普通菜豆Trihelix基因家族鉴定及表达分析

doi:10.16420/j.issn.0513-353x.2023-0972

摘要/Abstract

摘要：

对普通菜豆Trihelix基因家族进行了生物信息分析并利用转录组数据（RNA-seq）和荧光定量PCR（qRT-PCR）对其在逆境胁迫下的表达进行了分析。结果表明，普通菜豆全基因组中鉴定到40个PvTrihelixes，不均匀地分布在11条染色体上。系统进化分析将其分为5个亚族（GT-1、GT-2、GTγ、SIP1和SH4），亚族内成员的基因结构和保守基序基本相似。共线性分析发现，PvTrihelixes间存在5个串联重复和9个片段重复。启动子分析发现，PvTrihelix含有生长发育、光、激素和胁迫响应顺式作用元件。组织表达分析发现，PvTrihelixes在花中的表达水平较高的基因均属于GT-2亚家族和SIP1亚家族。qRT-PCR结果显示，PvTrihelix6/7/10/11在干旱胁迫（6% PEG）、PvTrihelix1/21/25/26/31/35在低温胁迫（4 ℃）和PvTrihelix21/23/25/27/28/40在盐胁迫（100 mmol · L^-1 NaCl）下显著上调表达。

关键词: 普通菜豆, Trihelix基因家族, 进化分析, 表达模式, 非生物胁迫

Abstract:

In this study，bioinformatics analysis of the common bean Trihelix gene family were performed，and the expression of the PvTrihelixes under adversity stress were examined using transcriptomic data（RNA-seq）and fluorescence quantitative PCR（qRT-PCR）. The results showed that 40 PvTrihelixes genes were identified in common bean genome，which unevenly distributed on 11 chromosomes. Phylogenetic analyses classified PvTrihelixes into five subfamilies（GT-1，GT-2，GTγ，SIP1 and SH4）. Five subfamilies were essentially similar in terms of gene structure and conserved motifs. Covariance analysis revealed there are 5 pairs of tandem repeat and 9 pairs of fragment repeat in PvTrihelixes. The promoter analysis revealed that PvTrihelixes contain cis-acting elements related to growth and development，light，hormones，and stress response. Tissue expression analysis revealed the presence of tissue expression specificity in PvTrihelix，and the genes with higher expression levels in flowers all belonged to members of the GT-2 subfamily and the SIP1 subfamily. The qRT-PCR results showed that PvTrihelix6/7/10/11 were significantly up-regulated under drought（6% PEG），PvTrihelix1/21/25/26/31/35 were up-regulated in response to low temperature（4 ℃），and PvTrihelix21/23/25/27/28/40were significantly up-regulated under salt（100 mmol · L^-1 NaCl）stress.

Key words: common bean, Trihelix gene family, evolutionary analysis, expression pattern, abiotic stress

罗新锐, 张晓旭, 王玉萍, 王智, 马媛媛, 周丙月. 普通菜豆Trihelix基因家族鉴定及表达分析[J]. 园艺学报, 2024, 51(12): 2775-2790.

LUO Xinrui, ZHANG Xiaoxu, WANG Yuping, WANG Zhi, MA Yuanyuan, ZHOU Bingyue. Identification and Expression Analysis of Trihelix Gene Family in Common Bean[J]. Acta Horticulturae Sinica, 2024, 51(12): 2775-2790.

