马铃薯SAT基因家族的鉴定和表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0133

摘要/Abstract

摘要：

【目的】丝氨酸乙酰转移酶（SAT）是硫同化为半胱氨酸（cysteine, Cys）的关键酶，参与植物多种生物过程，特别是在植物响应非生物胁迫中发挥着重要作用。但关于马铃薯SAT基因家族（StSAT）的分析尚未见报道。系统鉴定了马铃薯SAT基因家族，为深入了解StSAT基因家族的特征，进一步分析它们在马铃薯抵御非生物胁迫中的功能提供了理论依据。【方法】利用HMM 对马铃薯SAT基因家族进行鉴定，并对其染色体分布、基因结构、蛋白保守基序及物种间的共线性进行分析。利用PGSC下载的RNA-seq数据分析双单倍体（doubled-monoploid, DM）马铃薯中StSATs在不同组织部位、非生物胁迫和外源激素处理下的表达模式。通过qPCR（quantitative real-time PCR）分析四倍体马铃薯中StSATs在NaCl和PEG处理（0、1、3和24 h）下的相对表达水平。【结果】在马铃薯中鉴定出4个StSATs，它们分布在4条染色体上。根据系统发育特征，将4个StSATs分在3个亚族中。共线性分析发现，StSATs与拟南芥（Arabidopsis thaliana）、番茄（Solanum lycopersicum）、甘蓝（Brassica oleracea）、水稻（Oryza sativa）和玉米（Zea mays）中分别有4对、4对、2对、1对和1对直系同源基因。通过表达分析发现，四倍体马铃薯中4个StSATs随着NaCl和PEG处理时间的延长，其表达量显著升高（与0 h相比），很可能参与马铃薯对盐和渗透胁迫的响应。【结论】StSAT基因家族成员在马铃薯响应盐和渗透胁迫中发挥着重要作用。

关键词: 马铃薯, SAT基因家族, 顺式作用元件, 非生物胁迫, 表达分析

Abstract:

【Objective】 Serine acetyltransferase（SAT）is a critical enzyme in assimilating sulfur into cysteine（Cys）, participating in various biological processes in plants, especially in plant response to abiotic stress. However, analyses on the potato SAT gene family（StSAT）has not yet be reported. This study systematically and comprehensively identified the potato SAT gene family, providing a theoretical basis for further understanding the characteristics of the StSAT gene family and further analysis of their functions in potato resistance to abiotic stress. 【Method】HMM was used to identify the StSAT gene family in potatoes and analyze their chromosomal distribution, gene structure, protein-conserved motifs, and collinearity across species. Additionally, RNA-seq data downloaded from PGSC were used to analyze the expression patterns of StSATs in different tissue parts of doubled-monoploid（DM）potatoes under abiotic stress and exogenous hormone treatments. qPCR（quantitative real-time PCR）was to analyze the relative expressions of StSATs in tetraploid potatoes under NaCl and PEG treatment（0, 1, 3, and 24 h）【Result】Four StSATs were identified in potatoes, and they were distributed across four chromosomes. Based on phylogenetic characteristics, these four StSATs were classified into three subfamilies. According to synteny analysis, there were 4 orthologous SAT genes to Arabidopsis, 4 to tomato（Solanum lycopersicum）, 2 to cabbage（Brassica oleracea）, 1 to rice（Oryza sativa）, and 1 to corn（Zea mays）. Expression analysis showed that the expressions of all four StSATs significantly increased over time under NaCl and PEG treatments compared to 0 h, indicating their likely involvement in the potato's response to salt and osmotic stress.【Conclusion】 StSAT gene family members play an essential role in potato responses to salt and osmotic stress.

Key words: potato, SAT genes family, cis-acting element, abiotic stress, expression analysis

申鹏, 高雅彬, 丁红. 马铃薯SAT基因家族的鉴定和表达分析[J]. 生物技术通报, 2024, 40(9): 64-73.

SHEN Peng, GAO Ya-Bin, DING Hong. Identification and Expression Analysis of SAT Gene Family in Potato（Solanum tuberosum L.）[J]. Biotechnology Bulletin, 2024, 40(9): 64-73.

图/表 9

表1 qPCR引物信息

Table 1 Primer information for qPCR

基因ID Gene ID	正向引物Forward primer（5'-3'）	反向引物Reverse primer（5'-3'）
Soltu06G005680	GGTCGTGTTGAGACTGGTGTGATC	GCTTCGTGGTGCATCTCTACAGAC
Soltu02G022850	CTGTCGCTAACACCGCTTCTACTC	GCTCAATGCCCGTTCCAAACAATC
Soltu12G025770	GACACGGCGGAAGAAGCTATCTG	AGTGAGCGTTCAAGCGAAGAGTG
Soltu07G027060	CCAAACAAGCCCCAAATCGACAAC	GCAGGAAGCAAGGTCAGGGAAAG
Soltu10G001180	TGGTGCTGGTACTTGTGTTCTTGG	AGCAGTAGTTCTTGCAGGCACTTC

表2 StSATs的染色体定位及理化性质

Table 2 Chromosomal localization and physicochemical properties of StSATs

基因ID Gene ID	染色体定位 Chromosome localization		氨基酸长度 Amino acid length	相对分子量 Molecular weight/ kD	等电点 Point isoelectric（pI）
Soltu.DM.02G022850	chr02	36 554 173-36 560 967	349	37.43	6.41
Soltu.DM.07G027060	chr07	56 287 590-56 289 231	359	39.04	8.80
Soltu.DM.10G001180	chr10	978 595-980 158	365	40.23	6.50
Soltu.DM.12G025770	chr12	55 741 243-55 744 365	293	31.27	6.38

