黑莓果实发育过程中蔗糖磷酸合成酶基因的表达分析

doi:10.12302/j.issn.1000-2006.202007067

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南京林业大学学报（自然科学版） ›› 2022, Vol. 46 ›› Issue (1): 179-186.doi: 10.12302/j.issn.1000-2006.202007067

黑莓果实发育过程中蔗糖磷酸合成酶基因的表达分析

严志祥¹(), 杨海燕²^,^*(), 樊苏帆², 吴文龙², 闾连飞², 李维林¹^,^*()

1.南京林业大学林学院,南方现代林业协同创新中心,江苏南京 210037
2.江苏省中国科学院植物研究所,江苏南京 210014

收稿日期:2020-07-30 接受日期:2021-06-04 出版日期:2022-01-30 发布日期:2022-02-09
通讯作者: 杨海燕,李维林
基金资助:
国家自然科学基金项目(31700606)

Analysis of the expression of sucrose phosphate synthase genes during the development of blackberry fruit

YAN Zhixiang¹(), YANG Haiyan²^,^*(), FAN Sufan², WU Wenlong², LYU Lianfei², LI Weilin¹^,^*()

1. Co-Innovation Center for the Sustainable Forstry in Southern China, College of Forestry, Nanjing Forestry University, Nanjing 210037, China
2. Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing 210014, China

Received:2020-07-30 Accepted:2021-06-04 Online:2022-01-30 Published:2022-02-09
Contact: YANG Haiyan,LI Weilin

摘要/Abstract

摘要：

【目的】蔗糖磷酸合成酶(sucrose phosphate synthase, SPS)是调控植物蔗糖代谢合成的关键酶,在植物光合产物的积累与分配方面有重要作用。本研究旨在探讨黑莓3个SPS基因的系统发育关系、编码的蛋白特性、在不同发育时期、不同组织中的时空表达特性,并分析其与黑莓发育的关系。【方法】以黑莓栽培品种‘宝森’(‘Boysenberry’)为试材,从中克隆和鉴定了3个 SPS 基因家族成员,利用生物信息学和荧光定量聚合酶链式反应(qRT-PCR)等方法对3个黑莓SPS 基因RuSPS1、RuSPS2和RuSPS3的氨基酸序列、保守作用元件、编码的蛋白特性、蛋白结构及进化关系进行分析,并对这3个基因在黑莓中的时空表达情况与酶活性进行了相关性分析。【结果】多重氨基酸序列比对显示,黑莓SPS蛋白具有植物SPS家族特有的2个保守蛋白结构域及2个相对保守的蛋白磷酸位点;系统进化分析表明,RuSPS基因分为A、B两个亚族,其中RuSPS1和RuSPS3为A亚族成员,RuSPS2为B亚族成员;保守作用元件分析表明, 除RuSPS2含基本的蛋白保守元件外, RuSPS1和RuSPS3都存在不同程度的片段缺失;序列分析和比较揭示了黑莓SPS基因与其他家族的不同特征。qRT-PCR分析显示,3个RuSPS基因在黑莓各个组织器官中均有表达,其中RuSPS1在叶片和果实中表达量较高,在花中的表达量较低;RuSPS2在发育成熟的果实中有大量的表达,在其他器官中表达量较低;RuSPS3在各器官中的表达均较高,说明 SPS基因表达具有明显的组织特异性,3个RuSPS基因都随着果实发育进程在果实和叶片中表现了表达增加的趋势。果实和叶片中SPS酶活性的变化与RuSPS基因表达水平一致。相关分析表明,叶片中SPS活性与RuSPS2显著正相关(P<0.05),果实中SPS活性与RuSPS1显著负相关(P<0.05)。【结论】3个RuSPS基因与黑莓果实发育过程中的蔗糖合成与代谢关系密切,均参与了黑莓的生长发育调控,其中叶片中SPS活性的变化一定程度上是由RuSPS2调控,果实中SPS活性的变化则是由RuSPS1调控。

关键词: 黑莓, 果实发育, 蔗糖磷酸合成酶基因(SPS), 表达分析

Abstract:

