Genome-wide Identification and Expression Analysis of Key Chlorophyll Synthesis Related Gene CaPOR in Coffea arabica

doi:10.13304/j.nykjdb.2024.0365

Abstract

Abstract:

Chlorophyll plays a pivotal role in the process of photosynthesis and the accumulation of organic substances in plants. Pro tochlorophyllide oxidoreductase （POR） is an enzyme that catalyzes the conversion of prochlorophyllide， the precursor of chlorophyll， into chlorophyll a in the chloroplast. It is a critical enzyme for the light-induced greening of angiosperms and is indispensable for photosynthesis. To explore the genes involved in chlorophyll synthesis in Coffea arabica leaves， the transcriptome sequencing analysis was used to identify the chlorophyll synthesis-related genes in C. arabica. Furthermore， the real-time fluorescence quantitative analysis was used to examine the expression of key genes. The results showed that CaPOR gene family members shared similar physicochemical properties and conserved motifs. The promoter regions were primarily composed of light response elements and growth and development elements. Through a combination of chromosome localization， collinearity analysis， and codon usage bias analysis， CaPOR gene had formed a gene cluster in C. arabica， and there was an acceleration of gene duplication， which might be driven by the base mutation and natural selection. The expression levels of 7 selected CaPOR genes were markedly higher in mature leaves than those in young leaves， which was positively correlated with the change in chlorophyll synthesis during the growth and development of C. arabica leaves. Above results provided reference for the chlorophyll synthesis in C. arabica leaves and the functional exploration of POR genes.

Key words: Coffea arabica, chlorophyll, bioinformatic analysis, expression analysis

摘要：

叶绿素是植物进行光合作用、积累有机物的重要物质，其中POR（pro tochlorophyllide oxidoreductase）负责催化光合体中的光合色素前体原叶绿素酸转化为叶绿素a，是光诱导被子植物转绿的关键酶，对光合作用至关重要。为研究小粒咖啡叶片叶绿素合成POR基因，通过基因家族分析和转录组测序分析，筛选和鉴定小粒咖啡POR基因，并进一步利用实时荧光定量分析关键基因的表达。结果表明， CaPOR基因家族成员蛋白质的理化性质相似，所含保守基序大致相同，在启动子区域主要以光响应元件和生长发育类元件为主。结合染色体定位、共线性分析以及密码子偏好分析发现，CaPOR基因在小粒咖啡中已形成基因簇，且出现复制加快的现象，这一现象是由碱基突变以及自然选择共同影响所致。对筛选出的7个CaPOR基因的表达量进行分析发现，在小粒咖啡叶片的生长发育过程中，7个CaPOR基因的表达量均为成熟叶片显著高于嫩叶，与小粒咖啡叶片生长发育过程中叶绿素合成变化正相关。以上研究结果为进一步揭示小粒咖啡叶片叶绿素合成以及POR基因的功能研究提供参考。

关键词: 小粒咖啡, 叶绿素, 生物信息学分析, 表达分析

CLC Number:

S571.2

Zhenwei ZHANG, Xiangshu DONG, Jing YANG, Xuejun LI, Meijun QI, Kuaile JIANG, Yonglin YANG, Butian WANG, Xuedong SHI, Junchao QIU, Zhihua CHEN, Yu GE. Genome-wide Identification and Expression Analysis of Key Chlorophyll Synthesis Related Gene CaPOR in Coffea arabica[J]. Journal of Agricultural Science and Technology, 2024, 26(10): 83-97.

张振伟, 董相书, 杨婧, 李学俊, 杞美军, 蒋快乐, 杨永林, 王步天, 施学东, 邱俊超, 陈治华, 葛宇. 小粒咖啡叶绿素合成基因CaPOR的全基因组鉴定及表达分析[J]. 中国农业科技导报, 2024, 26(10): 83-97.

Figures/Tables 17

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基因名称 Gene name	正向引物序列 Forward primer sequence （5’-3’）	反向引物序列 Revers primer sequence （5’-3’）
24S	GCAGACAAGGCGGTGACTAT	CGGATCTTCTTGGCCCTGTT
CaPOR5	GAAGTCACGCAGGGGATTGA	TCGAATGTGGTCCCCATGAC
CaPOR6	TTAATTCGTCTGGCCGTCCC	GCTTTAACCGTCTGGCAAGC
CaPOR7	GAAGTCACGCAGGGGATTGA	TCGAATGTGGTCCCCATGAC
CaPOR8	TTAATTCGTCTGGCCGTCCC	GCTTTAACCGTCTGGCAAGC
CaPOR19	GGCAGTGCGAAATGTGGATG	CGGAAAGGGCTGTAGCTTGA
CaPOR20	GGCAGTGCGAAATGTGGATG	GCACGGAAAGGGCTTGATTG
CaPOR21	TCAAGCTACAGCCCTTTCCG	CAGCACCCTGTGGAATGGAT

基因名称 Gene name	正向引物序列 Forward primer sequence （5’-3’）	反向引物序列 Revers primer sequence （5’-3’）
24S	GCAGACAAGGCGGTGACTAT	CGGATCTTCTTGGCCCTGTT
CaPOR5	GAAGTCACGCAGGGGATTGA	TCGAATGTGGTCCCCATGAC
CaPOR6	TTAATTCGTCTGGCCGTCCC	GCTTTAACCGTCTGGCAAGC
CaPOR7	GAAGTCACGCAGGGGATTGA	TCGAATGTGGTCCCCATGAC
CaPOR8	TTAATTCGTCTGGCCGTCCC	GCTTTAACCGTCTGGCAAGC
CaPOR19	GGCAGTGCGAAATGTGGATG	CGGAAAGGGCTGTAGCTTGA
CaPOR20	GGCAGTGCGAAATGTGGATG	GCACGGAAAGGGCTTGATTG
CaPOR21	TCAAGCTACAGCCCTTTCCG	CAGCACCCTGTGGAATGGAT

