核桃SPL基因家族的系统进化和表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2023-1171

摘要/Abstract

摘要：

【目的】 SQUAMOSA promoter binding protein-like（SPL）转录因子在植物开花诱导中起重要的调控作用。从核桃基因组中鉴定SPL（JrSPL）基因家族成员并分析其表达特性，为探究SPL在核桃开花诱导中的功能研究提供参考。【方法】 采用生物信息学方法鉴定JrSPL基因家族成员，并预测其基本理化性质、保守结构域、进化关系、启动子顺式作用元件等；利用转录组测序和RT-qPCR技术分析JrSPL家族成员在核桃不同组织及嫁接诱导开花后的表达水平。【结果】 JrSPL家族有28个成员，其基因结构和蛋白结构均高度保守，分布在核桃14条不同的染色体，与拟南芥和毛果杨分别存在17处和24处共线性关系，根据系统发育关系可分为8组。JrSPL启动子存在大量的光响应元件、激素应答元件、胁迫响应元件等。转录组测序分析显示，JrSPL基因在核桃不同组织表达量有差异，在雄花、雌花、顶芽及叶中均有表达，多数基因在雌花中的表达量都比较高，有6个基因最为显著，它们可能在开花诱导中起关键作用。在嫁接诱导核桃开花材料中，检测的12个JrSPLs中除了JrSPL8外，在开花材料中的表达量均高于对照，JrSPL2和JrSPL25在混合花序的雄花中表达显著，JrSPL8在顶芽中高表达。【结论】 JrSPL基因在核桃成花中具有重要作用，其高表达是花期提前的主要因素。

关键词: 核桃, SPL基因, 全基因组鉴定, 组织特异性表达, 开花诱导

Abstract:

【Objective】 The SQUAMOSA promoter binding protein like（SPL）transcription factor is a crucial regulatory element in the induction of plant flowering. This study aims to identify the members of the SPL（JrSPL）gene family within the walnut genome and analyze their expression characteristics. The findings of this study will serve as a valuable reference for further investigations into the functional aspects of SPL in walnut flowering induction. 【Method】 The present study employed bioinformatics techniques to identify members of the JrSPL gene family, subsequently to predict their fundamental physicochemical properties, conserved domains, evolutionary relationships, and cis acting elements of promoters. Additionally, transcriptome sequencing and RT-qPCR technology were employed to investigate the expressions of JrSPL family members in various tissues of walnut, as well as following grafting-induced flowering. 【Result】 The JrSPL family consisted of 28 members, showing significant conservation in their gene and protein structures, which were dispersed across 14 distinct chromosomes within the walnut genome. Notably, there were 17 collinear relationships with Arabidopsis and 24 with Populus tomentosa, respectively. Through phylogenetic analysis, these members were categorized into 8 distinct groups. Additionally, the JrSPL promoter region encompassed a substantial quantity of light responsive elements, hormone responsive elements, stress responsive elements, and other similar regulatory motifs. The transcriptome analysis revealed that the JrSPL genes demonstrated varying expressions across different tissues of walnut, including male flowers, female flowers, apical buds, and leaves. Notably, the expressions of most genes were relatively high in female flowers, with 6 genes having significant expression, suggesting their potential involvement in the induction of flowering. In the context of grafting-induced walnut flowering materials, with the except of JrSPL8, the expressions of 12 JrSPLs observed in the flowering materials were found to be greater than those in the control group. Specifically, JrSPL2 and JrSPL25 showed significant expressions in the male flowers of mixed inflorescences, whereas JrSPL8 had high expression in the apical buds. 【Conclusion】 The JrSPL gene exerts a significant influence on the process of walnut flowering, with its high expression serving as the primary determinant of early flowering.

Key words: Juglans regia, SPL gene, genome-wide identification, tissue-specific expression, flowering induction

阿丽亚·外力, 陈永坤, 克拉热木·克里木江, 王宝庆, 陈凌娜. 核桃SPL基因家族的系统进化和表达分析[J]. 生物技术通报, 2024, 40(6): 180-189.

ALIYA Waili, CHEN Yong-kun, KELAREMU Kelimujiang, WANG Bao-qing, CHEN Ling-na. Phylogenetic Evolution and Expression Analysis of SPL Gene Family in Juglans regia[J]. Biotechnology Bulletin, 2024, 40(6): 180-189.

