白菜型油菜IPT基因家族鉴定及表达分析

doi:10.13304/j.nykjdb.2022.0373

中国农业科技导报 ›› 2024, Vol. 26 ›› Issue (2): 56-66.DOI: 10.13304/j.nykjdb.2022.0373

白菜型油菜IPT基因家族鉴定及表达分析

刘博¹^,²(), 王旺田¹^,²(), 马骊², 武军艳², 蒲媛媛², 刘丽君², 方彦², 孙万仓²(), 张岩¹^,², 刘睿敏¹, 曾秀存³

^1.甘肃农业大学生命科学技术学院，兰州 730070
^2.甘肃农业大学省部共建干旱生境作物学国家重点实验室，兰州 730070
^3.河西学院，甘肃张掖 734000

收稿日期:2022-05-05 接受日期:2022-07-23 出版日期:2024-02-15 发布日期:2024-02-04
通讯作者: 王旺田,孙万仓
作者简介:刘博 E-mail：2081826780@qq.com
基金资助:
甘肃农业大学干旱生境作物学重点实验室开放课题项目(GSCS-2020-06);甘肃省科技计划项目(21JR7RA835);国家现代农业产业技术体系建设项目(CARS-12);国家自然科学基金项目(31860388);甘肃省教育厅2021年度青年博士基金项目(2021QB-113);甘肃省高等学校创新基金项目(2022A-054)

Identification and Characterization of IPT Gene Family in Brassica rapa L.

Bo LIU¹^,²(), Wangtian WANG¹^,²(), Li MA², Junyan WU², Yuanyuan PU², Lijun LIU², Yan FANG², Wancang SUN²(), Yan ZHANG¹^,², Ruimin LIU¹, Xiucun ZENG³

^1.College of Life Science and Technology，Gansu Agricultural University，Lanzhou 730070，China
^2.State Key Laboratory of Aridland Crop Science，Gansu Agricultural University，Lanzhou 730070，China
^3.Hexi University，Gansu Zhangye 734000，China

Received:2022-05-05 Accepted:2022-07-23 Online:2024-02-15 Published:2024-02-04
Contact: Wangtian WANG,Wancang SUN

摘要/Abstract

摘要：

异戊烯基转移酶（isopentenyltransferase，IPT）是细胞分裂素生物合成的第一限速酶，为分析IPT基因家族在白菜型油菜生长中的功能，利用生物信息学方法从其基因组中鉴定出13个BrIPT基因，它们不均匀地分布在7条染色体上。BrIPTs可分为4个亚族，各家族成员均含有8~10个保守基序和1~2个UTR区。BrIPT基因的启动子区域包含众多响应元件。BrIPT基因家族成员受环境因子、生物激素及防御与应激调控。qRT-PCR结果显示，tRNA-IPT基因BrIPT4、BrIPT6、BrIPT9在白菜型油菜体内各个部位均有表达。相比于苗期，成熟期白菜型油菜IPT基因的表达量更高，为后续深入研究IPT基因家族成员的生理生化功能提供了理论依据。

关键词: 白菜型油菜, IPT基因家族, 细胞分裂素, 生物信息学分析

Abstract:

Isopentenyltransferase （IPT） is the first limiting enzyme for cytokinin biosynthesis， so to analyze the function of the IPT gene family in Brassica rapa L.， a total of 13 BrIPT genes were identified from the genome of Brassica rapa L. by bioinformatics， which were heterogeneously distributed on 7 chromosomes. These BrIPT geneswere divided into 4 substrates and each BrIPT gene included 8~10 conserved motifs and 1~2 UTR regions. The promoter regions of BrIPT genes contained numerous responsive elements. The BrIPT genes were regulated by environmental factors， biohormones， defense and stress. The qRT-PCR results showed that the tRNA-IPT genes BrIPT4， BrIPT6 and BrIPT9 were expressed in various parts of Brassica rapa L.. The expressions of IPT genes in Brassica rapa L. were higher at the mature stage than those at the seedling stage. The above results provided some basis for subsequent in-depth studies on the physiological and biochemical functions of IPT gene family members.

Key words: Brassica rapa L., IPT gene family, cytokinin, bioinformatics analysis

中图分类号:

S565.4

刘博, 王旺田, 马骊, 武军艳, 蒲媛媛, 刘丽君, 方彦, 孙万仓, 张岩, 刘睿敏, 曾秀存. 白菜型油菜IPT基因家族鉴定及表达分析[J]. 中国农业科技导报, 2024, 26(2): 56-66.

