2.5 构建BnaA02.CPSF6过表达纯合拟南芥株系

将构建好的pCAMBIA1305.1-35S-BnaA02.CPSF6- NOS重组载体转化根癌农杆菌GV3101。当野生型拟南芥(Col-0)生长约6周后, 待花序长出大量花蕾时, 采用花序浸染法转化野生型拟南芥。利用潮霉素和分子鉴定辅助筛选符合孟德尔遗传分离规律的单拷贝纯合株系, 获得8个BnaA02.CPSF6过表达纯合拟南芥株系, 选取基因表达量高且种子多的株系进行后续实验。

1.2.6 BnaA02.CPSF6转基因拟南芥表型观察

将适量的野生型拟南芥(Col-0)和BnaA02.CPSF6过表达纯合拟南芥种子分装在1.5 mL离心管中并用锡纸包裹, 置于4°C冰箱中3天。结束后, 将拟南芥种子均匀点播在培养基质中(营养土:珍珠岩:蛭石=3:1:1, v/v/v), 在同一钵中播种4株拟南芥, 分别位于钵中四角, 并用透明塑料盖暗光培养7天, 待其发芽后进行间苗和表型观察。培养条件: 光照强度为100 μmol·m^-2·s^-1, 光周期为16小时光照/8小时黑暗, 温度23°C。每隔3天浇水/施肥1次, 浇水与施肥交替进行, 采取由下至上的渗水灌溉方式。

2 结果与讨论

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2.1 BnaA02.CPSF6基因的克隆与生物信息学分析

根据BnTIR数据库中下载的BnaA02.CPSF6基因序列设计引物, 以中双11号甘蓝型油菜cDNA为模板, 通过PCR扩增得到全长为1 938 bp的CDS序列, 无内含子结构, 编码646个氨基酸残基, 分子量为69.67 kDa。使用DNAMAN软件对BnTIR数据库中下载的BnaA02.CPSF6同源基因序列进行比对分析(图1), 发现BnaCPSF6s在甘蓝型油菜品种中双11号中有6个同源基因, 本研究基因为BnaA02.CPSF6, 与其余5个基因BnaC02.CPSF6、BnaA10.CPSF6、BnaC09. CPSF6、BnaA03.CPSF6和BnaC03.CPSF6编码蛋白的氨基酸序列相似度分别为93%、74.46%、74.5%、70.48%和69.13%, 与拟南芥中同源基因AT5G55670的氨基酸序列相似度为74.75%。同时, 从BnTIR和TAIR网站下载BnaCPSF6s蛋白在不同物种中的同源序列, 使用MEGA-X软件以Neighbor-joining法构建系统进化树。结果显示, 与BnaA02.CPSF6基因亲缘关系由近至远的物种依次为白菜型油菜(B. rapa)、芥菜型油菜(B. juncea)、甘蓝(B. oleracea)、拟南芥和亚麻芥(Camelina sativa) (图2)。

利用PlantCare网站进行BnaA02.CPSF6基因启动子的调控元件预测, 在ATG起始密码子上游的2 000 bp内, 存在多个参与光响应的顺式作用元件、ABA响应元件和MYB转录因子家族结合位点(表2)。BnaA02.CPSF6基因启动子区域含有ABRE、MYB、ATC-motif、GT1-motif、Box 4、I-box、TCT-motif、MYC、CCAAT-box和G-box调控元件, 可能与调控植物生长发育及非生物胁迫响应密切相关。

2.2 BnaA02.CPSF6的亚细胞定位

将BnaA02.CPSF6基因与携带绿色荧光蛋白的pCAMBIA1305.1载体进行融合表达, 同时导入构建好的核定位标记融合蛋白OsD53-mCherry至菌株GV3101 (Liu et al., 2021), 随后利用农杆菌介导的瞬时表达系统, 将分别含有重组质粒pCAMBIA1305.1-sGFP- BnaA02.CPSF6和OsD53-mCherry的农杆菌株与番茄丛矮病毒p19蛋白菌株共同注射到烟草叶片下表皮中, 番茄丛矮病毒p19蛋白可有效抑制转录后基因沉默, 从而增强瞬时表达系统的活性。经过48小时后, 通过激光共聚焦显微镜观察到注射区域周边的烟草叶片有明显的绿色荧光信号, 且这些信号集中在细胞核区域(图3), 表明BnaA02.CPSF6蛋白定位于细胞核。

