芜菁酵母文库的构建和PSY潜在互作蛋白BrrCDPK家族成员鉴定

doi:10.16420/j.issn.0513-353x.2024-0833

摘要/Abstract

摘要：

以黄色芜菁W25和白色芜菁W21的不同组织为材料，构建芜菁cDNA酵母文库，库容1.5 × 10⁷ CFU，插入片段平均长度 > 1 000 bp。通过构建的诱饵载体pGBKT7-PSY与构建的cDNA文库杂交，共获得了18个阳性互作蛋白，通过酵母双杂交及双分子荧光互补（BiFC）验证，明确了候选蛋白CDPK8与PSY存在互作关系。对CDPK家族进行鉴定，共鉴定出50个含有STKc_CAMK protein kinase和2个EF-hand结构域的CDPK基因（命名为BrrCDPK1 ~ BrrCDPK50），结构分析显示CDPK编码氨基酸数在411 ~ 660个之间，平均等电点为8.79，各CDPK蛋白之间保守基序数和结构差异不明显，均含有motif 1、motif 2、motif 3和motif 6，染色体定位显示BrrCDPKs在7条染色体上的分布是非随机的；系统发育分析表明芜菁BrrCDPK与拟南芥AtCDPK被聚为不同类，暗示其在进化过程中的基因复制具有一定的种属特异性，基因表达谱分析鉴定出14个在黄色芜菁中高表达的CDPK基因。

关键词: 芜菁, 类胡萝卜素, 酵母双杂交, 互作蛋白, 钙依赖性蛋白激酶, 基因家族鉴定

Abstract:

Different tissues of yellow turnip W25 and white turnip W21 were used as plant materials to construct cDNA yeast library of turnip，whose library capacity was 1.5 × 10⁷ CFU and average length of insert frangment was greater than 1 000 bp. A total of 18 positive interacting proteins were obtained by hybridizing the constructed bait vector pGBKT7-PSY with the cDNA library. Then，a robust interaction between the candidate protein CDPK8 and PSY was confirmed by rigorous yeast two-hybrid and bimolecular fluorescence complementation（BiFC）experiments. The interacting protein CDPK was selected for gene family analysis，and a total of 50 CDPK genes（named BrrCDPK1-BrrCDPK50）members with STKc_CAMK protein kinase and 2 domains of EF-hand were identified. Structural analysis revealed that the number of amino acids encoded by CDPK genes ranged from 411 to 660，with an averagetheoretical pI of 8.79. No number and structural differences of conserved motifs among each CDPK proteins were found，and all proteins contained motif 1，motif 2，motif 3 and motif 6. Chromosome location showed that the distribution of BrrCDPKs on each chromosome was nonrandom. Phylogenetic analysis revealed that BrrCDPKs and AtCDPKs were clustered into different groups，indicating that CDPK gene duplication during evolution existed some species specificity. Gene expression profiling identified that 14 CDPK genes were highly expressed in yellow turnips than that in white turnip.

Key words: Brassica rapa ssp. rapifera, carotenoid, yeast two-hybrid, interactive protein, calcium-dependent protein kinase（CDPK）, gene family identification

边拴灵, 邓昌蓉, 樊露泽, 吴延寻, 任延靖. 芜菁酵母文库的构建和PSY潜在互作蛋白BrrCDPK家族成员鉴定[J]. 园艺学报, 2025, 52(10): 2625-2641.

BIAN Shuanling, DENG Changrong, FAN Luze, WU Yanxun, REN Yanjing. Construction of Yeast Library and Identification of the BrrCDPK Gene Family of PSY Potential Interacting Proteins in Turnip[J]. Acta Horticulturae Sinica, 2025, 52(10): 2625-2641.