Bo LIU, Wangtian WANG, Li MA, Junyan WU, Yuanyuan PU, Lijun LIU, Yan FANG, Wancang SUN, Yan ZHANG, Ruimin LIU, Xiucun ZENG. Identification and Characterization of IPT Gene Family in Brassica rapa L.[J]. Journal of Agricultural Science and Technology, 2024, 26(2): 56-66.

图/表 10

表1 试验所用引物序列

Table 1 Primer sequences used in the study

基因名称	上游引物序列	下游引物序列
Gene name	Forward primer sequence （5’-3’）	Reverse primer sequence （5’-3’）
BrIPT1	CAAGATTCTCCCAAGCCTCGTCTG	CGCCTGAGCAAGAAGTGGAAGTC
BrIPT2	TCCTTCATTCATGCCCTCCTTGTTG	CACCCAGAGGAAGCAGCAATCG
BrIPT3	GTGAACGGTTACACGGTGGATTAGG	CCTAACACATATCGGACGGCAACC
BrIPT4	AGGATTGTTCATCGGTGTTGGAGTG	GCAAGTGGTGTTACGTTGTTGTTCC
BrIPT5	CCTCACATTCCCTTCTTCCCTTCAC	GGTTGCTCCTAAGATGACGACGATC
BrIPT6	GCAACCGCCGTGCCTTATAC	GCCGGTGGGACCAGATATCA
BrIPT7	GCAGCTCGTTTCTCCGGAGA	TGCGGAGTAGATGGTGAGCG
BrIPT8	ATGGGTGCTACCGGAACAGG	GCAACTCTCCTCGGTCGTGA
BrIPT9	GCGGAATCGACGAAGCGAAG	CGCCAAGTCTACGGCTAGCT
BrIPT10	TCGCATGATGGAAGCAGGATTACTC	TGTACCACGGTCCACTAGAGATTCG
BrIPT11	GGAGAAGGAGGGAGAGTGGGATTC	CTTCACTACCTGTCTCTTCGCCAAC
BrIPT12	TCCGACAAGATCCAAGTTCACCAAG	GAGGCGGCAAAACACTGAGGAG
BrIPT13	ATCGTCACCAACAAAGTCACTCCAG	GTAATCCTCCGCCGTGAAATCCTC
Actin	TGTGCCAATCTACGAGGGTTT	TTTCCCGCTCGGCTGTTGT

表2 白菜型油菜IPT蛋白理化性质

Table 2 Protein physical and chemical properties of IPT in Brassica rapa L.

基因ID Gene ID	基因名 Gene name	氨基酸 Amino acids/aa	分子量 Molecular weight/Da	等电点PI	带负电荷残基数 Total number of negatively charged residues	带正电荷残基数 Total number of positively charged residue	不稳定指数Instability index	脂肪指数Aliphatic index
Brapa01T003069.1	BrIPT1	332	37 126.59	8.77	37	42	39.85	91.87
Brapa01T003426.1	BrIPT2	324	36 886.28	8.50	41	44	44.02	81.45
Brapa02T000813.1	BrIPT3	334	37 920.51	5.75	50	44	34.85	94.55
Brapa02T000886.1	BrIPT4	542	61 015.95	7.09	66	66	46.35	80.76
Brapa02T001911.1	BrIPT5	348	39 700.26	8.72	49	52	54.61	81.24
Brapa03T000921.1	BrIPT6	463	52 426.39	7.50	59	60	50.28	78.98
Brapa03T003913.1	BrIPT7	326	37 116.50	9.34	37	45	48.52	82.48
Brapa04T000031.1	BrIPT8	334	37 337.14	8.11	42	44	44.57	90.42
Brapa04T002174.1	BrIPT9	496	56 182.83	6.15	75	68	41.78	89.78
Brapa07T001138.1	BrIPT10	335	37 477.17	8.48	40	44	35.54	91.34
Brapa07T003284.1	BrIPT11	333	37 800.18	8.40	46	48	44.07	88.68
Brapa09T001382.1	BrIPT12	332	37 568.44	8.40	44	47	37.94	91.27
Brapa10T001241.1	BrIPT13	334	37 876.59	5.96	48	44	35.22	92.22

