马尾松MAPK级联途径应答信号物质基因的表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2021-1089

摘要/Abstract

摘要：

MAPK（mitogen-actived protein kinase）在响应和调控生物与非生物胁迫、激素调节等方面发挥重要作用。马尾松MAPK级联途径7个基因被鉴定,并进行分子特性和外源信号物质处理表达模式分析。结果表明,马尾松与火炬松、北美云杉等针叶裸子植物的关系较近,不同类型基因具有该类基因特有的MAPK结构域以及典型催化氨基酸基序,D（I/L/V）K是马尾松MAPK基因共有的活性基序,PmMAP4K与MAPK级联途径的3个成员未直接互作,MAPK级联途径基因可能通过磷酸化调控各种激素生物合成来响应非生物胁迫。7个基因均响应了MeJA、GA、SA、ABA、Ca²⁺处理,但模式不同。PmMAP4K基因对Ca²⁺处理响应明显,其次是PmMAP2K基因,其它基因对单独Ca²⁺处理响应不明显,4种激素和Ca²⁺共同处理均提高了基因表达量,MeJA+Ca²⁺处理MAPK基因表达量高于单独MeJA处理,而GA、SA、ABA单独处理基因的表达量高于其与Ca²⁺一起处理。以上结果为马尾松MAPK基因与信号物质途径互作应答非生物胁迫提供参考。

关键词: 针叶树, MAPK级联途径, 激素代谢, 基因调控

Abstract:

MAPK（mitogen-actived protein kinase）cascade pathway plays an important role in response and regulation of biotic,abiotic stresses,and hormone signals et al. In this study,7 genes related to MAPK cascade pathway in Pinus massoniana were identified. We analyzed their physical properties,functional domains,phylogenetic tree,conserved phosphorylation motifs,and gene interactions. We also tested the expression patterns of these genes under hormone and calcium treatment. The results showed that：these genes had close evolutionary relationships with gymnosperms,such as Pinus taeda and Picea asperata. These 7 genes had MAPK domain and typical catalytic amino acid motif. D（I/L/V）K was the common active motif of MAPKs genes of Pinus massoniana. PmMAP4K did not interact directly with other three members of MAPK cascade pathway. Genes in MAPK cascade might regulate the biosynthesis of various hormones by phosphorylation in response to abiotic stress. The results of gene expression showed that all the seven genes responded to MeJA,GA,SA,ABA and Ca²⁺ treatments,but the expression patterns were different. The results showed that the response of PmMAP4K to Ca²⁺ treatment was the most obvious followed by PmMAP2K gene,and other genes had no obvious response to Ca²⁺ treatment only. Four hormones and hormone-plus-Ca²⁺ treatments increased the expression of genes,respectively. The expression of PmMAPK in MeJA+Ca²⁺ treatment was higher than that in MeJA treatment alone,while the expression of genes under treatments of each GA,SA,and ABA was higher than that under treatments of each GA,SA,ABA plus Ca²⁺. This study provides theoretical support for the analysis of the interaction between MAPK cascade pathway genes and signal pathway in respond to abiotic stress.

Key words: conifer, MAPK cascade, hormone metabolism, gene regulation

陈虎, 杨章旗, 孙爽, 李鹏, 徐慧兰. 马尾松MAPK级联途径应答信号物质基因的表达分析[J]. 生物技术通报, 2022, 38(6): 187-197.

CHEN Hu, YANG Zhang-qi, SUN Shuang, LI Peng, XU Hui-lan. Expressions and of Genes Response to Signal Substances in MAPK Cascade Pathway Genes in Pinus massoniana[J]. Biotechnology Bulletin, 2022, 38(6): 187-197.

图/表 10

表1 荧光定量所用基因引物

Table 1 Primer used in the qRT-PCR

Gene	Forward primer（5'-3'）	Reverse primer（5'-3'）
PmCYP	CAAGGGTTCGTCGTTCCAC	TACGGCGAGAAGTTTGCC
PmMAPK2	CCATGGCGGTCAGTATGTGCAG	CATATGGCATTGTCTGCTCGGCA
PmMAPK6	CCGCAGCAGCTCCAGTTCAATC	CTCAGTCACGGCGGCCTCTT
PmMAPK9	GATCCGCAATGAGAAGGCCAGAAG	CCTTCGTCTGCTTGAGCGTATGC
PmMAPK15	CCTCATCAGAGCAGGCATCTTCAG	GAGAAGCAGCACCAAGACAGGAG
PmMAP2K	GTTGGAGTACATGGACGGTGGAAC	GGTTGCCAGACAGGTGCTGAAG
PmMAP3K	AGCAGCAGTCCTCCAGCGATC	GAGGTCTTGATTGCCGCCAACAA
PmMAP4K	ATGCTTCTCTGCCTCCTCTTCTCC	GACGATTCCGAAGTTCCGCAACA

