茶树SMAS基因家族的鉴定及互作分析

doi:10.13560/j.cnki.biotech.bull.1985.2022-1045

摘要/Abstract

摘要：

S-腺苷甲硫氨酸合成酶（S-adenosylmethionine synthase, SAMS）是催化甲硫氨酸和ATP合成S-腺苷甲硫氨酸（SAM）的唯一酶，研究表明SAMS参与木质素的生物合成。本研究对‘黄棪’茶树（Camellia sinensis）中鉴定的SAMS基因家族，进行表达模式及蛋白互作网络分析，挖掘可能参与木质素合成的 CsSAMS候选基因。以‘黄棪’茶树基因组为参考基因组，通过生物信息学鉴定CsSAMS基因家族成员，并分析其蛋白理化性质、系统进化树、染色体定位、基因结构、蛋白结构、表达模式，通过酵母双杂交技术（Y2H）研究其蛋白互作网络，通过紫外分光光度计方法对‘黄棪’‘铁观音’‘金观音’‘福鼎大毫茶’一芽二叶部位的木质素含量进行测定。生物信息学分析结果表明，‘黄棪’茶树中共鉴定到4个CsSAMS家族成员，其编码氨基酸个数为345-519，等电点为6.12-6.47。亚细胞定位预测结果表明，CsSAMS1定位于叶绿体，CsSAMS2、CsSAMS3定位于细胞质，CsSAMS4定位于细胞骨架。通过对不同茶树品种的CsSAMS表达量和木质素含量检测发现，CsSAMS2、CsSAMS3、CsSAMS4可能潜在调控木质素的含量，另外酵母双杂交结果表明，CsSAMS4可以与自身形成同源二聚体。本研究鉴定并分析了4个CsSAMS成员的理化性质并预测其功能，明确了不同组织部位和氮、氟处理下CsSAMS基因的表达模式，以及CsSAMS对木质素合成过程的潜在参与作用。

关键词: 茶树, SAMS, 生物信息学分析, 木质素, 表达分析, 互作蛋白

Abstract:

S-adenosylmethionine synthase（SAMS）is the only enzyme that catalyzes the synthesis of S-adenosylmethionine（SAM）from methionine and ATP. Research shows that SAMS participates in lignin biosynthesis. In this study, we analyzed the expression patterns and protein interaction networks of the SAMS gene family identified from ‘Huangdan’ tea plant（Camellia sinensis）to explore candidate genes of CsSAMS that may be involved in lignin synthesis. Then we identified the members of CsSAMS gene family by bioinformatics based on the genome of ‘Huangdan’, and further analyzed their protein physicochemical properties, phylogenetic tree, chromosome location, gene structure, protein structure and expression pattern. We also studied the protein interaction network by yeast two-hybrid technology（Y2H）. Finally, we determined the lignin contents of the first bud and the two leaves of ‘Haungdan’, ‘Tieguanyin’, ‘Jinguanyin’ and ‘Fuding Dahao’ by UV spectrophotometer. The results of bioinformatics analysis showed that 4 CsSAMS gene family members were identified in ‘Huangdan’. The number of encoded amino acids was 345-519, and the isoelectric point ranged from 6.12 to 6.47. The prediction results of subcellular localization revealed that CsSAMS1 was located in the chloroplast, CsSAMS2 and CsSAMS3 were located in the cytoplasm, and CsSAMS4 was located in the cytoskeleton. Expression of CsSAMS and lignin content detection of different tea varieties indicated that CsSAMS2, CsSAMS3 and CsSAMS4 might potentially regulate lignin content. In addition, Y2H results showed that CsSAMS4 formed homodimers with itself. In this study, the physicochemical properties of four CsSAMS members are identified and analyzed, and their functions are predicted. The expression patterns of CsSAMS genes in different tissue sites, under nitrogen and fluoride treatments, as well as the potential involvement of CsSAMS in lignin synthesis process, are clarified.