表3 氨基酸亲疏水性

Table 3 Amino acid hydrophilicity and hydrophobicity

基因ID Gene ID	基因名Gene name	最大亲水值Maximum hydrophilic value	位置Position/aa	最大疏水值Maximum hydrophobic value	位置Position/aa	亲水性平均系数 Grand average of hydropathicity
Brapa01T003069.1	BrIPT1	1.956	40	-2.922	276	-0.262
Brapa01T003426.1	BrIPT2	1.856	47	-2.311	214	-0.338
Brapa02T000813.1	BrIPT3	2.122	318	-2.489	263	-0.224
Brapa02T000886.1	BrIPT4	3.300	63，64	-3.100	124	-0.390
Brapa02T001911.1	BrIPT5	2.389	66	-3.411	57	-0.412
Brapa03T000921.1	BrIPT6	1.700	274	-3.489	50	-0.433
Brapa03T003913.1	BrIPT7	2.144	48	-2.356	214	-0.331
Brapa04T000031.1	BrIPT8	2.144	47	-2.522	153	-0.137
Brapa04T002174.1	BrIPT9	2.822	17	-3.744	482	-0.458
Brapa07T001138.1	BrIPT10	1.956	40	-2.756	276	-0.210
Brapa07T003284.1	BrIPT11	2.389	52	-3.078	308	-0.343
Brapa09T001382.1	BrIPT12	2.144	47	-2.456	153	-0.187
Brapa10T001241.1	BrIPT13	1.956	43	-2.489	263	-0.225

图1 IPT蛋白功能域比对

Fig. 1 Multiple alignment analyzing the binding domin of IPT proteins

图2 白菜型油菜IPT基因在染色体上的位置

Fig. 2 Position of IPT gene on chromosome in Brassica rapa L.

图3 白菜型油菜IPT基因的结构

Fig. 3 Structure of IPT gene in Brassica rapa L.

图4 白菜型油菜IPT蛋白保守基序的分布

Fig. 4 Distribution of conserved motifs of IPT protein in Brassica rapa L.

图5 白菜型油菜IPT蛋白家族系统进化树

Fig. 5 Phylogenetic tree of IPT protein family in Brassica rapa L.

图6 白菜型油菜IPT基因顺式作用元件位点图注：GT1-motif—光响应元件；LTR—低温响应元件；ABRE—脱落酸响应元件；O2-site—玉米醇溶蛋白响应元件；TGACG-motif—茉莉酸甲酯响应元件；TC-rich repeats—防御和应激反应响应元件；TATC-box—赤霉素响应元件；ARE—厌氧诱导响应元件；CGTCA-motif—茉莉酸响应元件；MRE—光响应元件；G-box—光响应元件；TCA-element—水杨酸响应元件；RY-element—种子特异性调控响应元件；G-Box—光响应元件；AuxRR-core—生长素响应元件；MBS—干旱诱导响应元件；ACE—光响应元件；circadian—昼夜节律控制响应元件；3-AF1 binding site—光响应元件；CAT-box—分生组织表达响应元件；MBSI—类黄酮生物合成基因调控响应元件；GC-motif—缺氧诱导响应元件；Sp1—光响应元件；GCN4-motif—胚乳表达响应元件。

Fig. 6 Site map of IPT gene element in Brassica rapa L.Note：GT1-motif—Light responsiveness； LTR—Low-temperature responsiveness； ABRE—Abscisic acid responsiveness； O2-site—Zein metabolism regulation； TGACG-motif—MeJA-responsiveness； TC-rich repeats—Defense and stress responsiveness； TATC-box—Gibberellin-responsiveness； ARE—Anaerobic induction； CGTCA-motif—MeJA-responsiveness； MRE—Light responsiveness； G-box—Light responsiveness； TCA-element—Salicylic acid responsiveness； RY-element—Seed-specific regulation； G-Box—Light responsiveness； AuxRR-core—Auxin responsiveness； MBS—Drought-induced response element； ACE—Light responsiveness； circadian—Circadian control； 3-AF1 binding site—Light responsive element； CAT-box—Meristem expression； MBSI—Flavonoid biosynthetic genes regulation； GC-motif—Anoxic specific inducibility； Sp1—Light responsive element； GCN4-motif—Endosperm expression.

图7 白菜型油菜IPT基因家族成员在不同组织中的表达注：cDNA1—苗期根；cDNA2—苗期下胚轴；cDNA3—苗期子叶；cDNA4—盛花期根；cDNA5—盛花期下胚轴；cDNA6—盛花期叶。

Fig. 7 Expression of IPT gene family in different tissuesNote：cDNA1—Seedling root；cDNA2—Seedling hypocotyl；cDNA3—Seedling cotyledons；cDNA4—Blooming root；cDNA5—Blooming hypocotyl；cDNA6—Blooming leaf.

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[6]	玉山江·麦麦提1,熊叶辉1,麦合木提江·米吉提1,Sehroon khan1,. 转IPT基因水稻的抗旱性研究[J]. , 2012, 14(6): 30-35.

白菜型油菜IPT基因家族鉴定及表达分析

Identification and Characterization of IPT Gene Family in Brassica rapa L.

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