生物技术通报 ›› 2023, Vol. 39 ›› Issue (4): 246-258.doi: 10.13560/j.cnki.biotech.bull.1985.2022-1045
王艺清1,2(), 王涛1,2, 韦朝领3, 戴浩民1,2, 曹士先4, 孙威江1,2(), 曾雯1,2()
收稿日期:
2022-08-23
出版日期:
2023-04-26
发布日期:
2023-05-16
通讯作者:
孙威江,男,博士,教授,研究方向:茶树种质资源创新与品质化学;E-mail: swj8103@126.com;作者简介:
王艺清,女,硕士研究生,研究方向:茶树分子生物学;E-mail: 784726205@qq.com
基金资助:
WANG Yi-qing1,2(), WANG Tao1,2, WEI Chao-ling3, DAI Hao-min1,2, CAO Shi-xian4, SUN Wei-jiang1,2(), ZENG Wen1,2()
Received:
2022-08-23
Published:
2023-04-26
Online:
2023-05-16
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。
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.
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 |
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 |
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 |
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
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 |
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
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