菠菜PSY基因家族的鉴定与表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2023-0392

摘要/Abstract

摘要：

八氢番茄红素合成酶（phytoene synthase, PSY）是植物类胡萝卜素合成途径中的第一个限速酶，对植物的生长发育具有重要作用。为了解菠菜（Spinacia oleracea L.）中PSY基因家族功能，本研究通过生物信息学方法对菠菜PSY基因家族成员的数目、结构、启动子元件、氨基酸特性和基因进化等进行鉴定分析，并利用实时荧光定量PCR技术（RT-qPCR）分析其在菠菜组织中及响应红蓝光比例变化的表达模式，同时测定R1B3（红光∶蓝光=1∶3）和R3B1（红光∶蓝光=3∶1）处理后菠菜总类胡萝卜素含量，进行SoPSYs基因表达量与总类胡萝卜素含量变化的相关性分析。结果显示，菠菜共有4个PSY基因，含有SQS_PSY保守结构域，SoPSY蛋白为亲水性蛋白。菠菜PSY1、PSY2和苋科的甜菜进化关系较近，PSY3、PSY4与番茄PSY3有较近的亲缘关系。菠菜PSY基因启动子序列上存在多种光响应和植物激素相关元件。SoPSY基因具有组织表达特异性，SoPSY1、SoPSY2、SoPSY3仅在叶片中表达，SoPSY4主要在根中表达。菠菜类胡萝卜素含量在R1B3和R3B1处理后均下降。不同基因型菠菜中，各SoPSY基因响应红蓝光比例变化的表达模式具有差异性，其中SoPSY1的表达在栽培类型菠菜中无显著变化，SoPSY2的表达模式在两种类型菠菜中相反，SoPSY3在野生型与栽培型菠菜中表达模式一致，且这3个基因的相对表达水平与总类胡萝卜素含量变化密切相关。本研究为深入了解PSY基因在菠菜中的生物学功能，以及响应红蓝光比例变化的调控机理奠定了基础。

关键词: PSY, 菠菜, 基因家族鉴定, 基因表达模式, 红蓝光, 类胡萝卜素, 相关性分析

Abstract:

PSY（phytoene synthase, PSY）is the first rate-limiting enzyme in the plant carotenoid synthesis pathway and plays an important role in plant growth and development. In order to understand the function of PSY gene family in spinach, the number, structure, promoter elements, amino acid characteristics and gene evolution of PSY gene family members in spinach were identified by bioinformatics method, and the expression pattern of PSY genes in spinach tissue and in response to the changes in the ratio of red to blue light was analyzed by real-time fluorescence quantitative PCR（RT-qPCR）. Meanwhile, the expressions of R1B3（red∶blue light = 1∶3）and R3B1（red light∶blue light = 3∶1）and the correlation between SoPSYs gene expression and total carotenoid content were analyzed. The results showed that there were 4 PSY genes in spinach, containing SQS_PSY conserved domain, and the SoPSY protein was a hydrophilic protein. PSY1 and PSY2 of spinach were closely related to beet in Amaranthaceae, and PSY3 and PSY4 were closely related to PSY3 in tomato. There were various light-responsive and phytohormone-related elements in the promoter sequence of spinach PSY genes. SoPSY genes had tissue expression specificity, SoPSY1, SoPSY2, and SoPSY3 were only expressed in the leaves, and SoPSY4 was mainly expressed in the roots. Spinach carotene content decreased after treatment with R1B3 and R3B1. Among spinach genotypes, the expression patterns of each SoPSY gene in response to the ratio of red to blue light were different. The expression of SoPSY1 was not significantly changed in cultivated spinach, the expression patterns of SoPSY2 were reversed in both spinach genotypes, and the relative expression patterns of SoPSY3 were consistent in the wild and cultivated spinach, and the relative expressions of these three genes was closely related to the change of total carotenoid content. This study lays the foundation for an in-depth understanding of the biological function of the PSY genes in spinach and their regulatory mechanism in response to changes in the ratio of red to blue light.

Key words: PSY, Spinacia oleracea, identification of gene family, gene expression pattern, red and blue light, carotenoids, correlation analysis

任丽, 乔舒婷, 葛晨辉, 魏梓桐, 徐晨曦. 菠菜PSY基因家族的鉴定与表达分析[J]. 生物技术通报, 2023, 39(12): 169-178.

