Whole-genome Identification and Bioinformatics of PnPAL Family in Black Peppers
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摘要:
目的 胡椒瘟病是影响胡椒产量的第一大病害，胡椒苯丙氨酸解氨酶（PnPAL）基因是胡椒抗瘟病的关键基因。探明胡椒PnPAL基因家族的基本特征、进化关系及表达模式，以便于其功能研究。 方法 通过全基因组查找、鉴定得到14条胡椒PnPAL家族成员，分别命名为PnPAL1～PnPAL14，并对这14条序列进行生物信息学及表达模式分析。 结果 蛋白质基本信息分析显示，该家族理论等电点在5.76～9.77，分子量在7.374 41～83.431 07 kDa。有7条胡椒的PnPAL基因含有8个motif和PLN02457保守结构域，该7条胡椒PnPAL家族成员被进一步鉴定为胡椒PnPAL家族成员。胡椒PnPAL家族成员含有诸多胁迫响应元件，特别是水杨酸、茉莉酸甲酯等与抵抗病原入侵的顺式作用元件。胡椒的PnPAL基因位于系统发生树基部位置，表明胡椒的PnPAL基因较为古老，与胡椒的系统地位相似。表达模式分析显示，PnPAL10在受病原诱导后基本呈上调趋势，表明PnPAL10可能是胡椒抗瘟病的关键基因。 结论 对胡椒PnPAL基因家族开展了生物信息学及表达模式分析，证明其在抗瘟病中的潜在作用，筛选到关键家族成员PnPAL3。 -
关键词:
- 胡椒 /
- 苯丙氨酸解氨酶(PAL) /
- 生物信息
Abstract:Objective Basic characteristics and evolutional relationship on the phenylpropane metabolic pathway associated with the pepper blast disease that ill-affects the cultivation and production of the globally important spice, black peppers, were studied. Method The phenylpropane metabolic pathway was believed to be key to the blast resistance of black peppers, and phenylalanine ammonialyase (PAL) to be the crucial, rate-limiting enzyme in the pathway. Hence, based on the transcriptomics of a resistant black pepper germplasm the sequences of 14 PALs were identified and named PnPAL1-PnPAL14. Their bioinformatics and expression patterns were analyzed. Result The gene family had a theoretical isoelectric point between pH 5.76 and 9.77 with a molecular weight between 7.37441 kDa and 83.43107 kDa. Seven of the identified PnPALs contained 8 motifs and PLN02457 conserved domains. They belonged to the PnPAL family that consisted of many stress response elements, especially salicylic acid, methyl jasmonate, and other cis-acting elements, which are known to resist the pathogenic invasion. The PnPALs were located at the base of the phylogenetic tree, being relatively old and holding a status like black peppers. PnPAL10 expressed in an up-regulation trend after a pathogenic induction suggesting a close relation of the gene to the blast resistance of the plant. Conclusion A critical role the PnPAL family played in the blast resistance of black peppers was positively identified. -
Key words:
- black pepper /
- phenylalanine ammonialyase /
- biological information
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图 2 胡椒PnPAL motif分析
注：A为胡椒PnPAL进化树，B为motif分析。
Figure 2. PnPAL motifs of black peppers
Note: A: PnPAL phylogenetic tree; B: motif analysis.
图 3 胡椒PnPAL domain分析
注：A为胡椒PnPAL进化树，B为domain分析。
Figure 3. PnPAL domain of black peppers
Note: A: PnPAL phylogenetic tree; B: domain analysis.
图 6 胡椒PnPAL3
基因相对表达分析 注：0 h、8 h、12 h、24 h、48 h表示接种病原菌不同时间点的表达模式；0 h-C、8 h-C、12 h-C、24 h-C、48 h-C 表示接种无菌水不同时间点的表达模式。图中不同小写字母表示接种病原、无菌水基因表达量的显著性水平（P＜0.05）；图7同此。
Figure 6. Relative expressions of PnPAL3 in black peppers
Note: 0 h, 8 h, 12 h, 24 h, and 48 h are expressions at respective time after pathogenic inoculation; 0 h-C, 8 h-C, 12 h-C, 24 h-C, and 48 h-C, expressions at respective time after sterile water inoculation. Data with different lowercase letters indicate significant difference in expressions between inoculation with pathogens and sterile water (P＜0.05). The same as Fig.7.
图 7 胡椒PnPAL10
基因相对表达分析 Figure 7. Analysis on relative expressions of PnPAL10 in black pepper
表 1 实时荧光定量PCR分析所用引物
Table 1. Primers used in RT-PCR analysis
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′)PnPAL1 GCAAAGGCCGTGACTTTCG TTCATCACCTCGCAGAAAACG PnPAL2 CGCGGCCAGCTTGAAGAG GCGAGGATGTTAGCGTCGTAGA PnPAL3 CCGACCACTTGACCCATAAGC GGTCCATCTCATGTGTCATTTGC PnPAL4 GCTAGGCCCACTCATAGAAGTGA AGTGTTGTCCATGGACACACCA PnPAL10 CAAAAGCTGAGAAATGTGCTGGT CCGACTTCCCGTTCTCATACTC PnPAL13 ACAACCAGGACGTCAACTCACTG ACGATGTGCTTGACCACCTCTC PnPAL14 GGCACAACTCCAAGGTTACGA GTTGGCGTCGTAGCAAACG 内参序列 GTTGGAGATAGGAGCAAGCAGA TCAAAGACCACCCCATCACCT 
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表 2 胡椒PnPAL基因家族成员的理化性质
Table 2. Physiochemical properties of PnPAL family in black peppers
名称
Gene name基因编号
Sequence
ID染色体定位
Chromosome
nambur染色体位置
Chromosome
location编码氨基酸个数
AA蛋白质分子量
Mw /kDa等电点
pI亚细胞定位
Subcellular
localizationPnPAL1 Pn2.1119 2 14526770-14528898- 652 70.242 67 5.84 细胞质 Cytoplasm PnPAL2 Pn2.1269 2 15529503-15531650+ 715 76.798 79 5.76 细胞质 Cytoplasm PnPAL3 Pn2.1585 2 18068679-18071787+ 711 77.464 65 5.93 细胞质 Cytoplasm PnPAL4 Pn2.1713 2 19171638-19174695+ 711 77.495 66 5.87 细胞质 Cytoplasm PnPAL5 Pn3.4768 3 37507112-37507766- 191 21.420 58 9.77 细胞核 Cytoplasm PnPAL6 Pn5.1823 5 20214336-20215764- 312 34.470 96 7.75 叶绿体 Chloroplast PnPAL7 Pn6.2505 6 23152346-23152648- 100 10.668 38 8.93 细胞核 Nucleus PnPAL8 Pn6.2507 6 23155823-23156041- 72 7.374 41 8.93 细胞核 Nucleus PnPAL9 Pn6.2506 6 23155093-23155674- 193 20.389 72 7.72 细胞核 Nucleus PnPAL10 Pn8.2617 8 29114372-29118271- 730 79.494 98 6.04 细胞质 Cytoplasm PnPAL11 Pn9.116 9 18656999-18659720- 147 16.374 80 7.77 叶绿体 Chloroplast PnPAL12 Pn18.280 18 240046-240597- 507 57.052 64 8.05 内膜系统 Endometrial system PnPAL13 Pn28.318 28 2239516-2241801- 761 83.431 07 6.08 质膜 Plasma membrane PnPAL14 Pn38.78 38 621136-623422+ 736 80.379 55 6.08 质膜 Plasma membrane
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