图/表 11

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基因 Gene	正向引物序列（5′-3′） Forward primer	反向引物序列（5′-3′） Reverse primer
PvTrihelix1	GCCGCTGCTGGAGGAAAGTTG	ATCGGTATCGCTCTCGGGATCAG
PvTrihelix2	GAATGAACGGAGGAGGCTGGAAATC	AGGTAGAAGAATGCTGCTGCTGATG
PvTrihelix3	TTGGTAGCAGCAGCCTTCGTTTC	GTCATTGTCCTTGAGTCCTCCAGTG
PvTrihelix4	GGACATCCTCTTGCCATTACCACAG	GCTTCTGGAGTGCCGTCAATACC
PvTrihelix5	AAGACGCTTTGGGAAGTGGTGAG	ATGGACATTGCCTGCTATGCTCTG
PvTrihelix6	TGGTCAAGATGGTGCCGAAACTG	CCTCATCCTCCTCTTCTTCCTCCTC
PvTrihelix7	CCATAGCTCGTGAATACCGCAAGG	GCAGCCGTGAGTCCAGCAATAG
PvTrihelix8	ACTCCTCCTGGGTCCACTTTAAGC	ATCATCGTCGTTGTCGTCATCATCG
PvTrihelix9	GCGTATCGTGGAGAATTGGAGGAAG	GTTGTGTGCCGAATGCCAGTTG
PvTrihelix10	ACCACCACCTCCACCTCTTCTTC	CCTAGTGAGCCTCCTGAAGTAGTCC
PvTrihelix11	TCCAGTTCTTCCTCCACCTCTTCC	CACTTCCTCTTCCTCTTGCGTCTTG
PvTrihelix12	GCGGGTGAAGTGGACTGATAAGATG	ACTGCTGAGGTGAAACACGATGAC
PvTrihelix13	GTGGTGCTGCTGCTGAGGATG	ATCATCTGCTGCCTCTTGCTGTTC
PvTrihelix14	ACCACCACCAACACCAACAATAGC	TGGAGGAGCAACAGAAGGAGGAG
PvTrihelix15	CTCCGCCTCACACAGCAAACC	TTCCTCCGACCAGCAGTCTTCC
PvTrihelix16	GGTCTCCTTGGCAGCGAATGAAG	CATCACCCACACAACTCACCACTG
PvTrihelix17	GGTGATGAGGAAGAGGAGGTAGAGG	CTGGCACACGAGGAGCAAGTTC
PvTrihelix18	CGACAACATTAGCAACTACGGCAAG	TCACCACCACCACCACCACTC
PvTrihelix19	TCTCTCCCTTCCGCCCTCAATG	ATCCTCATCTTCCTCCTCCGCATC