图1 多个物种中SAT蛋白的系统发育进化树以及StSAT基因家族成员的进化关系、基因结构和蛋白保守基序分析 A：多个物种中SAT蛋白的系统发育进化树，不同颜色的线代表3个亚族（蓝色代表C1亚族，红色代表C2亚族，绿色代表C3亚族），蓝色五角星表示马铃薯SAT家族成员（StSATs），橙色五角星表示番茄SAT家族成员（SlSATs），黄色圆圈表示玉米SAT家族成员ZmSATs，紫色圆圈表示水稻SAT家族成员（OsSATs），红色方块表示拟南芥SAT家族成员（AtSTAs），绿色方块表示甘蓝SAT家族成员（BoSATs）；B：StSAT基因家族的进化树，蓝色方块代表C1亚族，红色方块代表C2亚族，绿色方块代表C3亚族；C：StSATs的基因结构，蓝色框表示外显子，黑线表示内含子，红色方框表示上/下游区域；D：StSATs蛋白保守基序

Fig. 1 Phylogenetic tree of SAT proteins in multiple species and analysis in evolutionary relationship, gene structure and protein conserved motifs of StSAT gene family members A: Phylogenetic tree of SAT proteins in multiple species. The different colored lines represent the three subfamilies（blue for C1, red for C2, and green for C3）. The blue pentagrams indicate the potato SAT gene family member（StSATs）, orange pentagrams indicate the tomato SAT gene family member（SlSATs）, yellow circles indicate the corn SAT gene family member（ZmSATs）, purple circles indicate the rice SAT gene family member（OsSATs）, red squares indicate the Arabidopsis SAT gene family members（AtSTAs）, and green squares indicate the cabbage SAT gene family member（BoSATs）. B: Evolutionary tree of the StSAT gene family. The blue squares indicate the C1 subfamily, the red squares indicate the C2 subfamily, and the green squares indicate the C3 subfamily. C: Gene structure of StSATs. Blue boxes indicate exons, black lines indicate introns and red boxes indicate upstream/downstream regions. D: Protein conserved motif of StSATs

图2 多物种SAT基因的共线性分析不同的数字编号代表不同物种的染色体，红线表示物种间的直系同源基因

Fig. 2 Collinearity analysis of SAT genes in multiple species Different numerical numbers indicate the chromosomes of different species, and the red lines indicate orthologous gene pairs between different species

表3 马铃薯与其他物种间直系同源基因的Ka/Ks

Table 3 Ka/Ks ratios of orthologous genes between potato and other species

Gene ID1	Gene ID2	Ka	Ks	Ka/Ks
Soltu02G022850	Solyc02g082850	0.040 2	0.110 7	0.363 2
Soltu12G025770	Solyc12g008780	0.003 1	0.074 1	0.041 4
Soltu07G027060	Solyc07g065340	0.010 6	0.072 8	0.145 3
Soltu10G001180	Solyc10g006040	0.009 5	0.050 7	0.187 0
Soltu02G022850	AT2G17640	0.192 7	1.660 3	0.116 1
Soltu12G025770	AT5G56760	0.118 8	2.718 1	0.043 7
Soltu07G027060	AT3G13110	0.238 7	2.356 3	0.101 3
Soltu10G001180	AT1G55920	0.234 9	3.864 2	0.060 8
Soltu12G025770	Bo3g019690	0.122 7	3.050 0	0.040 2
Soltu07G027060	Bo6g035890	0.202 6	3.867 2	0.052 4
Soltu02G022850	Zm00001d027536	0.178 5	2.483 9	0.071 8
Soltu02G022850	Os03g04140	0.267 1	3.167 0	0.084 3

图3 启动子区域顺式作用元件预测

Fig. 3 Prediction of cis-acting elements in the promoter region of StSATs

图4 StSATs在DM马铃薯不同组织部位中的表达对4个StSATs在13个组织部位中的表达量取以2为底的对数，用log2FPKM绘制热图

Fig. 4 StSAT gene expressions in different tissues of DM potato The expressions of four StSATs in 13 tissue sites are logarithms base 2, and heat maps are plotted using log2FPKM

图5 StSATs在DM马铃薯非生物胁迫和激素处理下的表达在非生物胁迫（盐、甘露醇和热胁迫）中使用处理与对照的比值，取以2为底的对数，用log2FC绘制热图

Fig. 5 StSAT gene expressions in DM potato under abiotic stress and hormone treatment The ratios of treatment to control are used for abiotic stresses（salt, mannitol, and heat stress）, taken as base 2 logarithms and heat maps are plotted using log2FC

图6 StSAT基因在四倍体马铃薯NaCl和PEG处理下的相对表达水平对4个马铃薯StSAT基因在NaCl和PEG处理下的表达进行了qPCR 分析。数据为3 个独立生物学重复的平均值（±SE）。柱状图上方的不同字母表示P<0.05 时的显著差异

Fig. 6 Relative expressions of StSAT genes under NaCl and PEG treatment in tetraploid potato The relative expressions of four StSAT genes under NaCl and PEG treatment was analyzed by qPCR. Data are means（±SEs）from three independent biological replicates. Different letters above the bar chart indicate significant differences at P<0.05

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