基因名称 Gene name	蛋白序列ID Protein sequence ID	氨基酸数量 Number of amino acid/aa	相对分子量 Molecular weight/Da	理论等电点 Theoretical pI	不稳定系数 Instability index	脂肪系数 Aliphatic index	总平均亲水性 Grand average of hydropathicity
CaACSF1	XP_027071950.1	428	49 460.89	9.07	40.60	75.98	-0.340
CaACSF2	XP_027071221.1	427	49 387.84	8.91	38.47	76.39	-0.332
CaCAO1	XP_027093472.1	535	60 461.69	8.48	41.80	85.07	-0.279
CaCAO2	XP_027091395.1	535	60 451.67	8.14	42.68	85.25	-0.267

基因名称 Gene name	蛋白序列ID Protein sequence ID	氨基酸数量 Number of amino acid/aa	相对分子量 Molecular weight/Da	理论等电点 Theoretical pI	不稳定系数 Instability index	脂肪系数 Aliphatic index	总平均亲水性 Grand average of hydropathicity
CaACSF1	XP_027071950.1	428	49 460.89	9.07	40.60	75.98	-0.340
CaACSF2	XP_027071221.1	427	49 387.84	8.91	38.47	76.39	-0.332
CaCAO1	XP_027093472.1	535	60 461.69	8.48	41.80	85.07	-0.279
CaCAO2	XP_027091395.1	535	60 451.67	8.14	42.68	85.25	-0.267

基因名称 Gene name	蛋白序列ID Protein sequence ID	氨基酸数量 Number of amino acid/aa	相对分子量 Molecular weight/Da	理论等电点 Theoretical pI	不稳定系数 Instability index	脂肪系数 Aliphatic index	总平均亲水性 Grand average of hydropathicity
CaCHLD1	XP_027123386.1	763	83 861.49	6.18	46.55	88.64	-0.324
CaCHLD2	XP_027079730.1	688	75 126.52	5.47	41.55	91.06	-0.281
CaCHLG1	XP_027068575.1	380	40 758.37	7.58	22.00	111.16	0.386
CaCHLG2	XP_027072610.1	380	40 774.41	7.58	22.50	111.42	0.386
CaCHLH1	XP_027069536.1	1 382	153 535.80	5.74	39.12	88.81	-0.271
CaCHLH2	XP_027069535.1	1 382	153 367.80	5.85	37.72	89.44	-0.258
CaCHLH3	XP_027066034.1	1 155	127 594.20	6.53	38.64	92.68	-0.180
CaCHLI1	XP_027078841.1	429	46 808.50	6.10	40.88	96.99	-0.197
CaCHLI2	XP_027075032.1	429	46 886.54	6.10	42.42	96.55	-0.198
CaCHLM1	XP_027123226.1	276	29 896.53	8.27	40.56	95.11	-0.106
CaCHLM2	XP_027087377.1	321	34 625.96	8.57	38.44	90.90	-0.058
CaDVR1	XP_027116070.1	366	40 327.44	5.26	24.61	93.20	-0.061
CaDVR2	XP_027119467.1	415	45 457.33	6.21	25.54	89.98	-0.061
CaDVR3	XP_027119468.1	415	45 562.42	7.52	26.73	88.82	-0.092
CaDVR4	XP_027116071.1	366	40 320.50	5.74	24.87	92.16	-0.067
CaGSA1	XP_027127512.1	477	50 838.38	5.91	33.78	81.82	0.020
CaGSA2	XP_027087467.1	477	50 854.42	5.83	32.56	83.04	0.032
CaGSA3	XP_027098002.1	479	52 160.00	8.74	31.67	90.19	-0.096
CaGSA4	XP_027121611.1	479	52 206.09	8.70	30.44	90.19	-0.090
CaGSA5	XP_027126106.1	441	48 043.70	8.26	25.68	90.00	-0.008
CaGSA6	XP_027091382.1	484	52 975.94	8.27	32.52	88.04	-0.093
CaGSA7	XP_027094060.1	523	57 122.38	6.84	50.47	87.00	-0.115
CaGSA8	XP_027077773.1	475	52 482.79	8.13	37.84	91.20	-0.136
CaGSA9	XP_027074140.1	475	52 471.77	8.44	38.41	90.17	-0.152
CaGSA10	XP_027126874.1	468	51 593.81	8.31	38.51	91.30	-0.129
CaGSA11	XP_027090984.1	430	46 240.96	7.18	37.03	91.91	0.036
CaGSA12	XP_027087761.1	457	49 018.23	6.57	38.55	96.50	0.039
CaGSA13	XP_027087466.1	461	49 307.69	6.09	37.98	100.09	0.133
CaHEMA1	XP_027112743.1	571	61 845.26	8.44	42.32	95.36	-0.058
CaHEMA2	XP_027106141.1	571	61 889.21	8.44	42.85	94.85	-0.077
CaHEMA3	XP_027089895.1	556	61 269.68	9.06	38.49	90.27	-0.163
CaHEMA4	XP_027094467.1	555	61 104.53	9.20	38.37	90.43	-0.178
CaHEMB1	XP_027081050.1	429	46 855.52	6.36	50.45	86.67	-0.240
CaHEMB2	XP_027084484.1	429	46 697.38	6.36	50.45	85.78	-0.239
CaHEMC1	XP_027061577.1	382	41 061.84	6.76	34.87	90.73	-0.134
CaHEMC2	XP_027064519.1	383	41 150.93	6.80	33.88	91.51	-0.127
CaHEMD1	XP_027089872.1	299	31 787.38	8.50	51.65	94.95	0.040