Key words: Camellia sinensis, SAMS, bioinformatics analysis, lignin, expression analysis, interacting proteins

王艺清, 王涛, 韦朝领, 戴浩民, 曹士先, 孙威江, 曾雯. 茶树SMAS基因家族的鉴定及互作分析[J]. 生物技术通报, 2023, 39(4): 246-258.

WANG Yi-qing, WANG Tao, WEI Chao-ling, DAI Hao-min, CAO Shi-xian, SUN Wei-jiang, ZENG Wen. Identification and Interaction Analysis of SMAS Gene Family in Tea Plant（Camellia sinensis）[J]. Biotechnology Bulletin, 2023, 39(4): 246-258.

图/表 15

表1 引物序列

Table 1 Primer sequences

基因名称Gene name	引物序列Primer sequence（5'-3'）	备注Note
SAMS1	F CATGCCCAAGTGTAACAATAG R AGAGACGGAGAGAGAGAAGA	荧光定量 Quantitative real-time PCR
SAMS2	F CCACTGATGAAACACCTGAAC R GTATGAACTCTAATGGGGACC
SAMS3	F CACGGGGTTCTTTCTCTCTGTCT R GGTGTCCCTCATTCACTGATTCA
SAMS4	F AGCCCGTCATCCCTTCCCAATA R CTCCACCGTGAGCACCCCAACC
BD- SAMS2	F CATGGAGGCCGAATTCATGGATAGTTTCCTC R GCAGGTCGACGGATCCTCAAGCTTTGGGCT	酵母双杂交 Yeast two-hybrid
BD- SAMS4	F CATGGAGGCCGAATTCATGGAAACCTTCCTCT R GCAGGTCGACGGATCCTCAAGCTTTGGGCT
AD- SAMS2	F CATCGATACGGGATCATGGATAGTTTCCTCTT R ACGATTCATCTGCAGCTCAAGCTTTGGGCTTAA
AD- SAMS4	F CATCGATACGGGATCATGGAAACCTTCCTCT R ACGATTCATCTGCAGCTCAAGCTTTGGGCTTG

图1 CsSAMS家族基因的染色体分布

Fig. 1 Chromosomal distribution of CsSAMS family genes

表2 CsSAMS蛋白的基本理化性质

Table 2 Basic physicochemical properties of CsSAMS proteins

基因 Gene	序列号 Gene ID	蛋白长度 Amino acid/aa	分子量 Molecular weight/kD	等电点 pI	不稳定系数 Instability constant	亲水性 GRAVY	亚细胞定位 Subcellular localization	跨膜结构 Transmembrane domain	信号肽 Signal peptide
CsSAMS1	HD.05G0021870	519	56.98	6.47	28.69	-0.245	叶绿体Chloroplast	无 No	无 No
CsSAMS2	HD.06G0008900	390	42.75	6.24	25.34	-0.331	细胞质Cytoplasm	无 No	无 No
CsSAMS3	HD.08G0000160	345	37.79	6.42	18.7	-0.281	细胞质Cytoplasm	无 No	无 No
CsSAMS4	HD.11G0014340	390	42.68	6.12	27.59	-0.294	细胞骨架Cytoskeleton	无 No	无 No

图2 CsSAMS进化树（A）、保守基序（B）、内含子和外显子结构分布（C）

Fig. 2 Phylogenetic tree（A）, conserved motifs（B）, intron and exon structure distribution（C）of CsSAMS