图/表 9

表1 生物信息学软件及使用目的

Table 1 Bioinformatics software and purpose of usage

软件或在线工具名称Name of the software or online tool	用途Usage
ProtParam在线工具（https://web.expasy.org/protparam/）	蛋白理化性质预测Prediction of protein physicochemical properties
ProtComp9.0（http://www.softberry.com/berry.phtml?group=programs&subgroup=proloc&topic=protcompan）	亚细胞定位预测Prediction of subcellular localization
MEME程序（http://meme-suite.org）	保守基序分析Conservative motif analysis
ClustalW2（v2.1）	多重序列比对Multiple sequence alignment
PlantCARE（http://bioinformatics.psb.ugent.be/webtools/plantcare/html/）	启动子顺式作用元件预测Prediction of promoter cis-acting elements

表2 实时荧光定量PCR引物序列

Table 2 RT-qPCR primer sequence

引物名称 Primer name	引物序列 Primer sequence（5'-3'）	引物名称 Primer name	引物序列 Primer sequence（5'-3'）
JrSPL1-F	ATCCCCCTCGCAATGGGAGTGG	JrSPL16-R	CCTCCAAAGGCTTGTTCTTCTCCGC
JrSPL1-R	CAGAGAAGCATGTCCCAAGTCTGG	JrSPL20-F	GCAGAGCCGAGGGAAAGAGGAG
JrSPL2-F	ATCCCCCTCGCAATGGGAGTGG	JrSPL20-R	CCGCCTGGCAAGTAATCAGCGTTG
JrSPL2-R	CAGAGAAGCATGTCCCAAGTCTGG	JrSPL21-F	CGCCGATGCCAAGCCGTACC
JrSPL3-F	CTCTCTGGCCGAGTCGGGAG	JrSPL21-R	GTCGCCTCTCATTGTGTCCGGC
JrSPL3-R	CACCACTCCTCACCTTCTTCGGC	JrSPL22-F	CTCTCTTCTGTCATCACCTCCGGC
JrSPL4-F	CGTCTGCTTCTCCACCCATCTCG	JrSPL22-R	CGGATCCCATGTGAAACCTTCCGC
JrSPL4-R	CAGCACACCACCACCCCTCACC	JrSPL23-F	GTGCCAGGTGTGCAGTTGTTGCAAG
JrSPL8-F	ACAGGCCGGATGCCATAGTCGAG	JrSPL23-R	CCCACTTGAATGGGGCAACCGAG
JrSPL8-R	CTGCCTGGCAACATGGCGGTG	JrSPL25-F	GCTATGAGTGCTGCGGATTTGCGG
JrSPL14-F	GGCTCGTGGTGTGGCAGTTCATC	JrSPL25-R	TTGAAGAGCCCCCGGTCAGAGG
JrSPL14-R	CTGAGGGTGTAGGGACTCGGGA	Jractin-F	ATGATGTCAAGGAAGGACTC
JrSPL16-F	GGCACCGCAGCGAATGATGACAC	Jractin-R	CACAATGATCTCAGCTCCG

图1 JrSPL保守基序（A）和基因结构分析（B）

Fig. 1 Conserved protein motif（A）and gene structure analysis（B）of JrSPL

图2 JrSPL基因物种内共线性分析

Fig. 2 Collinearity analysis of JrSPL gene in inter-species

图3 JrSPL基因与AtSPL和PtSPL基因共线性分析 Pt：毛果杨；At：拟南芥；Jr：核桃

Fig. 3 Collinearity analysis between SPL in J. regia and AtSPL and PtSPL gene Pt: P. trichocarpa; At: A. thaliana; Jr: J. regia

图4 核桃、拟南芥、番茄和毛果杨SPL家族成员系统进化关系 Sl：番茄；Pt：毛果杨；At：拟南芥；Jr：核桃

Fig. 4 Phylogenetic relation of SPL family members of J. regia, A. thaliana, S. lycopersicum and P. trichocarpa Sl: S. lycopersicum; Pt: P. trichocarpa; At: A. thaliana; Jr: J. regia

图5 JrSPL基因在核桃不同组织中的表达分析图例表示对FPKM值进行log2对数变换的结果；红色表示高表达水平，蓝色表示低表达水平；S：茎； L：叶；M：雄花；F：雌花；E：胚；K：果壳；I：总苞

Fig. 5 Expression analysis of JrSPL genes in different tissues The legend indicates the log2 logarithmic transformation on FPKM value. Red indicates high expression levels and blue indicates low expression levels. S: Stem;L: leaf; M: male flower; F: female flower; E: embryo; K: kernel; I: involucre

图6 JrSPL基因启动子顺式元件分析

Fig. 6 Cis-element analysis of JrSPL genes promoter region

图7 核桃嫁接诱导开花后JrSPL基因的相对表达量 CK代表晚实核桃嫁接后的再生枝条茎尖；Jf1、Jf2、Jf3、Jm、Js1、Js2分别代表嫁接诱导后‘新新2号’开花材料中的纯雌花（Jf1，Jf2）、混合花序的雌花（Jf3）及雄花（Jm）、茎尖（Js1，Js2）；*代表与对照比较相对表达量有极显著差异（P<0.01）

Fig. 7 Relative expression of JrSPL gene in J. regia after grafting-induced flowering CK refers to the stem tips of regenerated branches following the grafting of late fruiting walnut trees. Jf1, Jf2, Jf3, Jm, Js1, and Js2 correspondingly denote distinct floral structures within the flowering material of 'Xinxin 2' post-grafting induction, including pure female flowers (Jf1, Jf2), mixed female flowers (Jf3), male flowers (Jm), and stem tips (Js1, Js2). * indicates a significant difference in relative expression levels compared to the control（P < 0.01）

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