PvTrihelix20	ACGAACGATTGGATGCTCTGATTGG	GAAGGAGACGGCGAGGAGGAAG
PvTrihelix21	GGGAAAGAGAGACGCTGAGAACAAG	CGACCTGACCCGACCCTAATGG
PvTrihelix22	GTGGTGGAAGTGGAAGTGGAAGAAG	AACAACCTGACAAGAAGTGCCTCTC
PvTrihelix23	GCCGCCGTCATGTCATTCCTTC	TGTTGGTGTGGTGGTTGCTGTAC
PvTrihelix24	TTCCTTCAACCACCGCCTTCAC	GCAGTAGCAGCAGTTGTAGGAGTAG
PvTrihelix25	CACCTCCTCCACCACCTCCAC	ACTGTGTTCCGTGCCTGATGATTC
PvTrihelix26	CTAATACTGCCACCGCTGCTGTC	TCAAACCCAAACCACCCTTTCTCTG
PvTrihelix27	CCACTCCTCCTTCTCTGACGACTC	CGCTCCAATCTTCTCTATCCTTCCG
PvTrihelix28	GTTCGATGGAGTACCTGAGCAGTTC	GGGTTGGAGTGGCAGTTGATGG
PvTrihelix29	AGCCTCAGCCTCACCAACTCC	CCACTCCTTCTGCTTCTCCACAAG
PvTrihelix30	ACCACCACCTCCACCAACCTTAC	CTTCTCACTGCCGCCATCGTC
PvTrihelix31	AGCAGCAGCCTCAGCAACAAC	TCCTCCTCATCCGAATCATCCTCAG
PvTrihelix32	CGGTTCGGCGTTCGGATTGAG	TCTCTGACCTGCAACTCCCACTC
PvTrihelix33	CGTGAGGATGGAGCAGATGAAGATG	AGGGTTGCGACGAGGGAGAAG
PvTrihelix34	AGCGATAGAGACAGAGGCGATGAG	CTCCTTCTGCTTCTGCCCACAAC
PvTrihelix35	ACCCAGAAACCACTACGACAACAAC	AAGCACTTACGGAACACGGAACC
PvTrihelix36	CAACGACCTCAATCTCCGCTTCAC	CCTTCATTTCGCCTGGCTCTTCC
PvTrihelix37	TGGTGGTGGTAGCAGCAGTTTG	ACTCCATCTCCTTCAACCTCCTCTC
PvTrihelix38	CCGACGAGGACGCCAATTATCAG	AACCCTCTTTCACCGCTTCTCATTC
PvTrihelix39	GAGGCGTATGAACATGCGGAGAG	TGGTGCTATGGTGATTGTTGCTAGG
PvTrihelix40	ACCAACCACAACAACAACCTAGACC	ACACTGCTCCTTCACTCCATTCATC
Actin	GAAGTTCTCTTCCAACCATCC	TTTCCTTGCTCATTCTGTCCG

