甜樱桃GH3基因家族全基因组鉴定与表达分析

doi:10.16420/j.issn.0513-353x.2020-0911

摘要/Abstract

摘要：

以甜樱桃（Prunus avium L.）为材料,对IAA酰胺合成酶（Gretchen Hagen 3,GH3）基因家族全基因组进行鉴定及生物信息分析;同时,分析其在不同组织中的表达及对外源GA₃、ABA、MeJA、IAA的响应。结果表明,甜樱桃基因组中共存在8个GH3基因,根据在染色体的位置依次命名为PavGH3.1 ~ PavGH3.8,编码区长度为1 683 ~ 1 851 bp,预测大多数基因定位在叶绿体中;PavGH3外显子数3 或 4,且有较多保守基序;除5号和7号染色体外,其他染色体均有分布;启动子存在ABA、MeJA等6种激素响应元件;PavGH3在进化上分为2组,且PavGH3.5和PavGH3.6与拟南芥GH3一些成员存在共线性。表达分析显示,PavGH3.2、PavGH3.6和PavGH3.7为3个主要表达基因。PavGH3.4和PavGH3.5在第2次生理落果中较第1次上调表达,与正常果实相比,第1次生理落果中PavGH3.5和PavGH3.7下调表达,PavGH3.4和PavGH3.6上调表达。PavGH3.2、PavGH3.3、PavGH3.6和PavGH3.7为主要响应激素的基因。推测PavGH3基因家族广泛参与甜樱桃的生长发育,且可能与落果关系密切。

关键词: 甜樱桃, IAA酰胺合成酶, 基因家族, 生长素响应, 表达分析

Abstract:

Auxin plays a crucial role in regulating the growth and development of plants. The acyl acid amido synthetase Gretchen Hagen 3（GH3）may modulate auxin levels via conjugting both indole-3-acetic acid（IAA）and salicylic acid（SA）in response to environment changes. In the current investigation,genome-wide identification and comprehensive analysis of the GH3 gene family in sweet cherry（Prunus avium）were conducted using the genomic sequence. Totally,eight members of PavGH3 genes were identified from the whole genome of sweet cherry,which are not evenly distributed on the chromosomes,with the coding sequence of 1 683-1 851 bp,most of which were forecasted in chloroplast. The analysis of gene structure and domain revealed that PavGH3 demonstrated high similarity among the members,and the number of exons was three or four. A total of 16 conserved domains were obtained,and the number of the conserved domains varied among the members. These gene members were clustered into two major groups（I and II）,based on phylogenetic analysis. Sequence analysis of promoters showed many cis-regulatory elements which presumably responsed to phytohormones such as abscisic acid,methyl jasmonate etc. Synteny block indicated that PavGH3.5 and PavGH3.6 showed collinearity with GH3 in Arabidopsis thaliana,suggesting that they were evolutionarily conserved. Multiple sequence alignment demonstrated that these members are highly similar and conserved. As detected by qRT-PCR in 17 tissues. PavGH3.2,PavGH3.3 and PavGH3.6 were highy expressed in these tissues. PavGH3.4 and PavGH3.5 were up-regulated in the dropped-fruit of the second physiological abscission in comparison with that of the first one. Compared with retention fruits,surprisingly,PavGH3.5 and PavGH3.7 were significantly down-regulated in the first physiological fruit drops,PavGH3.4 and PavGH3.6 were up-regulated. IAA,GA₃,ABA and MeJA were used to foliar application,and PavGH3.2,PavGH3.3,PavGH3.6 and PavGH3.7 were differentially expressed in response to the type and application stage of the phytohormones,however,no responses were observed in the others. Therefore,it is speculated that the PavGH3 gene family is considerably involved in the regulation of the growth and development of sweet cherry,and probably in the fruit abscission.

Key words: Prunus avium, Gretchen Hagen 3, gene family, auxin response, expression analysis

中图分类号:

S662.5

侯黔东, 沈天娇, 余欢欢, 仇志浪, 文壮, 张惠敏, 吴亚维, 文晓鹏. 甜樱桃GH3基因家族全基因组鉴定与表达分析[J]. 园艺学报, 2021, 48(12): 2360-2374.

HOU Qiandong, SHEN Tianjiao, YU Huanhuan, QIU Zhilang, WEN Zhuang, ZHANG Huimin, WU Yawei, WEN Xiaopeng. Genome-wide Identification and Expression Analysis of Prunus avium Gretchen Hagen 3（GH3）Gene Family[J]. Acta Horticulturae Sinica, 2021, 48(12): 2360-2374.

图/表 9

表1 甜樱桃GH3基因家族荧光定量引物

Table 1 Quantitative real-time PCR primer of Prunus avium GH3 gene family

基因 Gene	正向引物（5′-3′） Forward primer	反向引物（5′-3′） Reverse primer	扩增产物/bp PCR product	退火温度/℃ T_m
PavGH3.1	CAGTTGCAGAAATGCTGTGTCC	TCATGTGGCTTGCTTAGTGGGT	276	61
PavGH3.2	GGTCCTCAGCAGCCGAATCAC	CCACCCTTCAGAAGCGCCATA	265	64
PavGH3.3	ACTTCTTTGGTGCCCCTTGCTTC	GTGGTCTGTGCGCTATGGTGTGT	179	65
PavGH3.4	CTCTTACACCCTCATTCCCTCCAT	CGACTCTCAGCACATCTCCAACT	224	59
PavGH3.5	CATAGAGGAGACGACCAGGAACA	GGAACTCAGAGATGGGATGAGAGCT	239	62
PavGH3.6	CTCCCATCTTGTCAGCTCACCCA	GCTCACGAACAGAAAGTACAGGCCC	197	62
PavGH3.7	CTGTTGAGGTTCTTGGTGGGC	AGTTTTCTCGGGGGGTTGCA	263	60
PavGH3.8	CTCCCTTGCTTTCTCTGCCTAAT	GACCACAGCCTCAATGTACTTG	125	60
PavEF1-α2	ATCCAGAGTAGCAGAACCAATCAC	GTTAGGCATCCAGTCCCAGAAT	127	58
PavRSP3	TCAAGGTCAGGTAAGGGGGTC	GTGAGGTGATTGTTAGTGGAAAGC	223	56.5