图3 CsSAMS系统进化树分析

Fig. 3 Phylogene tree of CsSAMS CsSAM1-4：茶树 Camellia sinensis；AtMAT1（At1g02500）、AtMAT2（At4g01850）、AtMAT3（At2g36880）AtMAT4（At3g17390）：拟南芥 Arabidopsis thaliana；SlSAMS1（NP.001234425）、SlSAMS2（NP.001296305）、SlSAMS3（NP.001234004）、SlSAMS4（XP.010312254）：番茄 Solanum lycopersicum；ZmSAMS1（NP.001130734）、ZmSAMS2（NP.001146249）、ZmSAMS3（NP.001148708）、ZmSAMS4（NP.001132867）：玉米 Zea mays；MtSAMS1（XP.003626035）、MtSAMS2（XP.003609861）、MtSAMS3a（XP.003625682）、MtSAMS3b（XP.013468134）、MtSAMS4（XP.013463713）：蒺藜苜蓿 Medicago truncatula；TuSAMS（XP.020178978）、TuSAMS1（EMS55466）、TuSAMS4（EMS52834）：乌拉尔图小麦 Triticum urartu；OsSAMS1（NP.001389410）、OsSAMS2（NP.001393223）、OsSAMS3（XP.015614349）：水稻 Oryza sativa；DcSAMS（AAA33274）：康乃馨 Dianthus caryophyllus；LcSAMS（AAP13994）：荔枝 Litchi chinensis；PcSAMS（AAG17036）：扭叶松 Pinus contorta；RpSAMS（AIT39705）：刺槐 Robinia pseudoacacia；LrSAMS（AFC88125）：石蒜 Lycoris radiata；BjSAMS（AAK71234）：芥菜 Brassica juncea；CcSAMS（KYP71740）：木豆 Cajanus cajan

图4 CsSAMS种内共线性分析

Fig. 4 Intraspecies collinearity analysis of CsSAMS

表3 CsSAMS同源基因的Ka/Ks分析

Table 3 Ka/Ks analysis of CsSAMS homologous genes

基因对名称 Gene pair name	非同义突变频率Nonsynonymous mutation Ka	同义突变频率Synonymous mutation Ks	Ka/Ks
CsSAMS1/CsSAMS3	0.033 115 095	1.036 155 399	0.031 959 584
CsSAMS2/CsSAMS4	0.022 137 640	0.526 911 233	0.042 013 983

图5 ‘黄棪’‘铁观音’CsSAMS共线性分析

Fig. 5 Collinearity analysis of CsSAMS within species of HD and TGY

图6 CsSAMS启动子顺式作用元件预测 I：激素响应；Ⅱ：非生物胁迫响应；Ⅲ：光响应；Ⅳ：转录因子识别、结合位点；Ⅴ：组织特异性元件；Ⅵ：生长发育相关元件；Ⅶ：核心元件

Fig. 6 Analysis of cis-elements in the upstream region of CsSAMS family genes in tea plants I: Phytohormone response. Ⅱ: Abiotic stress response. Ⅲ: Light response. Ⅳ: TF recongnition and binding site. Ⅴ: Tissue specificity. Ⅵ: Plant growth. Ⅶ: Core

图7 CsSAMS蛋白二级结构、三级结构预测

Fig. 7 Secondary and tertiary structure prediction of CsSAMS

图8 CsSAMS基因表达模式 A：CsSAMS 在不同组织部位中的表达模式；B：CsSAMS 在低氮处理下的表达模式；C：CsSAMS 在高氮处理下的表达模式；D：CsSAMS 在氟处理下的表达模式

Fig. 8 Expression patterns of CsSAMS A: Expression pattern of CsSAMS in different tissue. B: Expression pattern of CsSAMS under low nitrogen treatment. C: Expression pattern of CsSAMS under high nitrogen treatment. D: Expression pattern of CsSAMS under fluorine treatment

图9 不同茶树品种木质素含量大写字母表示在P<0.01水平差异显著

Fig. 9 Lignin content of different tea varieties Capital letters indicate significance at the P<0.01 level

图10 不同茶树品种CsSAMS表达模式

Fig. 10 Expression pattern of CsSAMS gene in different tea varieties

图11 不同茶树品种木质素含量和CsSAMS表达量相关性分析 *表示在0.05水平上显著相关，**表示在0.01水平上显著相关

Fig. 11 Correlation analysis between the lignin content and CsSAMS genes expression in different tea plant varieties * indicates significant correlation at the 0.05 level, and ** indicates significant correlation at the 0.01 level

图12 SAMS酵母双杂交结果

Fig. 12 Results of Y2H for SAMS

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