白菜型油菜开花调控基因BrFT的生物信息学特性和表达分析

doi:10.3969/j.issn.1004-1524.2023.05.03

浙江农业学报 ›› 2023, Vol. 35 ›› Issue (5): 992-1000.DOI: 10.3969/j.issn.1004-1524.2023.05.03

白菜型油菜开花调控基因BrFT的生物信息学特性和表达分析

姚彦林¹^,²(), 马骊², 刘丽君², 蒲媛媛¹^,², 李学才¹^,², 王旺田³, 方彦¹^,², 孙万仓¹^,², 武军艳¹^,²^,^*()

1.甘肃农业大学农学院/甘肃省油菜工程技术研究中心,甘肃兰州730070
2.省部共建干旱生境作物学国家重点实验室,甘肃兰州730070
3.甘肃农业大学生命科学技术学院,甘肃兰州 730070

收稿日期:2022-03-10 出版日期:2023-05-25 发布日期:2023-06-01
作者简介:姚彦林(1989—),女,甘肃会宁人,硕士研究生,研究方向为作物遗传育种。E-mail: 1083737026@qq.com
通讯作者: *武军艳,E-mail: 94790094@qq.com
基金资助:
国家现代农业产业技术体系(CARS-12);省部共建干旱生境作物学国家重点实验室(甘肃农业大学)主任基金课题(GSCS-2020-Z1)

Bioinformatics and expression analysis of flowering regulation gene BrFT in Brassica rapa L.

YAO Yanlin¹^,²(), MA Li², LIU Lijun², PU Yuanyuan¹^,², LI Xuecai¹^,², WANG Wangtian³, FANG Yan¹^,², SUN Wancang¹^,², WU Junyan¹^,²^,^*()

1. Rapeseed Engineering Research Center of Gansu Province/College of Agronomy, Gansu Agricultural University, Lanzhou 730070, China
2. Gansu Provincial Key Laboratory of Aridland Crop Science, Lanzhou 730070, China
3. College of Life Science and Technology, Gansu Agricultural University, Lanzhou 730070, China

Received:2022-03-10 Online:2023-05-25 Published:2023-06-01

摘要/Abstract

摘要：

FT基因是调控植物开花的重要基因,为探究FT基因在白菜型油菜光周期途径中调控开花的作用,本研究以拟南芥FT蛋白序列为索引从白菜型油菜基因组中检索到3个BrFT基因,利用生物信息学方法对基因序列特征进行分析,通过qRT-PCR技术研究基因的器官特异性表达,筛选基因在不同白菜型油菜中克隆并检测其在4 ℃,16 h光照/8 h黑暗条件下的表达模式。结果表明,3个BrFT均由4个外显子和3个内含子组成,分布于A02和A07染色体上,其中BrFT1和BrFT2存在明显的共线关系,3个BrFT蛋白都包含PEBP保守基序。基因上游2 000 bp启动子区域均含有大量的光应答元件,一些参与厌氧诱导、防御和应激反应的调控元件,另外BrFT1和BrFT2含有参与植物生长发育、激素诱导的元件。qRT-PCR检测发现,BrFT在白菜型油菜根、茎、叶、花蕾和花中均有表达,在叶中表达量高于其他器官,且BrFT1表达量高于其余两个成员,克隆到陇油6号、陇油17号、天油2号中的BrFT1基因,序列比对相似性为99.5%。在4 ℃,16 h光照/8 h黑暗条件下不同白菜型油菜中BrFT1随处理时间均呈现上调表达,但品种间存在差异,结合对不同白菜型油菜开花的观察,表明BrFT1基因的过量表达能够促进白菜型油菜提早开花。

关键词: 白菜型油菜, FT基因, 生物信学分析, 基因克隆, 表达分析

Abstract:

FT gene is an important gene associated with flowering in plants. In order to explore the role of FT gene in regulating flowering in photoperiod pathway of Brassica rapa L. In this study, 3 BrFT genes were identified from the winter rapeseed genome using Arabidopsis FT genes as queries,bioinformatics methods were used to analyze the gene sequence characteristics, and the organ specific expression of the gene was studied by qRT-PCR. Screening genes were cloned from different Brassica rapa L., and the expression patterns of genes under 4 ℃, 16 h light/8 h darkness conditions were detected. The results showed that 3 of which contained 4 exons and 3 introns, and distributed on chromosomes A02 and A07, among which BrFT1 and BrFT2 had obvious collinear relationship, and all the three BrFT proteins contained PEBP conserved motif. The 2 000 bp upstream promoter region of the gene all contained light response elements,some regulatory elements involved in anaerobic induction, defense and stress response,and BrFT1 and BrFT2 contain action elements involved in plant growth and development and hormone induction. qRT-PCR detection showed that BrFT could be expressed in roots, stems, leaves, flower buds and flowers,and the expression level in leaves was higher than that in other organs, the expression level of BrFT1 was higher than that of the other two members. So the BrFT1 gene in Longyou 6, Longyou 17, Tianyou 2 were cloned and the sequence alignment similarity was 99.5%. BrFT1 in Brassica rapa L. showed an up-regulation trend at 4 ℃, 16 h light/8 h darkness, but there were differences among varieties. Combined with the observation of flowering in different types of Brassica rapa L. with different temperature, it showed that excessive expression of BrFT1 gene could promote the early flowering of Brassica rapa L.

Key words: Brassica rapa L., FT gene, bioinformatics analysis, gene clone, expression analysis

中图分类号:

S565.4

姚彦林, 马骊, 刘丽君, 蒲媛媛, 李学才, 王旺田, 方彦, 孙万仓, 武军艳. 白菜型油菜开花调控基因BrFT的生物信息学特性和表达分析[J]. 浙江农业学报, 2023, 35(5): 992-1000.