2.3 BnaA02.CPSF6的组织特异性表达

将甘蓝型油菜幼苗期的根、茎、叶、花和不同发育时期的种子(授粉后第15、20、25、30、35、40、45和50天)样品进行RNA提取并反转录, 以cDNA为模板进行荧光定量PCR, 检测不同组织中BnaA02. PSF6的表达量(图4)。以叶片为对照, 发现BnaA02. PSF6在授粉后第15、20、25、30和35天的种子中高表达, 其中授粉后第15天的种子中BnaA02.CPSF6的表达量最高, 相较于叶片提高了2.24倍。

2.4 非生物胁迫和外源激素处理下BnaA02.CPSF6基因的表达模式

研究表明, BnaA02.CPSF6基因启动子区域存在多个与MYB结合的位点及响应脱落酸的顺式作用元件(表2)。为进一步明确BnaA02.CPSF6基因对非生物胁迫与外源激素的响应情况, 对生长至第2片真叶的甘蓝型油菜中双11号进行盐、干旱、低磷、ABA、IAA、GA₃、SA和MeJA处理, 以未处理的野生型作为对照。结果表明, 在盐胁迫条件下, BnaA02.CPSF6表达量随处理时间的延长逐渐升高, 处理24小时后, 表达量提升至对照组的1.64倍; 在干旱胁迫条件下, 处理24小时后, BnaA02.CPSF6表达量达到最高, 为对照组的1.28倍; 在ABA、IAA、GA₃、SA和MeJA处理条件下, BnaA02.CPSF6表达量随处理时间的延长先降低后升高, 处理12小时后, BnaA02.CPSF6表达量分别降低至对照组的0.78、0.56、0.73、0.65和0.63倍; 在低磷胁迫条件下, BnaA02.CPSF6表达量的变化不显著(图5A-H)。

2.5 BnaA02.CPSF6过表达调控拟南芥抽薹及开花时间

为阐明BnaA02.CPSF6基因对拟南芥抽薹及开花时间的影响, 我们构建了植物过表达载体pCAMBIA1305.1-35S-BnaA02.CPSF6-NOS, 并采用含15 μg∙mL^-1潮霉素的1/2MS固体培养基进行筛选, 转基因株系均可生长。同时, 通过PCR鉴定以及潮霉素抗性筛选符合3:1分离比的阳性株系, 共获得8个T₃代纯合单拷贝插入的BnaA02.CPSF6过表达拟南芥株系。在正常生长条件下, 从拟南芥种子春化播种后开始计算天数, 记录Col-0野生型及BnaA02.CPSF6过表达拟南芥株系的抽薹开花时间及对应莲座叶数量, 发现Col-0野生型抽薹时间为33天, 莲座叶数量为12.6片, 而BnaA02. CPSF6过表达拟南芥株系#8、#51和#64抽薹时间分别为24.6、25.0和22.9天, 莲座叶数量分别为8.7、8.3和8.0片, 相较于Col-0野生型抽薹时间分别提前8.4、8.0和10.1天, 莲座叶数量分别减少了3.9、4.3和4.6片(图6A-D)。上述结果表明, BnaA02.CPSF6可能是营养生长向生殖生长转变的促进因子, 显著缩短了拟南芥抽薹开花时间。

2.6 讨论

本研究从甘蓝型油菜中克隆了BnaA02.CPSF6基因, 利用烟草瞬时表达系统, 通过激光共聚焦显微镜观察, 结果显示BnaA02.CPSF6蛋白定位于细胞核, 表明其可能参与相关基因的转录调控。BnaA02.CPSF6基因组成型表达, 且在发育前期种子中高表达, 表明BnaA02.PSF6基因可能参与种子发育。PlantCare预测分析表明, 该启动子具有多个与光响应相关的元件(Box 4和I-box等)。I-box主要存在于重要蛋白基因的启动子中, 参与光合作用和光形态建成等过程(崔国新等, 2010)。BnaA02.CPSF6启动子区域还存在MYB结合位点, 通过与MYB转录因子结合响应环境胁迫和调控生长发育过程。

