桃XTH家族基因鉴定及其在桃果实贮藏过程中的表达特性

doi:10.3864/j.issn.0578-1752.2022.23.011

摘要/Abstract

摘要： 目的通过桃木葡聚糖内糖基转移/水解酶（xyloglucan endotransglucosylase/hydrolase，XTH）基因家族鉴定与不同肉质桃贮藏过程中相关基因的表达分析，发掘PpXTHs家族成员中参与桃果实软化的重要候选基因，为深入解析PpXTHs在果实采后贮藏过程中的功能研究奠定基础。方法根据XTH蛋白保守结构域Glyco_hydro_16 domain和XET_C domain，利用Hmmer 3.1软件对桃蛋白质数据库进行搜索，鉴定桃XTH基因家族成员；利用在线软件ProtParam预测其分子量、理论等电点等理化性质；利用在线分析工具Plant-mPLoc预测其亚细胞定位；MEGA11软件构建系统进化树；运用在线工具MEME对其保守motif进行分析，Tbtools呈现蛋白保守结构域和基因结构图谱；MapChart软件绘制基因在染色体上的分布图；利用实时荧光定量PCR(quantitative reverse transcription PCR，qRT-PCR)技术检测PpXTHs在不同肉质桃贮藏过程中的表达特性。结果在桃基因组共鉴定XTH基因家族成员27个，分布在7条染色体上。系统进化树显示，PpXTHs家族成员可分为祖先类群、Ⅰ/Ⅱ亚家族以及ⅢA和ⅢB亚家族。蛋白结构域分析显示所有PpXTHs成员均含有Glyco_hydro_16和XET_C两个蛋白保守结构域。qRT-PCR结果表明，属于ⅢB亚家族的PpXTH33随着溶质桃贮藏期延长，表达量上调，且表达量显著高于同期硬质桃贮藏过程的表达水平；PpXTH33克隆测序结果显示其CDS序列与桃参考基因组一致，长度为924 bp，编码307个氨基酸；激光共聚焦显微镜观察发现PpXTH33与GFP融合蛋白可能主要于细胞壁与细胞核上产生绿色荧光信号。结论桃27个PpXTHs家族成员蛋白结构均含有2个XTH蛋白保守结构域，不均匀分布在7条染色体上。PpXTHs在溶质桃和硬质桃贮藏过程中的表达特性显示，PpXTH33与桃采后果实软化密切相关。

关键词: 桃, 果实软化, XTH基因家族, 基因表达, 亚细胞定位

Abstract:

【Objective】 The aim of this study was to identify members of the XTH gene family from peach, and to analyze the expression of PpXTHs in peach fruit with different textures during storage, which not only provided data for the research on the candidate PpXTHs involved in peach fruit softening, but also laid the foundation for further study on the PpXTHs function in peach fruit softening.【Method】 The HMM profiles of the Glyco_hydro_16 domain and XET_C domain were used to search all XTH proteins with the Hmmer 3.1 software in the peach protein database. The molecular weight, theoretical isoelectric point and other physicochemical properties were then predicted by the online tool ProtParam. PpXTHs subcellular localization were predicted by the online software Plant-mPLoc. The MEGA 11 software was used to construct a phylogenetic tree. The online tool MEME was used to analyze conserved motifs, the conserved motifs, conserved protein domains and gene structure maps were draw by Tbtools. According to the PpXTH gene family location information, chromosome mapping was carried out with MapChart software. The expressions of PpXTHs in peach fruit with different textures during storage were monitored by qRT-PCR..【Result】 A total of 27 PpXTH genes were systematically identified from peach, which were distributed on seven chromosomes. Based on the phylogenetic tree, the ancestral group, Ⅰ/Ⅱ subfamily, ⅢA subfamily and ⅢB subfamily were classified. In addition, according to the analysis of protein domains, all PpXTH gene family proteins had Glyco_hydro_16 and XET_C conserved domain. The results from qRT-PCR analysis showed that PpXTH33 belonging to the ⅢB subfamily was upregulated as the storage period increased in melting peach fruit, with the expression being markedly higher than that during the storage period of stony hard peach fruit. The positive clone sequencing was consistent with the coding sequence of the Prunus persica reference genome with a length of 924 bp for a 307 amino acid sequence. The PpXTH33 combined with green fluorescent protein may mainly located in the cell wall and nucleus detected by confocal laser scanning microscopy..【Conclusion】 All 27 PpXTHs protein structures contained two highly XTH conserved domains and the genes were distributed on seven chromosomes. The expression characteristics of PpXTH33 in peach fruit with different flesh textures during storage suggested that PpXTH33 was closely associated with peach fruit softening during storage.