图/表 11

参考文献 37

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引物名称Primer name	引物序列（5′-3′）Primer sequence
CDPK8-F	ATGGGAGGTTGTACCTCCA
CDPK8-R	TCACCGAGTTTTAGCTAAAGG
pGBKT7-CDPK8-F	TGGCCATGGAGGCCGAATTCATGGGAGGTTGTACCTCCA
pGBKT7-CDPK8-R	CGCTGCAGGTCGACGGATCCTCACCGAGTTTTAGCTAAAGG
pGADT7-CDPK8-F	CCATGGAGGCCAGTGAATTCATGGGAGGTTGTACCTCCA
pGADT7-CDPK8-R	AGCTCGAGCTCGATGGATCCTCACCGAGTTTTAGCTAAAGG
pGADT7-PSY-F	CCATGGAGGCCAGTGAATTCATGTCTTCTGTAGCAGTGTTA
pGADT7-PSY-R	AGCTCGAGCTCGATGGATCCTTAAGTTGTTCCTCTTGAACTT
G2-PSY-F	AGCTCGAGCTCGATGGATCCATGGGAGGTTGTACCTCCA
G2-PSY-R	CTACCCGGGAGCGGTACCCTCGAGTCACCGAGTTTTAGCTAAAGG
G4-CDPK8-F	AGCTCGAGCTCGATGGATCCATGTCTTCTGTAGCAGTGTTA
G4-CDPK8-R	GAGCTCCTACCCGGGAGCGGTACCTTAAGTTGTTCCTCTTGAACTT

引物名称Primer name	引物序列（5′-3′）Primer sequence
CDPK8-F	ATGGGAGGTTGTACCTCCA
CDPK8-R	TCACCGAGTTTTAGCTAAAGG
pGBKT7-CDPK8-F	TGGCCATGGAGGCCGAATTCATGGGAGGTTGTACCTCCA
pGBKT7-CDPK8-R	CGCTGCAGGTCGACGGATCCTCACCGAGTTTTAGCTAAAGG
pGADT7-CDPK8-F	CCATGGAGGCCAGTGAATTCATGGGAGGTTGTACCTCCA
pGADT7-CDPK8-R	AGCTCGAGCTCGATGGATCCTCACCGAGTTTTAGCTAAAGG
pGADT7-PSY-F	CCATGGAGGCCAGTGAATTCATGTCTTCTGTAGCAGTGTTA
pGADT7-PSY-R	AGCTCGAGCTCGATGGATCCTTAAGTTGTTCCTCTTGAACTT
G2-PSY-F	AGCTCGAGCTCGATGGATCCATGGGAGGTTGTACCTCCA
G2-PSY-R	CTACCCGGGAGCGGTACCCTCGAGTCACCGAGTTTTAGCTAAAGG
G4-CDPK8-F	AGCTCGAGCTCGATGGATCCATGTCTTCTGTAGCAGTGTTA
G4-CDPK8-R	GAGCTCCTACCCGGGAGCGGTACCTTAAGTTGTTCCTCTTGAACTT