基因 Gene	正向引物序列（5′-3′） Forward primer	反向引物序列（5′-3′） Reverse primer
PvTrihelix1	GCCGCTGCTGGAGGAAAGTTG	ATCGGTATCGCTCTCGGGATCAG
PvTrihelix2	GAATGAACGGAGGAGGCTGGAAATC	AGGTAGAAGAATGCTGCTGCTGATG
PvTrihelix3	TTGGTAGCAGCAGCCTTCGTTTC	GTCATTGTCCTTGAGTCCTCCAGTG
PvTrihelix4	GGACATCCTCTTGCCATTACCACAG	GCTTCTGGAGTGCCGTCAATACC
PvTrihelix5	AAGACGCTTTGGGAAGTGGTGAG	ATGGACATTGCCTGCTATGCTCTG
PvTrihelix6	TGGTCAAGATGGTGCCGAAACTG	CCTCATCCTCCTCTTCTTCCTCCTC
PvTrihelix7	CCATAGCTCGTGAATACCGCAAGG	GCAGCCGTGAGTCCAGCAATAG
PvTrihelix8	ACTCCTCCTGGGTCCACTTTAAGC	ATCATCGTCGTTGTCGTCATCATCG
PvTrihelix9	GCGTATCGTGGAGAATTGGAGGAAG	GTTGTGTGCCGAATGCCAGTTG
PvTrihelix10	ACCACCACCTCCACCTCTTCTTC	CCTAGTGAGCCTCCTGAAGTAGTCC
PvTrihelix11	TCCAGTTCTTCCTCCACCTCTTCC	CACTTCCTCTTCCTCTTGCGTCTTG
PvTrihelix12	GCGGGTGAAGTGGACTGATAAGATG	ACTGCTGAGGTGAAACACGATGAC
PvTrihelix13	GTGGTGCTGCTGCTGAGGATG	ATCATCTGCTGCCTCTTGCTGTTC
PvTrihelix14	ACCACCACCAACACCAACAATAGC	TGGAGGAGCAACAGAAGGAGGAG
PvTrihelix15	CTCCGCCTCACACAGCAAACC	TTCCTCCGACCAGCAGTCTTCC
PvTrihelix16	GGTCTCCTTGGCAGCGAATGAAG	CATCACCCACACAACTCACCACTG
PvTrihelix17	GGTGATGAGGAAGAGGAGGTAGAGG	CTGGCACACGAGGAGCAAGTTC
PvTrihelix18	CGACAACATTAGCAACTACGGCAAG	TCACCACCACCACCACCACTC
PvTrihelix19	TCTCTCCCTTCCGCCCTCAATG	ATCCTCATCTTCCTCCTCCGCATC
PvTrihelix20	ACGAACGATTGGATGCTCTGATTGG	GAAGGAGACGGCGAGGAGGAAG
PvTrihelix21	GGGAAAGAGAGACGCTGAGAACAAG	CGACCTGACCCGACCCTAATGG
PvTrihelix22	GTGGTGGAAGTGGAAGTGGAAGAAG	AACAACCTGACAAGAAGTGCCTCTC
PvTrihelix23	GCCGCCGTCATGTCATTCCTTC	TGTTGGTGTGGTGGTTGCTGTAC
PvTrihelix24	TTCCTTCAACCACCGCCTTCAC	GCAGTAGCAGCAGTTGTAGGAGTAG
PvTrihelix25	CACCTCCTCCACCACCTCCAC	ACTGTGTTCCGTGCCTGATGATTC
PvTrihelix26	CTAATACTGCCACCGCTGCTGTC	TCAAACCCAAACCACCCTTTCTCTG
PvTrihelix27	CCACTCCTCCTTCTCTGACGACTC	CGCTCCAATCTTCTCTATCCTTCCG
PvTrihelix28	GTTCGATGGAGTACCTGAGCAGTTC	GGGTTGGAGTGGCAGTTGATGG
PvTrihelix29	AGCCTCAGCCTCACCAACTCC	CCACTCCTTCTGCTTCTCCACAAG
PvTrihelix30	ACCACCACCTCCACCAACCTTAC	CTTCTCACTGCCGCCATCGTC
PvTrihelix31	AGCAGCAGCCTCAGCAACAAC	TCCTCCTCATCCGAATCATCCTCAG
PvTrihelix32	CGGTTCGGCGTTCGGATTGAG	TCTCTGACCTGCAACTCCCACTC
PvTrihelix33	CGTGAGGATGGAGCAGATGAAGATG	AGGGTTGCGACGAGGGAGAAG
PvTrihelix34	AGCGATAGAGACAGAGGCGATGAG	CTCCTTCTGCTTCTGCCCACAAC
PvTrihelix35	ACCCAGAAACCACTACGACAACAAC	AAGCACTTACGGAACACGGAACC
PvTrihelix36	CAACGACCTCAATCTCCGCTTCAC	CCTTCATTTCGCCTGGCTCTTCC
PvTrihelix37	TGGTGGTGGTAGCAGCAGTTTG	ACTCCATCTCCTTCAACCTCCTCTC
PvTrihelix38	CCGACGAGGACGCCAATTATCAG	AACCCTCTTTCACCGCTTCTCATTC
PvTrihelix39	GAGGCGTATGAACATGCGGAGAG	TGGTGCTATGGTGATTGTTGCTAGG
PvTrihelix40	ACCAACCACAACAACAACCTAGACC	ACACTGCTCCTTCACTCCATTCATC
Actin	GAAGTTCTCTTCCAACCATCC	TTTCCTTGCTCATTCTGTCCG