qRT-PCR结果表明, 在盐胁迫条件下, BnaA02. CPSF6表达量随处理时间的延长逐渐升高; 在干旱、IAA、GA₃、SA和MeJA处理条件下, BnaA02.CPSF6的表达水平随处理时间的延长先受抑制后上调。盐胁迫作为本研究中最快上调BnaA02.CPSF6表达的处理, 通过分析启动子区域, 发现其中存在多个与MYB转录因子家族结合的位点, 而MYB转录因子家族在植物对盐胁迫响应中发挥重要作用(Wang et al., 2021)。推测甘蓝型油菜在受到盐胁迫时, MYB转录因子与该位点结合, 并上调下游BnaA02.CPSF6基因的表达。植物激素是植物开花过程中的重要调控因子。例如, 赤霉素促进植物开花, 通过调节DELLA蛋白的活性促进花的形成(李巧峡等, 2019; Bao et al., 2020); 而生长素通常在植物顶端分生组织中发挥作用, 促进侧生器官的起始(Dai et al., 2023)。后续将深入研究BnaA02.CPSF6基因是否能感知植物体内激素水平的变化, 从而调控开花时间。

真核生物的mRNA的3′末端普遍会发生可变多聚腺苷酸化, 这一过程主要通过核酸内切酶切割mRNA, 随后由多聚腺苷酸化聚合酶加poly(A)尾完成, 可变剪接对基因的表达调控具有重要意义。本研究构建了BnaA02.CPSF6过表达纯合拟南芥株系, 初步分析了BnaA02.CPSF6基因在拟南芥开花调控中的作用, 通过在拟南芥中过表达BnaA02.CPSF6基因, 使其开花提前, 莲座叶数目减少, 说明BnaA02.CPSF6基因在拟南芥中正调控开花, 这与前人对同一家族CPSF30-L的研究结果相似(Song et al., 2021), 即CPSF30-L基因功能缺失会导致开花延迟。后续将从AraShare网站购买atcpsf突变体, 比较其与Col-0野生型和Bna02.CPSF6过表达纯合拟南芥的抽薹及开花时间, 并深入探究BnaA02.CPSF6是否通过调节前体mRNA影响开花调控模块基因FCA/FPA/FLC的功能和表达, 并与CFI_m复合体共同调控植物开花时间, 为后续甘蓝型油菜CPSF家族基因功能研究以及甘蓝型油菜早熟新品种选育奠定理论基础。

作者贡献声明

收起

李青洋: 实验设计, 完成实验和数据分析, 论文初稿写作; 刘翠: 参与载体构建和拟南芥遗传转化材料筛选及鉴定, 提供论文写作意见; 何李和彭姗: 参与表达模式、非生物胁迫和激素处理实验及结果分析; 马嘉吟和胡子祎: 参与拟南芥培养和表型分析; 刘宏波: 研究整体构思及总负责人, 指导实验设计、数据分析、论文写作与修改。全体作者已阅读并同意论文最终版本。

基金

收起

国家自然科学基金(32071929)
浙江省自然科学基金(LY21C130001)
浙江省“十四五”农业新品种选育重大科技专项子课题(2021C02064-2-1)
浙江农林大学科研发展基金(2021FR044)

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2025年第60卷第1期

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文章信息

doi: 10.11983/CBB24068

接收时间：2024-05-05
首发时间：2026-10-27
出版时间：2025-01-10

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收稿日期：2024-05-05
录用日期：2024-07-23

基金

国家自然科学基金(32071929)

浙江省自然科学基金(LY21C130001)

浙江省“十四五”农业新品种选育重大科技专项子课题(2021C02064-2-1)

浙江农林大学科研发展基金(2021FR044)