序号No.	样本名Sample name	基因注释Gene annotation	登录号GenBank	基因ID Gene ID
C1	1237-BD-1-1 1237-BD-4 1237-BD-10	白菜低分子量热休克蛋白（BcHSP17.6）mRNA，完整CDS Brassica rapa low molecular weight heat-shock protein（BcHSP17.6）mRNA，complete CDS	AF022217.1	BraA06g020450.3C
C2	1237-BD-2 1237-BD-6 1237-BD-18	白菜核前体mRNA的调节域含蛋白1A（LOC103855848），转录本变体X2 Brassica rapa regulation of nuclear pre-mRNA domain-containing protein 1A（LOC103855848），transcript variant X2	XM_009132891.3	BraA03g004120.3C
C3	1237-BD-3-2	白菜嘌呤渗透酶1（LOC103838976），mRNA Brassica rapa purine permease 1（LOC103838976）	XM_009115443.3	BraA09g036510.3C
C4	1237-BD-8-1	白菜Golgi SNAP受体复合体成员1-2（LOC103858165），转录变体X2 Brassica rapa Golgi SNAP receptor complex member 1-2（LOC103858165），transcript variant X2	XM_018657146.2	BraA03g023640.3C
C5	1237-BD-9	白菜translin（LOC103857651），转录变体X1 Brassica rapa translin（LOC103857651），transcript variant X1	XM_009134861.3	BraA03g019260.3C
C6	1237-BD-11	白菜17.6 kDⅠ类热休克蛋白2-like（LOC103843285） Brassica rapa 17.6 kD classⅠheat shock protein 2-like （LOC103843285）	XM_009119989.3	BraA05g014680.3C
C7	1237-BD-12	甘蓝型油菜多细胞器RNA编辑因子1，mitochondrial（LOC106437506） Brassica napus multiple organellar RNA editing factor 1，mitochondrial（LOC106437506）	XM_013878402.3	BraA03g049050.3C
C8	1237-BD-13	白菜RNA聚合酶Ⅱ转录亚基26的推定介质（LOC103833180） Brassica rapa putative mediator of RNA polymeraseⅡtranscription subunit 26（LOC103833180）	XM_033282535.1	BraA10g022490.3C
C9	1237-BD-14-2	甘蓝型油菜CDPK相关激酶2（LOC106362920），转录变体X2 Brassica napus CDPK-related kinase 2（LOC106362920），transcript variant X2	XM_022695160.2	BraA01g033890.3C
C10	1237-BD-15-1	甘蓝型油菜formyltetrahydrofolate deformylase1，线粒体（LOC106402097），转录变体X4 Brassica napus formyltetrahydrofolate deformylase 1，mitochondrial（LOC106402097），transcript variant X4	XM_048781377.1	BraA06g041130.3C
C11	1237-BD-15-2	白菜 patellin-4（LOC103840314），转录变体X2 Brassica rapa patellin-4（LOC103840314），transcript variant X2	XM_009116810.2	BraA09g034310.3C
C12	1237-BD-17-1	甘蓝型油菜硫氧还蛋白O2，线粒体（LOC106352370），转录变体X1 Brassica napus thioredoxin O2，mitochondrial（LOC106352370），transcript variant X1	XM_048739086.1	BraA09g033920.3C
C13	1237-BD-17-2	白菜同源箱亮氨酸拉链蛋白ATHB-6（LOC103837893） Brassica rapa homeobox-leucine zipper protein ATHB-6（LOC103837893）	XM_009114280.3	BraA09g054870.3C
C14	1237-BD-20	白菜未鉴定的LOC103863008（LOC103863008），转录变体X3 Brassica rapa uncharacterized LOC103863008（LOC103863008），transcript variant X3	XM_033289696.1	BraA04g002940.3C
C15	1237-BD-21	白菜蛋白MET1，chloroplastic（LOC103829781） Brassica rapa protein MET1，chloroplastic（LOC103829781）	XM_009105478.3	BraA05g016190.3C
C16	1237-BD-22	白菜转座酶蛋白At4g04430（LOC103847917），转录变体X2 Brassica rapa probable transposase-like protein At4g04430（LOC103847917），transcript variant X2	XM_018655836.2	未检索到 Not retrieved
C17	1237-BD-23	甘蓝型油菜高迁移率B型蛋白2（LOC106416143），转录变体X2 Brassica napus high mobility group B protein 2（LOC106416143），transcript variant X2	XM_048738078.1	BraA08g027710.3C
C18	1237-BD-24	白菜E3泛素蛋白连接酶BRE1-like 1（LOC103866184） Brassica rapa E3 ubiquitin-protein ligase BRE1-like 1（LOC103866184）	XM_009144061.3	BraA04g030860.3C