基因 Gene	基因序列号 Gene ID	相对分子质量/Da Molecular weight	等电点 pI	氨基酸数 Amino acid	不稳定系数 Instability index	亲水性 Hydro- pathicity	亚细胞定位 Subcellular localization
PvTrihelix1	Phvul.001G077900	34942.98	4.86	310	58.84	-0.711	叶绿体，线粒体 Chloroplast，mitochondria
PvTrihelix2	Phvul.001G185100	51846.89	6.28	450	59.03	-1.102	细胞核Nuclear
PvTrihelix3	Phvul.001G187000	68096.08	6.32	592	54.91	-1.126	细胞核Nuclear
PvTrihelix4	Phvul.001G258700	43075.53	5.83	377	52.11	-0.775	细胞质Cytoplasm
PvTrihelix5	Phvul.002G126400	36335.79	8.66	309	66.06	-1.115	细胞核Nuclear
PvTrihelix6	Phvul.002G281800	40517.74	6.51	359	57.47	-0.764	细胞核Nuclear
PvTrihelix7	Phvul.003G007800	38333.55	8.22	340	64.78	-0.932	细胞核Nuclear
PvTrihelix8	Phvul.003G097500	33089.51	9.42	294	62.37	-0.938	细胞核Nuclear
PvTrihelix9	Phvul.003G209600	96885.44	8.76	870	40.28	-0.351	叶绿体Chloroplast
PvTrihelix10	Phvul.004G095200	67136.46	5.48	590	53.08	-1.094	细胞核Nuclear
PvTrihelix11	Phvul.004G095400	72575.65	5.40	656	65.95	-0.954	细胞核Nuclear
PvTrihelix12	Phvul.004G095500	50753.47	6.17	442	48.63	-1.054	细胞核Nuclear
PvTrihelix13	Phvul.004G161800	40924.74	4.60	354	50.82	-1.188	细胞核Nuclear
PvTrihelix14	Phvul.005G025700	38695.34	9.31	349	59.73	-0.952	细胞核Nuclear
PvTrihelix15	Phvul.005G095000	34005.92	9.69	300	63.97	-1.030	细胞核Nuclear
PvTrihelix16	Phvul.005G148400	50793.91	5.70	448	49.20	-0.910	细胞核Nuclear
PvTrihelix17	Phvul.005G171600	45586.93	4.81	400	47.87	-1.130	细胞核Nuclear
PvTrihelix18	Phvul.006G100300	64796.82	6.20	568	66.90	-1.068	细胞核Nuclear
PvTrihelix19	Phvul.006G139200	37809.28	6.06	329	66.93	-1.055	细胞核Nuclear
PvTrihelix20	Phvul.006G176000	34015.83	5.16	301	66.03	-0.937	细胞核，线粒体 Nuclear，mitochondria
PvTrihelix21	Phvul.007G000400	39649.55	6.94	356	62.13	-0.843	细胞核Nuclear
PvTrihelix22	Phvul.007G079600	51421.22	6.27	450	46.34	-1.041	细胞核Nuclear
PvTrihelix23	Phvul.007G079700	79820.62	6.33	718	62.20	-0.797	叶绿体Chloroplast
PvTrihelix24	Phvul.007G079800	71489.66	5.64	635	58.93	-0.954	细胞核Nuclear
PvTrihelix25	Phvul.007G100500	38928.46	9.23	342	75.23	-1.049	细胞核Nuclear
PvTrihelix26	Phvul.007G104900	59282.82	5.90	517	51.79	-0.931	细胞核Nuclear
PvTrihelix27	Phvul.007G105700	52373.71	6.92	482	34.19	-0.380	细胞核Nuclear
PvTrihelix28	Phvul.007G186700	67151.82	6.87	587	58.76	-1.016	细胞核Nuclear
PvTrihelix29	Phvul.007G222800	69360.44	6.04	599	52.63	-1.152	细胞核Nuclear
PvTrihelix30	Phvul.007G224400	52633.09	6.40	482	37.37	-0.399	细胞核Nuclear
PvTrihelix31	Phvul.007G224600	54549.65	6.33	473	61.79	-1.219	细胞核Nuclear
PvTrihelix32	Phvul.008G019400	35835.89	5.31	325	49.25	-0.782	细胞核Nuclear
PvTrihelix33	Phvul.008G049300	35030.59	9.63	316	58.32	-0.838	细胞核Nuclear
PvTrihelix34	Phvul.008G098300	34431.83	6.97	285	57.52	-1.329	细胞核Nuclear
PvTrihelix35	Phvul.009G147500	58890.97	6.58	514	59.74	-0.993	细胞核Nuclear
PvTrihelix36	Phvul.009G167400	96180.81	8.59	871	36.28	-0.403	叶绿体Chloroplast
PvTrihelix37	Phvul.010G000200	32237.22	6.65	271	55.61	-1.204	细胞核Nuclear
PvTrihelix38	Phvul.010G018700	30096.12	7.03	260	51.29	-0.849	线粒体Mitochondria
PvTrihelix39	Phvul.010G126400	40737.45	9.66	375	50.80	-0.919	细胞核Nuclear
PvTrihelix40	Phvul.011G205900	38051.82	9.18	338	57.31	-0.976	细胞核Nuclear