YAO Yanlin, MA Li, LIU Lijun, PU Yuanyuan, LI Xuecai, WANG Wangtian, FANG Yan, SUN Wancang, WU Junyan. Bioinformatics and expression analysis of flowering regulation gene BrFT in Brassica rapa L.[J]. Acta Agriculturae Zhejiangensis, 2023, 35(5): 992-1000.

图/表 11

表1 引物序列

Table 1 Primer sequence

引物名称	引物序列	引物用途
The name of the primer	Primer sequence (5'→3')	Use of primers
BrFT1-F	ATGTCTTTAAGTAATAGAGATCCTCTTGTGGT	基因克隆
BrFT1-R	CTAACTTCTTCGTCCTCCGCAG	Gene clone
BrFT1-F	ACGAGAGTCCAAGGCCCAAC	实时荧光定量PCR
BrFT1-R	TGGCGCCATCCTGGTTCATA	Real-time fluorescence quantitative PCR
BrFT2-F	TCCCTGCGACAACTGGAACAAAC	实时荧光定量PCR
BrFT2-R	CGGAACAATACCAGCACGAGACG	Real-time fluorescence quantitative PCR
BrFT3-F	AGGTTCAACACTCGTGAGTTCGC	实时荧光定量PCR
BrFT3-R	CTTCTTCCTCCGCAGCCATTCTC	Real-time fluorescence quantitative PCR
L17-BrFT1-F	ACGAGAGTCCAAGGCCCAAC	实时荧光定量PCR
L17-BrFT1-R	TGGCGCCATCCTGGTTCATA	Real-time fluorescence quantitative PCR
Actin-F	TGTGCCAATCTACGAGGGTTT	实时荧光定量PCR
Actin-R	TTTCCCGCTCGGCTGTTGT	Real-time fluorescence quantitative PCR

表2 BrFT基因家族成员基本信息

Table 2 Basic information of BrFT gene family members

基因	基因ID	开放阅读框	氨基酸长度	分子量	等电点	不稳定系数	亲水性	α-螺旋	β-折叠	延伸链	无规则卷曲
Gene	Gene ID	Open reading frame/bp	Amino acid length/aa	Molecular weight/ku	pI	Unstable coefficient	Hydro- philicity	Alpha helix/%	Beta turn/%	Extended strand/%	Random coil/%
BrFT1	Brapa02T001728	528	175	19.81	7.75	46.66	-0.34	14.86	3.43	22.86	58.85
BrFT2	Brapa02T003364	528	175	19.77	7.82	38.39	-0.34	17.14	4.00	21.71	57.15
BrFT3	Brapa02T004252	531	176	19.93	7.72	38.47	-0.29	13.64	3.98	25.57	56.81

图1 BrFT基因保守结构域、基因结构图

Fig.1 The conserved domain and gene structure of BrFT gene

图2 BrFT基因在染色体上的位置信息及共线性关系背景区域为白菜型油菜基因组中的所有共线性部分,红色线条代表存在共线性关系的BrFT基因对,不同颜色的半圈代表染色体。

Fig.2 Location information and collinearity of BrFT gene on chromosomes The background area is all the collinearity parts in B. rapa genome. The red lines represent the contributing BrFT gene pairs, and the half circles with different colors represent chromosomes.

图3 BrFT基因顺式作用元件分析

Fig.3 Cis-acting element analysis of BrFT gene

图4 陇油7号在4 ℃,16 h光照/8 h黑暗处理30 d时BrFT的表达模式

Fig.4 Expression pattern of BrFT in Longyou 7 at 4 ℃, 16 h light/8 h darkness for 30 days

图5 BrFT基因在陇油7号(A)和天祝小油菜(B)各器官中的表达不同数据柱上没有相同小写字母的表示差异显著(P<0.05)。下同。

Fig.5 Expression of BrFT genes in organs of Longyou 7(A) and Tianzhu small rape (B) The bars with different lowercase letters showed the significant difference (P<0.05). The same as below.

图6 BrFT1基因扩增产物电泳图 L6,陇油6号;L17,陇油17号;T2,天油2号。

Fig.6 Electrophoretic image of BrFT1 gene amplification product L6, Longyou 6; L17, Longyou 17; T2, Tianyou 2.

图7 不同品种白菜型冬油菜BrFT1基因氨基酸序列比对分析

Fig.7 Alignment analysis of the amino acid sequence of BrFT1 gene in different varieties of winter rapeseed

图8 BrFT1基因在不同白菜型冬油菜4 ℃,16 h光照/8 h黑暗处理中的荧光定量表达

Fig.8 Quantitative expression of BrFT1 gene in different varieties of winter rapeseed at 4 ℃, 16 h light/8 h darkness

表3 不同处理时间对白菜型油菜开花株率的影响

Table 3 Effect of different treatment time on flowering plant rate of Brassica rapa L.

时间 Time/d	开花株率 Flowering plant rate/%
时间 Time/d	天油2号 Tianyou 2	陇油6号 Longyou 6	陇油17号 Longyou 17	陇油7号 Longyou 7
0	0	0	0	0
20	46.8	15.6	23.2	13.3
30	88.9	26.6	46.7	39.6
40	98.3	40.0	73.3	46.6
50	100	73.3	93.3	71.6
60	100	86.1	93.3	86.6
70	100	95.4	97.1	94.3

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白菜型油菜开花调控基因BrFT的生物信息学特性和表达分析

Bioinformatics and expression analysis of flowering regulation gene BrFT in Brassica rapa L.

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