序号No.	样本名Sample name	基因注释Gene annotation	登录号GenBank	基因ID Gene ID
C1	1237-BD-1-1 1237-BD-4 1237-BD-10	白菜低分子量热休克蛋白（BcHSP17.6）mRNA，完整CDS Brassica rapa low molecular weight heat-shock protein（BcHSP17.6）mRNA，complete CDS	AF022217.1	BraA06g020450.3C
C2	1237-BD-2 1237-BD-6 1237-BD-18	白菜核前体mRNA的调节域含蛋白1A（LOC103855848），转录本变体X2 Brassica rapa regulation of nuclear pre-mRNA domain-containing protein 1A（LOC103855848），transcript variant X2	XM_009132891.3	BraA03g004120.3C
C3	1237-BD-3-2	白菜嘌呤渗透酶1（LOC103838976），mRNA Brassica rapa purine permease 1（LOC103838976）	XM_009115443.3	BraA09g036510.3C
C4	1237-BD-8-1	白菜Golgi SNAP受体复合体成员1-2（LOC103858165），转录变体X2 Brassica rapa Golgi SNAP receptor complex member 1-2（LOC103858165），transcript variant X2	XM_018657146.2	BraA03g023640.3C
C5	1237-BD-9	白菜translin（LOC103857651），转录变体X1 Brassica rapa translin（LOC103857651），transcript variant X1	XM_009134861.3	BraA03g019260.3C
C6	1237-BD-11	白菜17.6 kDⅠ类热休克蛋白2-like（LOC103843285） Brassica rapa 17.6 kD classⅠheat shock protein 2-like （LOC103843285）	XM_009119989.3	BraA05g014680.3C
C7	1237-BD-12	甘蓝型油菜多细胞器RNA编辑因子1，mitochondrial（LOC106437506） Brassica napus multiple organellar RNA editing factor 1，mitochondrial（LOC106437506）	XM_013878402.3	BraA03g049050.3C
C8	1237-BD-13	白菜RNA聚合酶Ⅱ转录亚基26的推定介质（LOC103833180） Brassica rapa putative mediator of RNA polymeraseⅡtranscription subunit 26（LOC103833180）	XM_033282535.1	BraA10g022490.3C
C9	1237-BD-14-2	甘蓝型油菜CDPK相关激酶2（LOC106362920），转录变体X2 Brassica napus CDPK-related kinase 2（LOC106362920），transcript variant X2	XM_022695160.2	BraA01g033890.3C
C10	1237-BD-15-1	甘蓝型油菜formyltetrahydrofolate deformylase1，线粒体（LOC106402097），转录变体X4 Brassica napus formyltetrahydrofolate deformylase 1，mitochondrial（LOC106402097），transcript variant X4	XM_048781377.1	BraA06g041130.3C
C11	1237-BD-15-2	白菜 patellin-4（LOC103840314），转录变体X2 Brassica rapa patellin-4（LOC103840314），transcript variant X2	XM_009116810.2	BraA09g034310.3C
C12	1237-BD-17-1	甘蓝型油菜硫氧还蛋白O2，线粒体（LOC106352370），转录变体X1 Brassica napus thioredoxin O2，mitochondrial（LOC106352370），transcript variant X1	XM_048739086.1	BraA09g033920.3C
C13	1237-BD-17-2	白菜同源箱亮氨酸拉链蛋白ATHB-6（LOC103837893） Brassica rapa homeobox-leucine zipper protein ATHB-6（LOC103837893）	XM_009114280.3	BraA09g054870.3C
C14	1237-BD-20	白菜未鉴定的LOC103863008（LOC103863008），转录变体X3 Brassica rapa uncharacterized LOC103863008（LOC103863008），transcript variant X3	XM_033289696.1	BraA04g002940.3C
C15	1237-BD-21	白菜蛋白MET1，chloroplastic（LOC103829781） Brassica rapa protein MET1，chloroplastic（LOC103829781）	XM_009105478.3	BraA05g016190.3C
C16	1237-BD-22	白菜转座酶蛋白At4g04430（LOC103847917），转录变体X2 Brassica rapa probable transposase-like protein At4g04430（LOC103847917），transcript variant X2	XM_018655836.2	未检索到 Not retrieved
C17	1237-BD-23	甘蓝型油菜高迁移率B型蛋白2（LOC106416143），转录变体X2 Brassica napus high mobility group B protein 2（LOC106416143），transcript variant X2	XM_048738078.1	BraA08g027710.3C
C18	1237-BD-24	白菜E3泛素蛋白连接酶BRE1-like 1（LOC103866184） Brassica rapa E3 ubiquitin-protein ligase BRE1-like 1（LOC103866184）	XM_009144061.3	BraA04g030860.3C