表2 MAPKs蛋白生物信息学分析

Table 2 Bioinformatics analysis of MAPKs protein in P. massoniana

基因 Gene	开放阅读框 Open reading frame	氨基酸序列Amino acid sequence/aa	蛋白分子量Protein mass/kD	等电点 Protein isoelectric point	亚细胞定位Subcellular localization	跨膜结构Transmembrane	磷酸化位点Kinase phosphorylation（Ser,Thr,Tyr）	二级结构Secondary structure/%
基因 Gene	开放阅读框 Open reading frame	氨基酸序列Amino acid sequence/aa	蛋白分子量Protein mass/kD	等电点 Protein isoelectric point	亚细胞定位Subcellular localization	跨膜结构Transmembrane	磷酸化位点Kinase phosphorylation（Ser,Thr,Tyr）	α螺旋 Alpha helix	延伸链 Extended strand	β转角 Beta turn	无规卷曲 Random coil
PmMAPK6	1 173	390	44.49	5.38	细胞核	无	8,10,8	42.31	13.59	4.36	39.74
PmMAPK9	1 839	612	69.41	9.11	细胞核	无	44,23,8	44.77	10.46	4.25	40.52
PmMAPK2	1 119	372	42.95	5.28	细胞核	无	11,11,9	43.82	14.25	5.91	36.02
PmMAPK15	1 731	576	65.7	9.22	细胞核	无	30,16,11	38.54	12.85	5.73	42.88
PmMAP2K	1 014	337	37.62	9.02	细胞核	无	15,4,3	34.42	16.02	5.64	43.92
PmMAP3K	1 962	653	72.33	5.86	细胞核	无	56,26,7	28.18	9.8	4.44	57.58
PmMAP4K	2 748	915	100.62	5.12	细胞核	无	77,41,19	29.4	12.13	4.92	53.55

表3 MAPKs蛋白功能结构域分析

Table 3 Analysis functional domain of MAPKs protein in P. massoniana

基因名/结构域Gene/Domain	MAPK	MAP2K	MAP3K	MAP4K	SPS1	PknB	BREX-PgiW
PmMAPK6	50-386				63-377	111-260
PmMAPK9	91-428				92-384	89-380
PmMAPK2	32-368				42-344	95-242
PmMAPK15	24-361				25-317	24-233	71-246
PmMAP2K		56-323			59-336	112-258	64-258
PmMAP3K			40-303		41-396	40-298
PmMAP4K				299-554	299-558	300-549

图1 马尾松与其他物种MAPK途径基因进化树分析

Fig.1 Phylogenetic relationship of P. massoniana and other plants MAPK genes

图2 马尾松MAPKs基因氨基酸序列比对

Fig. 2 Alignment of amino acid sequences of MAPKs in P. massoniana

图3 马尾松与拟南芥相关MAPKs基因互作网络分析 A：马尾松MAPK级联途径相关基因一级互作网络;B：马尾松MAPK级联途径相关基因二级互作网络;C：马尾松MAPK级联途径相关基因三级互作网络;D：PmMAP4K基因互作网络

Fig. 3 Interaction network analysis of MAPKs proteins identified in P. massoniana and Arabidopsis A：A first-order interaction network of genes related to MAPK cascade pathway in P. massoniana;B：secondary interaction network of genes related to MAPK cascade pathway in P. massoniana;C：a three-level interaction network of genes related to MAPK cascade pathway in P. massoniana;D：the PmMAP4K gene interaction network

图4 马尾松MAPK级联途径基因在MeJA处理下表达模式不同字母表示0.05水平上有显著差异,下同

Fig. 4 Expressions of MAPKs genes in response to MeJA treatments in P. massoniana Different letters indicate significant differences at the 0.05 level. The same below

图5 马尾松MAPK级联途径基因在GA处理下表达模式

Fig. 5 Expressions of MAPKs genes in response to GA treatments in P. massoniana

图6 马尾松MAPK级联途径基因在ABA处理下表达模式

Fig. 6 Expressions of MAPK genes in response to ABA treatments in P. massoniana

图7 马尾松MAPK级联途径基因在SA处理下表达模式

Fig. 7 Expressions of MAPKs genes in response to SA treatments in P. massoniana

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