【Objective】Sucrose phosphate synthase (SPS) is a key enzyme that regulates the synthesis pathway of sucrose metabolism in plants. Although it plays an important role in the accumulation and distribution of photosynthetic products, systematic research on SPS genes in blackberry has rarely been carried out. This study aimed to investigate the phylogenetic relationship between SPS genes in blackberry, the protein properties SPS expression pattern in different tissues at various developmental stages, and its relationship with blackberry development.【Method】 The blackberry cultivar, ‘Boysenberry’, was used in the tests, where three SPS gene family members were cloned and identified. The amino acid sequences, conservative elements, encoded protein characteristics, protein structure, evolutionary relationships, and spatio-temporal expression of RuSPS1, RuSPS2 and RuSPS1 genes were analyzed using bioinformatics and quantitative real-time polymerase chain reaction (qRT-PCR). Correlation analysis was conducted between gene expressions and phosphate synthase activity.【Result】The multiple amino acid sequence comparisons showed that the blackberry SPS protein has two conserved domains and two relatively conserved unique protein phosphate sites to the plant SPS family. Evolutionary analysis showed that the three RuSPS genes were divided into two subfamilies, RuSPS1 and RuSPS3 belong to subfamily A RuSPS2 becong to subfamily B. The conserved elements analysis showed that the RuSPS1 and RuSPS3 genes exhibited fragment deletions to different degrees, with the exception of the basic conserved elements of RuSPS2. The different characteristics of blackberry SPS genes were revealed with other families using sequence analysis and comparison of blackberry SPS genes. The qRT-PCR analysis showed that RuSPS were expressed in all organs. RuSPS1 expression in leaves and fruits was higher than in flowers, RuSPS2 expression was high in mature fruits although much lower in other tissues, while RuSPS3 was highly expressed in all organs. These results indicate that SPS genes had tissue-specific expression in blackberry. Additionally, the expression of three RuSPS genes had increased during fruit and leaf development. Correlation analysis showed that the expression of RuSPS2 showed a significant positive correlation with leaf SPS activity (P < 0.05), and the expression of RuSPS1 was significantly negatively correlated (P < 0.05) with fruit SPS activity.【Conclusion】 There is a close relationship among RuSPS genes, sucrose synthesis and metabolism during blackberry fruit development. This is indicative of the involvement of blackberry SPS genes in the regulation of blackberry growth and development. It is inferred that the change of SPS enzyme activity in leaves was regulated by RuSPS2, while changes to SPS activity in fruits was regulated by RuSPS1. These results lay a foundation for in-depth analysis of the function of the blackberry SPS family genes in future.

Key words: blackberry, fruit development, sucrose phosphate synthase gene (SPS), expression analysis

中图分类号:

S66
Q786

严志祥,杨海燕,樊苏帆,等. 黑莓果实发育过程中蔗糖磷酸合成酶基因的表达分析[J]. 南京林业大学学报（自然科学版）, 2022, 46(1): 179-186.

YAN Zhixiang, YANG Haiyan, FAN Sufan, WU Wenlong, LYU Lianfei, LI Weilin. Analysis of the expression of sucrose phosphate synthase genes during the development of blackberry fruit[J].Journal of Nanjing Forestry University (Natural Science Edition), 2022, 46(1): 179-186.DOI: 10.12302/j.issn.1000-2006.202007067.

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引物 primers	序列(5'-3') sequences (5'-3')	预期片段/bp predicted length	退火温度/℃ annealing temperature
SPS1-F	CCACAGGAGAGGAACACCAGATTG	214	59.4
SPS1-R	CACCATGAGCCGATACATCAGTCA	214	59.4
SPS2-F	CGACGGCGATTGAGGAACAGA	233	58.7
SPS2-R	CCACTCCAACTGCTTCTTCTTACG	233	58.7
SPS3-F	GGTGACTCTGCCGCTCTTCTATC	166	59.0
SPS3-R	GCTCCTCTGCTTCTATCCTTCTCA	166	59.0
Actin-F	GGTGTCAGCCATACAGTCCC	783	64.0
Actin-R	CAGTGAGGTCACGACCAGCA	783	64.0

基因 gene	基因长度/bp gene length	开放阅读框长度/bp length in ORF	蛋白长度/aa protein length	分子质量/ ku molecular weight	等电点 IP
RuSPS1	3 030	3 027	1 009	113.53	6.16
RuSPS2	3 207	3 204	1 068	119.97	6.00
RuSPS3	2 622	2 553	851	93.98	5.96

基因 gene	果实蔗糖磷酸合成酶活 sucrose phosphate synthase activity in fruits				叶片蔗糖磷酸合成酶活 sucrose phosphate synthase activity in leaves
基因 gene	21 d	27 d	33 d	39 d	21 d	27 d	33 d	39 d
RuSPS1	-0.016^*	-0.530^*	-0.228^**	-0.381^*	-0.406^**	0.230^*	0.250^*	-0.135^*
RuSPS2	0.066	-0.268^*	-0.696^**	-0.085	0.186^**	0.023^*	0.021^**	0.159^*
RuSPS3	-0.197	-0.322^*	0.137	-0.189	0.097	0.131	0.329^*	-0.370^*

黑莓果实发育过程中蔗糖磷酸合成酶基因的表达分析

Analysis of the expression of sucrose phosphate synthase genes during the development of blackberry fruit

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