基因名 Gene name	基因编号 Gene ID	登录号 Accession number	cDNA长度/bp cDNA length	氨基酸数 Number of amino acids	分子量/kD Molecular weight	等电点 Theoretical pI	脂肪系数 Aliphatic index	疏水性 GRAVY
BrrCDPK1	Cluster-30924.46777	XM_009103718.3	1 737	578	64.58	8.95	83.39	-0.317
BrrCDPK2	Cluster-30924.137521	XM_013807283.3	1 743	580	64.88	9.07	83.28	-0.324
BrrCDPK3	Cluster-30924.143914	XM_013807283.3	1 731	576	64.47	9.01	83.68	-0.318
BrrCDPK4	Cluster-30924.13630	XM_048738123.1	1 755	584	65.72	8.88	81.70	-0.392
BrrCDPK5	Cluster-30924.67984	XM_010509132.2	1 806	601	67.46	8.99	88.80	-0.318
BrrCDPK6	Cluster-30924.158029 Cluster-30924.77034 Cluster-30924.77038 Cluster-30924.80376	XM_009113894.3	1 785	594	66.74	9.06	83.43	-0.374
BrrCDPK7	Cluster-30924.58681	XM_009113894.3	1 785	594	66.76	9.12	82.61	-0.375
BrrCDPK8	Cluster-30924.142390	XM_009113894.3	1 785	594	66.77	9.06	83.60	-0.373
BrrCDPK9	Cluster-30924.157993	XM_009113894.3	1 785	594	66.70	9.06	84.09	-0.372
BrrCDPK10	Cluster-30924.131630	XM_013879035.3	1 704	567	63.43	8.84	86.84	-0.218
BrrCDPK11	Cluster-30924.136201	XM_013792411.3	1 707	568	63.43	8.84	86.88	-0.205
BrrCDPK12	Cluster-30924.24510	XM_013879035.3	1 704	567	63.59	8.67	85.47	-0.219
BrrCDPK13	Cluster-30924.57260	XM_009140827.3	1 728	575	64.34	8.61	86.16	-0.238
BrrCDPK14	Cluster-30924.150034	XM_013886734.3	1 713	570	63.80	8.85	86.74	-0.251
BrrCDPK15	Cluster-30924.150032 Cluster-30924.150033	XM_009140827.3	1 728	575	64.38	8.70	86.16	-0.243
BrrCDPK16	Cluster-30924.14001	XM_033289955.1	1 239	412	46.61	8.43	86.09	-0.229
BrrCDPK17	Cluster-30924.24231	XM_013871943.3	1 236	411	46.25	8.00	88.42	-0.203
BrrCDPK18	Cluster-30924.82876	XM_013895504.3	1 791	596	66.44	8.95	83.98	-0.351
BrrCDPK19	Cluster-30924.151434	XM_013895504.3	1 791	596	66.45	8.88	84.80	-0.343
BrrCDPK20	Cluster-30924.142865	XM_013895504.3	1 791	596	66.49	9.01	83.98	-0.344
BrrCDPK21	Cluster-30924.142866	XM_009144190.3	1 791	596	66.45	8.95	83.98	-0.356
BrrCDPK22	Cluster-30924.146395 Cluster-30924.169091	XM_013895504.3	1 791	596	66.44	8.88	83.98	-0.350
BrrCDPK23	Cluster-30924.52514	XM_018622041.2	1 809	602	67.28	9.16	82.66	-0.366
BrrCDPK24	Cluster-30924.140928 Cluster-30924.155408	XM_009144190.3	1 791	596	66.46	8.95	83.98	-0.356
BrrCDPK25	Cluster-30924.122791	XM_013781701.1	1 734	577	64.40	8.52	84.85	-0.260