作者信息

浙江农林大学现代农学院, 杭州 311300

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* E-mail: hbliu@zafu.edu.cn

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Primer name	Sequence (5′-3′)	Purpose
BnaA02.CPSF6-FP	GGAGCTTTAAGATGGATGAAGGAG	Gene cloning
BnaA02.CPSF6-RP	CCTAGTCATTATTCAGTTGTAAGCCG
35S-GFP-BnaA02.CPSF6-FP	GACAGCCCAGATCAACTAGTATGGATGAAGGAGATGGGAGAGATG	Subcellular localization
35S-GFP-BnaA02.CPSF6-RP	CTTGCTCACCATGGATCCTTCAGTTGTAAGCCGCCTTCGTTTC
BnaA02.CPSF6-qRT-FP	GGAACAGAAGTCGATCGTCCAGAG	qRT-PCR
BnaA02.CPSF6-qRT-RP	GAGAGCCTTCTTTCTGTTACAAGGC
BnaUBC9-FP	GCATCTGCCTCGACATCTTGA	Reference gene
BnaUBC9-RP	CGATAGCAGCACCTTGGAGATA
35S-BnaA02.CPSF6-NOS-FP	GGGACTCTTGACCATGGATGGATGAAGGAGATGGGAGAGATG	Vector construction
35S-BnaA02.CPSF6-NOS-RP	CAATTCACACGTGACGCGTTTATTCAGTTGTAAGCCGCCTTCG

Primer name

Sequence (5′-3′)

Purpose

BnaA02.CPSF6-FP

GGAGCTTTAAGATGGATGAAGGAG

Gene cloning

BnaA02.CPSF6-RP

CCTAGTCATTATTCAGTTGTAAGCCG

35S-GFP-BnaA02.CPSF6-FP

GACAGCCCAGATCAACTAGTATGGATGAAGGAGATGGGAGAGATG

Subcellular localization

35S-GFP-BnaA02.CPSF6-RP

CTTGCTCACCATGGATCCTTCAGTTGTAAGCCGCCTTCGTTTC

BnaA02.CPSF6-qRT-FP

GGAACAGAAGTCGATCGTCCAGAG

qRT-PCR

BnaA02.CPSF6-qRT-RP

GAGAGCCTTCTTTCTGTTACAAGGC

BnaUBC9-FP

GCATCTGCCTCGACATCTTGA

Reference gene

BnaUBC9-RP

CGATAGCAGCACCTTGGAGATA

35S-BnaA02.CPSF6-NOS-FP

GGGACTCTTGACCATGGATGGATGAAGGAGATGGGAGAGATG

Vector construction

35S-BnaA02.CPSF6-NOS-RP

CAATTCACACGTGACGCGTTTATTCAGTTGTAAGCCGCCTTCG

Element name	Sequence	Quantity	Function	Position (bp)
ATC-motif	AGTAATCT	1	Light responsive element	-160 (-)
GT1-motif	GGTTAA	6	Light responsive element	-368 (-); -705 (-); -889 (-); -1225 (-); -1803 (-)
Box 4	ATTAAT	9	Light responsive element	-407 (+/-); -566 (+/-); -928 (+/-); -1087 (+/-); -1292 (+/-); -1443 (+/-); -1689 (+/-); -1842 (+/-); -1992 (+/-)
I-box	AAGATAAGGCT	1	Light responsive element	-473 (-)
TCT-motif	TCTTAC	3	Light responsive element	-1274 (+); -1372 (+); -1770 (+)
G-box	CACGTT	3	Light responsive element	-687 (-); -1207 (-); -1603 (-)
MYB-like sequence	TAACCA	4	MYB-like binding site	-367 (+); -888 (+); -1404 (+); -1802 (+)
CCAAT-box	CAACGG	1	MYBHv1 binding site	-1591 (-)
ABRE	ACGTG	3	Abscisic acid responsiveness	-687 (+)

Element name

Sequence

Quantity

Function

Position (bp)

ATC-motif

AGTAATCT

Light responsive element

-160 (-)

GT1-motif

GGTTAA

Light responsive element

-368 (-); -705 (-); -889 (-); -1225 (-); -1803 (-)

Box 4

ATTAAT

Light responsive element

-407 (+/-); -566 (+/-); -928 (+/-); -1087 (+/-);
-1292 (+/-); -1443 (+/-); -1689 (+/-); -1842 (+/-); -1992 (+/-)

I-box

AAGATAAGGCT

Light responsive element

-473 (-)

TCT-motif

TCTTAC

Light responsive element

-1274 (+); -1372 (+); -1770 (+)

G-box

CACGTT

Light responsive element

-687 (-); -1207 (-); -1603 (-)

MYB-like sequence

TAACCA

MYB-like binding site

-367 (+); -888 (+); -1404 (+); -1802 (+)

CCAAT-box

CAACGG

MYBHv1 binding site

-1591 (-)

ABRE

ACGTG

Abscisic acid responsiveness

-687 (+)