Key words: peach, fruit softening, XTH gene family, gene expression, subcellular localization

郭绍雷, 许建兰, 王晓俊, 宿子文, 张斌斌, 马瑞娟, 俞明亮. 桃XTH家族基因鉴定及其在桃果实贮藏过程中的表达特性[J]. 中国农业科学, 2022, 55(23): 4702-4716.

GUO ShaoLei, XU JianLan, WANG XiaoJun, SU ZiWen, ZHANG BinBin, MA RuiJuan, YU MingLiang. Genome-Wide Identification and Expression Analysis of XTH Gene Family in Peach Fruit During Storage[J]. Scientia Agricultura Sinica, 2022, 55(23): 4702-4716.

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https://www.chinaagrisci.com/CN/Y2022/V55/I23/4702

图/表 8

表1

表2

桃XTH基因家族成员特征分析"

命名方式 Symbol	基因号 Gene ID	氨基酸数量 Number of amino acid	分子量 Mw (Da)	理论等电点 pI	亚细胞定位 Subcellular localization
PpXTH1a	Prupe.8G200900.1	287	33055.89	6.19	细胞壁 Cell wall
PpXTH1b	Prupe.8G201000.1	289	33545.54	6.31	细胞壁 Cell wall
PpXTH5	Prupe.1G210900.1	332	38568.82	8.73	细胞壁 Cell wall, 细胞质 Cytoplasm
PpXTH6	Prupe.6G213600.1	291	33271.63	7.60	细胞壁 Cell wall
PpXTH8	Prupe.4G025600.1	300	34771.06	4.92	细胞壁 Cell wall
PpXTH9	Prupe.3G223200.1	296	33606.76	5.73	细胞壁 Cell wall
PpXTH10	Prupe.8G152500.1	295	34300.00	9.35	细胞壁 Cell wall
PpXTH16a	Prupe.4G072800.1	282	32460.74	9.44	细胞壁 Cell wall, 细胞质 Cytoplasm
PpXTH16b	Prupe.1G169700.1	277	31640.95	9.28	细胞壁 Cell wall, 细胞质 Cytoplasm
PpXTH16c	Prupe.3G172100.1	269	30385.02	8.58	细胞壁 Cell wall
PpXTH16d	Prupe.3G171500.1	285	32329.43	8.36	细胞壁 Cell wall, 细胞质 Cytoplasm
PpXTH16e	Prupe.3G171600.1	285	32263.44	8.91	细胞壁 Cell wall, 细胞质 Cytoplasm
PpXTH16f	Prupe.3G171700.1	282	32083.21	8.98	细胞壁 Cell wall, 细胞质 Cytoplasm
PpXTH16g	Prupe.3G172000.1	285	32135.26	8.70	细胞壁 Cell wall, 细胞质 Cytoplasm
PpXTH16h	Prupe.3G171800.1	288	32353.36	8.48	细胞壁 Cell wall, 细胞质 Cytoplasm
PpXTH25a	Prupe.1G088500.1	297	33791.75	4.80	细胞壁 Cell wall
PpXTH25b	Prupe.1G069800.1	285	31908.85	8.25	细胞壁 Cell wall, 细胞质 Cytoplasm
PpXTH25c	Prupe.1G088800.1	284	32099.38	8.49	细胞壁 Cell wall, 细胞质 Cytoplasm
PpXTH25d	Prupe.1G088600.1	293	32945.77	5.12	细胞壁 Cell wall, 细胞质 Cytoplasm
PpXTH25e	Prupe.1G089000.1	299	33370.47	5.49	细胞壁 Cell wall, 细胞质 Cytoplasm
PpXTH25f	Prupe.1G088900.1	296	32990.73	6.74	细胞壁 Cell wall, 细胞质 Cytoplasm
PpXTH26	Prupe.7G225000.1	290	32996.23	8.20	细胞壁 Cell wall
PpXTH28	Prupe.1G337000.1	338	38610.43	6.94	细胞壁 Cell wall
PpXTH30	Prupe.5G027800.1	359	41218.83	8.96	细胞壁 Cell wall
PpXTH32a	Prupe.7G128000.1	292	33720.45	9.52	细胞壁 Cell wall
PpXTH32b	Prupe.6G110300.1	351	39654.76	5.97	细胞壁 Cell wall
PpXTH33	Prupe.1G255100.1	307	34235.96	8.39	细胞壁 Cell wall