BrrCDPK26	Cluster-30924.24346	XM_013892390.3	1 734	577	64.41	8.52	84.33	-0.263
BrrCDPK27	Cluster-30924.67985	XM_009147578.3	1 983	660	74.20	9.02	85.41	-0.248
BrrCDPK28	Cluster-30924.67983 Cluster-30924.92828	XM_009147578.3	1 983	660	74.31	9.07	86.15	-0.246
BrrCDPK29	Cluster-30924.11427	XM_009147578.3	1 983	660	74.30	9.07	85.56	-0.252
BrrCDPK30	Cluster-30924.24345	XM_009147578.3	1 983	660	74.33	9.07	84.97	-0.254
BrrCDPK31	Cluster-30924.77035	XM_013732238.1	1 791	596	66.91	9.09	83.79	-0.362
BrrCDPK32	Cluster-30924.36432	XM_013851068.3	1 791	596	66.95	9.13	83.79	-0.355
BrrCDPK33	Cluster-30924.77036	XM_013893888.3	1 791	596	66.82	9.19	83.31	-0.363
BrrCDPK34	Cluster-30924.77037	XM_013851068.3	1 791	596	66.91	9.13	83.79	-0.362
BrrCDPK35	Cluster-30924.114427	XM_009147578.3	1 983	660	74.32	9.07	86.15	-0.244
BrrCDPK36	Cluster-69076.1 Cluster-69076.4	XM_013786949.3	1 776	591	65.92	8.66	83.55	-0.309
BrrCDPK37	Cluster-69076.2	XM_013786949.3	1 776	591	65.93	8.52	84.53	-0.295
BrrCDPK38	Cluster-69076.0	XM_009151493.3	1 776	591	65.90	8.66	84.38	-0.306
BrrCDPK39	Cluster-30924.23018	XM_013786949.3	1 776	591	65.95	8.77	83.89	-0.314
BrrCDPK40	Cluster-30924.12640	XM_013787387.3	1 803	600	66.93	8.51	82.48	-0.343
BrrCDPK41	Cluster-30924.75342	XM_048769751.1	1 242	413	47.04	6.68	93.27	-0.195
BrrCDPK42	Cluster-30924.119657 Cluster-30924.139625	XM_013880416.3	1 791	596	66.84	8.98	81.88	-0.355
BrrCDPK43	Cluster-30924.119656 Cluster-30924.139627	XM_013880416.3	1 791	596	66.91	8.98	81.39	-0.360
BrrCDPK44	Cluster-30924.139626 Cluster-30924.16433	XM_013880416.3	1 791	596	66.87	8.98	81.22		-0.356
BrrCDPK45	Cluster-30924.139628	XM_013880416.3	1 791	596	66.87	8.91	81.71		-0.360
BrrCDPK46	Cluster-30924.74759	XM_013880416.3	1 791	596	66.85	8.98	81.88		-0.354
BrrCDPK47	Cluster-30924.114809 Cluster-30924.114810 Cluster-30924.123336 Cluster-30924.127303 Cluster-30924.130998 Cluster-30924.49496	XM_048739418.1	1 743	580	65.01	8.91	80.88		-0.359
BrrCDPK48	Cluster-30924.101435 Cluster-30924.124946	XM_048739418.1	1 743	580	65.00	8.91	80.71		-0.363
BrrCDPK49	Cluster-30924.155459	XM_048769751.1	1 242	413	47.04	6.44	93.51		-0.188
BrrCDPK50	Cluster-30924.148411	XM_048741021.1	1 785	594	66.10	8.76	82.64		-0.314