REN Li, QIAO Shu-ting, GE Chen-hui, WEI Zi-tong, XU Chen-xi. Identification and Expression Analysis of Spinach PSY Gene Family[J]. Biotechnology Bulletin, 2023, 39(12): 169-178.

图/表 10

表1 实时荧光定量PCR引物

Table 1 Primers used for quantitative real-time PCR

引物名称 Primer name	上游引物 Upstream primer（5'-3'）	下游引物 Downstream primer（5'-3'）
18S	CTGAGAAACGGCTACCACA	CCCAAGGTCCAACTACGAG
SoPSY1	CGGTGTAGTGAAGTTTGTGCT	TGCCCAAATGGCTTTTCTCTTT
SoPSY2	CGGTGTAGTGAAGTTTGTGCTGA	TATAGCCCAAATGGCTTTTCGCTTC
SoPSY3	GCTCTTTCCTTGGGTACTGCT	GAGCTCGTCTTGTGGAAGGT
SoPSY4	ATGGCCGAACTTCGTTGATG	CCCTTATTGGATAGAGCAAGAAAGC

表2 菠菜PSY基因家族的鉴定及理化性质

Table 2 Identification and characteristic features of PSY gene family in spinach

基因名称 Gene name	基因ID Gene ID	染色体编号 Chrom No.	氨基酸长 Length/aa	分子质量 Molecular weight/kD	等电点 pI	重力值 GRAVY	亚细胞定位 Location
SoPSY1	Spo03700	SpoScf_03339	418	47.15	8.88	-0.262	Chloroplast
SoPSY2	Spo13982	SpoScf_02365	418	47.14	8.89	-0.262	Chloroplast
SoPSY3	Spo00660	chr4	359	41.48	9.17	-0.494	Chloroplast
SoPSY4	Spo15086	chr4	351	40.24	9.11	-0.363	Chloroplast

图1 菠菜PSY基因结构分析

Fig. 1 Gene structure analysis of PSY in spinach

图2 PSY基因家族的系统进化关系、保守结构域和保守基序分析

Fig. 2 Phylogenetic relationship, conserved domain and conserved motifs analysis of PSY gene family

图3 菠菜PSY基因启动子区域顺式调节元件分析

Fig. 3 Characterization of cis-acting regulatory elements in the promoter region of spinach PSY genes

图4 菠菜不同组织中PSY基因的相对表达量图中误差线表示标准偏差； *P<0.05，** P<0.01和*** P<0.001；下同

Fig. 4 Relative expressions of PSY genes in the different tissues of spinach The error line in the figure refers to the standard deviation ; *P<0.05, ** P<0.01 and *** P<0.001. The same below

图5 红蓝光处理对‘US362’菠菜PSY基因表达特性的影响

Fig. 5 Effects of red and blue light treatment on the expression characteristics of PSY genes in ‘US362’ spinach

图6 红蓝光处理对‘KS4’菠菜PSY基因表达特性的影响

Fig. 6 Effects of red and blue light treatment on the expression characteristics of PSY gene in ‘KS4’ spinach

图7 红蓝光处理下菠菜总类胡萝卜素含量变化

Fig. 7 Changes of total carotenoid content in spinach under red and blue light treatment A: ‘US362’; B: ‘KS4’

表3 红蓝光处理下菠菜总类胡萝卜素含量变化与PSY基因相对表达量的相关性分析

Table 3 Correlation analysis of total carotenoid content and PSY gene relative expression in spinach under red and blue light treatment

处理 Treatment	含量变化量 Content variation	24 h基因相对表达量24 h relative expression				12 d基因相对表达量12 d relative expression
处理 Treatment	含量变化量 Content variation	SoPSY1	SoPSY2	SoPSY3	SoPSY4	SoPSY1	SoPSY2	SoPSY3	SoPSY4
CK	US362	-0.849	0.615	-0.102	-1.000**	0.999*	-0.593	0.745	-0.999*
CK	KS4	-0.362	-0.475	-0.104	-0.920	-0.754	-0.686	-0.348	-0.146
R1B3	US362	-0.058	0.749	0.673	0.570	-0.042	0.999*	0.953	0.765
R1B3	KS4	-0.476	-0.490	-0.479	-0.726	-0.978	-0.955	-0.011	0.011
R3B1	US362	-0.999*	-0.485	0.954	-0.485	-0.970	0.492	-0.970	-0.338
R3B1	KS4	0.128	-0.380	0.534	0.765	-0.697	0.260	-0.061	-0.156

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