甘蓝型油菜<i>BnaFIL</i>基因启动子的克隆与表达分析

doi:10.7668/hbnxb.20192788

摘要/Abstract

摘要：

为了进一步了解启动子在甘蓝型油菜FIL基因(BnaFIL)表达调控中的作用,根据甘蓝型油菜基因组数据,以甘蓝型油菜叶片提取的DNA为模板,对甘蓝型油菜BnaFIL基因的启动子序列pBnaFIL进行克隆,长度为1 326 bp。采用PlantCARE在线分析软件对该启动子序列进行生物信息学序列分析,结果表明,该序列含有参与光反应的部分保守DNA模块以及CAAT-box和TATA-box等核心启动子必备元件,与分生组织表达有关的顺式作用的调控元件CAT-box以及光敏反应元件。通过该启动子序列替换pBI121植物表达载体上的CaMV35S启动子,使该启动子与GUS基因融合获得pBnaFIL-GUS表达载体,将载体通过农杆菌花序浸染的方法转入拟南芥中,获得了早花启动子重组质粒阳性转基因株系和晚花启动子重组质粒阳性转基因株系。之后对转基因拟南芥植株进行GUS染色分析,对启动子的表达效果进行了检测,最终在不同的转基因拟南芥植株中均发现了GUS基因的表达。结果表明,早花材料与晚花材料中启动子表达强弱存在差异,早花材料启动子的驱动基因表达效果比晚花材料启动子的驱动效果要好,由此推断,启动子的驱动效果对油菜开花早晚进行了调控,导致FIL基因在不同材料中的表达效果不一致。

关键词: 甘蓝型油菜, BnaFIL基因, 启动子, GUS染色, 表达载体

Abstract:

To further understand the role of the promoter in the regulation of the expression of the Brassica napus FIL gene(BnaFIL),it used the DNA extracted from the leaves of Brassica napus as a template based on the genomic data of Brassica napus.For cloning,the length was 1 326 bp.Using PlantCARE online analysis software to perform bioinformatics sequence analysis on the promoter sequence,the results showed that the sequence contained some conserved DNA modules involved in the photoreaction and essential elements for core promoters such as CAAT-box and TATA-box.Expression related cis-acting regulatory elements CAT-box and photosensitive response elements.Replace the CaMV35S promoter on the pBI121 plant vector with the promoter sequence,fused the promoter with the GUS gene to obtain the pBnaFIL-GUS expression vector,and transfer the vector into Arabidopsis thaliana by the method of Agrobacterium inflorescence.Flower promoter recombinant plasmid-positive transgenic lines and late flower promoter recombinant plasmid-positive transgenic lines.After that,GUS staining analysis was performed on the transgenic Arabidopsis plants,and the expression effect of the promoter was tested.Finally,the GUS gene expression was found in different transgenic Arabidopsis plants.The results showed that there were differences in promoter expression between early-flowering materials and late-flowering materials.The promoters of early-flowering materials could drive gene expression better than those of late-flowering materials.It was inferred from this that the driving effect of the promoter regulates the early and late flowering of rape,resulting in different expression effects of the FIL gene in different materials.

Key words: Brassica napus, BnaFIL gene, Promoter, GUS staining, Expression vector

陈静, 胡蓉, 刘勇, 秦艺, 熊兴华. 甘蓝型油菜BnaFIL基因启动子的克隆与表达分析[J]. 华北农学报, 2022, 37(3): 53-59. doi: 10.7668/hbnxb.20192788.

CHEN Jing, HU Rong, LIU Yong, QIN Yi, XIONG Xinghua. Cloning and Expression Analysis of BnaFIL Gene Promoter in Brassica napus L.[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(3): 53-59. doi: 10.7668/hbnxb.20192788.

http://www.hbnxb.net/CN/Y2022/V37/I3/53

图/表 10

表1 引物序列

Tab.1 The primer sequences

引物名称 Primer name	引物序列(5'-3') Primer sequence(5'-3')	退火温度/℃ Annealing temperature
BnaFIL-P-F	ACATATTTTCAGAGGTTCGTC	52
BnaFIL-P-R	GATCTTCTTATTTTGGGAGTG
ScaⅠ-P-F	AAAAGTACTACATATTTTCAGAGGTTCGTC	57
BamHⅠ-P-R	CGCGGATCCGATCTTCTTATTTTGGGAGTG
P-pBI121-F	CATCGTTTGTTTCGGACCACT	57
P-pBI121-R	CACTTCCTGATTATTGACCCA

图1 BnaFIL基因的启动子PCR扩增 1.晚花材料;2.早花材料;M.DL2000 Marker。图3同。

Fig.1 PCR amplification of the promoter of BnaFIL gene 1.Late flower material;2.Early flower material; M.DL2000 Marker.The same as Fig.3.

图2 BnaFIL基因的启动子顺式作用元件分析 1,2.SNP位点。

Fig.2 Analysis of cis-acting elements of the promoter of BnaFIL gene 1,2.SNP sites.

表2 启动子元件

Tab.2 Promoter elements

所含元件 Contains components	功能 Function
ATCT-motif、BOX4	参与光响应的部分保守DNA模块
CAAT-BOX	在启动子及增强子区域共有的顺式作用元件
CAT-BOX	顺式作用的调控元件与分生组织表达有关
TATA-BOX	核心启动子元件
TCA-element	参与水杨酸反应的顺式作用元件
TCCC-motif、TCT-motif	光敏元件的一部分

图3 启动子扩增

Fig.3 Promoter amplification

图4 植物表达载体构建过程

Fig.4 Flow chart for construction of the plant expression vector

图5 重组质粒双酶切鉴定 1.晚花材料重组质粒;2.早花材料重组质粒;M.Trans 5k DNA Marker。

Fig.5 Double enzyme digestion identification of recombinant plasmid 1.Recombinant plasmid of late flowering material;2.Recombinant plasmid of early flower material;M.Trans 5k DNA Marker.

图6 农杆菌菌落PCR检测 A.转早花启动子菌落PCR;B.转晚花启动子菌落PCR; M.DL2000 Marker。

Fig.6 PCR detection of Agrobacterium tumefaciens A.Transforming early flowering promoter colony PCR; B.Transforming late flowering promoter colony PCR;M.DL2000 Marker.

图7 转基因拟南芥阳性鉴定 1-3.早花重组质粒抗性苗;4-8.晚花重组质粒抗性苗; 9.野生型拟南芥。

Fig.7 Positive identification of transgenic Arabidopsis thaliana 1-3.Early-flowering recombinant plasmid resistant seedlings; 4-8.Late-flowering recombinant plasmid resistant seedlings; 9.Wild type Arabidopsis thaliana.

图8 GUS染色结果 A.野生型拟南芥;B.含pBI121载体拟南芥;C.含晚花启动子重组质粒的转基因拟南芥;D.含早花启动子重组质粒的转基因拟南芥。

Fig.8 GUS staining results A.Wild-type Arabidopsis;B.Vector Arabidopsis containing pBI121;C.Transgenic Arabidopsis containing late-flowering promoter recombinant plasmid;D.Transgenic Arabidopsis containing early-flowering promoter recombinant plasmid.

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甘蓝型油菜BnaFIL基因启动子的克隆与表达分析

Cloning and Expression Analysis of BnaFIL Gene Promoter in Brassica napus L.

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