基因 Gene	基因序列号 Gene ID	相对分子质量/Da Molecular weight	等电点 pI	氨基酸数 Amino acid	不稳定系数 Instability index	亲水性 Hydro- pathicity	亚细胞定位 Subcellular localization
PvTrihelix1	Phvul.001G077900	34942.98	4.86	310	58.84	-0.711	叶绿体，线粒体 Chloroplast，mitochondria
PvTrihelix2	Phvul.001G185100	51846.89	6.28	450	59.03	-1.102	细胞核Nuclear
PvTrihelix3	Phvul.001G187000	68096.08	6.32	592	54.91	-1.126	细胞核Nuclear
PvTrihelix4	Phvul.001G258700	43075.53	5.83	377	52.11	-0.775	细胞质Cytoplasm
PvTrihelix5	Phvul.002G126400	36335.79	8.66	309	66.06	-1.115	细胞核Nuclear
PvTrihelix6	Phvul.002G281800	40517.74	6.51	359	57.47	-0.764	细胞核Nuclear
PvTrihelix7	Phvul.003G007800	38333.55	8.22	340	64.78	-0.932	细胞核Nuclear
PvTrihelix8	Phvul.003G097500	33089.51	9.42	294	62.37	-0.938	细胞核Nuclear
PvTrihelix9	Phvul.003G209600	96885.44	8.76	870	40.28	-0.351	叶绿体Chloroplast
PvTrihelix10	Phvul.004G095200	67136.46	5.48	590	53.08	-1.094	细胞核Nuclear
PvTrihelix11	Phvul.004G095400	72575.65	5.40	656	65.95	-0.954	细胞核Nuclear
PvTrihelix12	Phvul.004G095500	50753.47	6.17	442	48.63	-1.054	细胞核Nuclear
PvTrihelix13	Phvul.004G161800	40924.74	4.60	354	50.82	-1.188	细胞核Nuclear
PvTrihelix14	Phvul.005G025700	38695.34	9.31	349	59.73	-0.952	细胞核Nuclear
PvTrihelix15	Phvul.005G095000	34005.92	9.69	300	63.97	-1.030	细胞核Nuclear
PvTrihelix16	Phvul.005G148400	50793.91	5.70	448	49.20	-0.910	细胞核Nuclear
PvTrihelix17	Phvul.005G171600	45586.93	4.81	400	47.87	-1.130	细胞核Nuclear
PvTrihelix18	Phvul.006G100300	64796.82	6.20	568	66.90	-1.068	细胞核Nuclear
PvTrihelix19	Phvul.006G139200	37809.28	6.06	329	66.93	-1.055	细胞核Nuclear
PvTrihelix20	Phvul.006G176000	34015.83	5.16	301	66.03	-0.937	细胞核，线粒体 Nuclear，mitochondria
PvTrihelix21	Phvul.007G000400	39649.55	6.94	356	62.13	-0.843	细胞核Nuclear
PvTrihelix22	Phvul.007G079600	51421.22	6.27	450	46.34	-1.041	细胞核Nuclear
PvTrihelix23	Phvul.007G079700	79820.62	6.33	718	62.20	-0.797	叶绿体Chloroplast
PvTrihelix24	Phvul.007G079800	71489.66	5.64	635	58.93	-0.954	细胞核Nuclear
PvTrihelix25	Phvul.007G100500	38928.46	9.23	342	75.23	-1.049	细胞核Nuclear
PvTrihelix26	Phvul.007G104900	59282.82	5.90	517	51.79	-0.931	细胞核Nuclear
PvTrihelix27	Phvul.007G105700	52373.71	6.92	482	34.19	-0.380	细胞核Nuclear
PvTrihelix28	Phvul.007G186700	67151.82	6.87	587	58.76	-1.016	细胞核Nuclear
PvTrihelix29	Phvul.007G222800	69360.44	6.04	599	52.63	-1.152	细胞核Nuclear
PvTrihelix30	Phvul.007G224400	52633.09	6.40	482	37.37	-0.399	细胞核Nuclear
PvTrihelix31	Phvul.007G224600	54549.65	6.33	473	61.79	-1.219	细胞核Nuclear
PvTrihelix32	Phvul.008G019400	35835.89	5.31	325	49.25	-0.782	细胞核Nuclear
PvTrihelix33	Phvul.008G049300	35030.59	9.63	316	58.32	-0.838	细胞核Nuclear
PvTrihelix34	Phvul.008G098300	34431.83	6.97	285	57.52	-1.329	细胞核Nuclear
PvTrihelix35	Phvul.009G147500	58890.97	6.58	514	59.74	-0.993	细胞核Nuclear
PvTrihelix36	Phvul.009G167400	96180.81	8.59	871	36.28	-0.403	叶绿体Chloroplast
PvTrihelix37	Phvul.010G000200	32237.22	6.65	271	55.61	-1.204	细胞核Nuclear
PvTrihelix38	Phvul.010G018700	30096.12	7.03	260	51.29	-0.849	线粒体Mitochondria
PvTrihelix39	Phvul.010G126400	40737.45	9.66	375	50.80	-0.919	细胞核Nuclear
PvTrihelix40	Phvul.011G205900	38051.82	9.18	338	57.31	-0.976	细胞核Nuclear