表2

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基因 Gene	正向引物 Forward primer (5′-3′)	反向引物 Reverse primer (5′-3′)
PpXTH1a	ATGGCTTTCTCAGTGGTTCGT	AAAGCCGGACTTCTCTTCCA
PpXTH1b	GCAATCACGCTGCAGACAAA	TAGGCTGTGTTGGGTAGGGA
PpXTH5	CCGCAGCTTACCACTCCTAC	AGAATTTGGCCTCCACCGAG
PpXTH6	GAGTTCTTGGGGAACCGGAC	GGGTTGGAGCTTGGGGTATG
PpXTH8	GATGGGTGCCAATGGGAAGA	GGCAAAGTTGGGTAGCGTTC
PpXTH9	CAAGGACCAAGCTATGGGGG	CTGCTGCACTTCTTTGCGTT
PpXTH10	GGAAATGTGGCAGGCAAACC	AGCTGTCGCCATTCCATAGG
PpXTH16a	GCCAAACCGCGATGAGATTG	ACGCTGAGGGTTCCAAAGAA
PpXTH16b	AAACGGACTGGTCAAAGGCA	CACTCAACTGGAGGGCCTTG
PpXTH16c	AAGACTGACTGGAGCCATGC	CGGGAACTTAGGACGGTCAG
PpXTH16d	CTGGAACTCTCAGCGCATC	AGGAGCTTGTGTCCAGTCAG
PpXTH16e	TCTTCTCAAGGGCCAACCCA	GGCTGTTCCAGACAATGGAGT
PpXTH16f	ATGACTTCTTCCAAGGTCACTGT	TGAGAAGCTGTCCTCCGTTG
PpXTH16g	CCCTACACTCTCCACACAAAC	GGGTTGGCTTTTGGGAAATGG
PpXTH16h	GGTGCTCATTCTTTCTTTGGTC	TCTGCATGTCAATCCTGCCC
PpXTH25a	CCACAAGAGGTGGACTGGTG	ACCCATGCCATCTTCCCTTG
PpXTH25b	CAGGCTCTTCCCGTTGTTCT	GTGGGAAGCGCTTGGTATCT
PpXTH25c	AACGCCAATGCTTGCGTATG	TTCAGGAGGGAGGCCTTGT
PpXTH25d	ACTGGGCTACAAGAGGAGGT	TCTTGGCCGGTGGAGTCTAA
PpXTH25e	AATGCCTGCGTATGGTCCTC	AGCCTTGGGGAAATCTCTTGG
PpXTH25f	AGAGCTCCTTTCACTGCTTCC	CAGCCTCTCCTGCCTTGTTA
PpXTH26	CAGGGGATTGCTTACCCCAA	TGTCTCCGTTCCACTTGCAG
PpXTH28	CTACACGGCTGTGCAGTTGA	TGTGACGGGGTCAAACACTC
PpXTH30	CAGGTTCAGGCAGCGATACA	GATTGCGATTGCGGGTTCTC
PpXTH32a	CTGGGGTCCTCAGCATCAAA	GTCATGGTACCCCGGATGAG
PpXTH32b	TGTGGGGTCCACAGATCAC	CCTGATCAAACCCTACTGTGCT
PpXTH33	CTACACAAGCCCAAGCGTTC	ACAACCCCAGAAGTGAGACC
TEF2	GGTGTGACGATGAAGAGTGATG	TGAAGGAGAGGGAAGGTGAAAG
pCAMBIA1302-PpXTH33-GFP	AACACGGGGGACTCTTGAccatgg ATGGCATTTTTGCAGGGAAAAC TCC	GAAAAGTTCTTCTCCTTTactagt TTTGCACTCTGGTGGCATGAC