表2 甜樱桃PavGH3基因家族成员信息

Table 2 Member information of PavGH3 gene family in Prunus avium

基因 Gene	基因ID Gene ID	染色体 chromosome	CDS/bp	氨基酸长度/aa Length	蛋白分子量 Molecular weight	理论等电点 Theoretical pI	亲水性 GRAVY	亚细胞定位预测 Subcellular localization
PavGH3.1	Pav_sc0001540.1_g090.1.mk	PAV_r1.0chr1	1 851	616	70 875.15	5.68	-0.295	细胞质,细胞核 Cytoplasm,nucleus
PavGH3.2	Pav_sc0000254.1_g140.1.mk	PAV_r1.0chr2	1 683	560	62 622.79	5.80	-0.136	叶绿体 Chloroplast
PavGH3.3	Pav_sc0002360.1_g300.1.mk	PAV_r1.0chr3	1 794	597	67 453.09	6.45	-0.212	叶绿体 Chloroplast
PavGH3.4	Pav_sc0000269.1_g440.1.mk	PAV_r1.0chr4	1 845	614	69 182.12	5.91	-0.272	叶绿体 Chloroplast
PavGH3.5	Pav_sc0000049.1_g070.1.mk	PAV_r1.0chr6	1 800	599	67 477.05	5.81	-0.265	叶绿体 Chloroplast
PavGH3.6	Pav_sc0001422.1_g120.1.mk	PAV_r1.0chr8	1 803	600	67 636.32	5.56	-0.258	叶绿体 Chloroplast
PavGH3.7	Pav_sc0003065.1_g090.1.mk	PAV_r1.0chr8	1 725	574	64 563.26	5.92	-0.096	叶绿体 Chloroplast
PavGH3.8	Pav_sc0000848.1_g820.1.mk	PAV_r1.0chr8	1 806	601	68 250.59	5.87	-0.196	叶绿体 Chloroplast

图1 甜樱桃PavGH3基因家族进化树（a）、基因结构（b）、保守结构域（c）及Motif（d）

Fig. 1 Phylogenetic tree（a）,gene structure（b）,conserved domain（c）and Motif Logo（d）of PavGH3 gene family in Prunus avium

图2 甜樱桃、拟南芥、小麦、苹果及水稻GH3基因家族系统进化树

Fig. 2 Phylogenetic tree of Prunus avium,Arabidopsis thaliana,Triticum aestivum,Malus domestica and Oryza sativa GH3 gene families

图3 甜樱桃PavGH3基因家族上游植物激素相关的顺式作用元件

Fig. 3 Phytohormone related cis-acting elements in upstream of gene family in Prunus avium

图4 甜樱桃PavGH3基因家族染色体定位箭头标注基因方向。

Fig. 4 Chromosome mapping of PavGH3 gene family in Prunus avium The gene direction is marked with an arrow.

图5 甜樱桃不同组织中的PavGH3半定量 A：不同时期花和果实;B：PavGH3半定量。YL：幼叶;ML：成熟叶;OL：老叶;FL1：休眠花芽;FL2：小花蕾;FL3：开花前花蕾;FL4：盛开花朵;FR1：小果;FR2：中果;FR3：转色期果;FR4：成熟果;AS：一年生茎;BS：二年生茎;CA：果柄;AFS：脱落果柄;FAb1：小果落果;FAb2：中果落果。

Fig. 5 PavGH3 semi-quantitative in different tissue of sweet cherries A：Flowers and fruit;B：Semi-quantitative in sweet cherries. YL：Young leaf;ML：Mature leaf;OL：Old leaf;FL1：Dormant flower bud;FL2：Flower bud;FL3：Flower bud before flowering;FL4：Blooming flowers;FR1：Small fruit;FR2：Middle fruit;FR3：Fruit of red-fleshed;FR4：Ripe fruit;AS：Annual stem;BS：Biennial stem;CA：Carpopodium;AFS：Abscising fruit carpopodium;FAb1：Fruit abscising of small fruit;FAb2：Fruit abscising of middle fruit.

图6 甜樱桃PavGH3基因表达热图 YL：幼叶;ML：成熟叶;OL：老叶;FL1：休眠花芽;FL2：小花蕾;FL3：开花前花蕾;FL4：盛开花朵;FR1：小果;FR2：中果;FR3：转色期果;FR4：成熟果;AS：一年生茎;BS：二年生茎;CA：果柄;AFS：脱落果柄;FAb1：小果落果;FAb2：中果落果。

Fig. 6 PavGH3 gene expression heat map of sweet cherries YL：Young leaf;ML：Mature;OL：Old leaf;FL1：Dormant flower bud;FL2：Flower bud;FL3：Flower bud before flowering;FL4：Blooming flowers;FR1：Small fruit;FR2：Middle fruit;FR3：Fruit of red-fleshed;FR4：Ripe fruit;AS：Annual stem;BS：Biennial stem;CA：Carpopodium;AFS：Abscising fruit carpopodium;FAb1：Fruit abscising of small fruit;FAb2：Fruit abscising of middle fruit.

图7 甜樱桃4个GH3基因对外源GA3、MeJA、ABA和IAA的响应模式

Fig. 7 Response patterns of GH3 gene family in Prunus avium to GA3,MeJA,ABA and IAA treatment

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