苹果MLP家族基因鉴定及非生物胁迫响应分析

doi:10.16420/j.issn.0513-353x.2020-0276

摘要/Abstract

摘要：

在苹果中鉴定了13个Major Latex Protein（MLP）家族基因MdMLP。序列比对及构建蛋白同源模型发现,MdMLP蛋白含有Bet_v_1典型的Gly-rich loop区域结构,且为MLP家族特有的GxxxxxG结构。经多物种MLP系统发育及共线性比较分析,MdMLP与其他蔷薇科物种MLP具有相似基因结构和蛋白质保守结构域。qRT-PCR分析表明,MdMLP 在‘新疆1号’苹果14个器官组织中均有不同程度的表达,对ABA、NaCl、PEG、低温（4 ℃）和高温（40 ℃）有一定响应,且同一亚族基因表达情况呈现相似趋势。String构建蛋白互作网络发现,MdMLP可能通过与PRSP、SNRK1/2、bHLH等应激、ABA相关转录蛋白互作,参与苹果对非生物胁迫的防御。

关键词: 苹果, MLP 基因, 全基因组鉴定, 非生物胁迫分析

Abstract:

In the apple,13 Major Latex Protein（MLP）genes MdMLP were identified,and through sequence alignment and construction of the MdMLP protein homologous model,it was found that MdMLP contained a typical Gly-rich loop region structure of Bet_v_1 proteins, which was a unique GxxxxxG structure in the MLP family. Comparison of MLP phylogeny and collinearity in different species indicated that MdMLP had similar gene structure,conserved domain and high homology with other Rosaceae MLP members. qRT-PCR analysis showed that all MdMLP genes had different levels of expression in 14 organs of apple‘Xinjiang 1’. At the same time,MdMLP genes responded to ABA,NaCl,PEG,cold（4 ℃）and heat（40 ℃）stress treatments,and the same subgroup genes showed a similar expression trend when exposed to abiotic stress. It suggested that MdMLP may interact with PRSP,SNRK1/2,bHLH and ABA-related transcriptional proteins to participate in the defense against abiotic stress in apple.

Key words: Malus × domestica, MLP genes, genome-wide identification, abiotic stress analysis

中图分类号:

S661.1

李欣欣, 侯鸿敏, 徐吉花, 孙晓红, 张玉刚. 苹果MLP家族基因鉴定及非生物胁迫响应分析[J]. 园艺学报, 2021, 48(1): 1-14.

LI Xinxin, HOU Hongmin, XU Jihua, SUN Xiaohong, ZHANG Yugang. Genome-wide Identification and Abiotic Stress Response Analysis of MLP Family Genes in Apple[J]. Acta Horticulturae Sinica, 2021, 48(1): 1-14.

图/表 9

表1 荧光定量PCR引物

Table 1 Primers for qRT-PCR

基因名称 Gene name	正向引物序列（5′-3′） Forward primer sequence	反向引物序列（5′-3′） Revers primer sequences
MdMLP1	TAGCGATTGTGGTAAGCTGG	GGAGATGTGGTGTGGTTTGT
MdMLP2	ACGAGAAGTGGGAGGGTAAA	GGAGATGTGGTGTGGTTTGT
MdMLP3	TAGCGATTGTGGTAAGCTGG	GGAGATGTGGTGTGGTTTGT
MdMLP4	GCACGGGAACACAAATGATG	AGACCTCAACTGCAAACTGG
MdMLP5/13	GGCGGTTGCTTATAAGGTGA	AACCTGTTCGTGTGCTTTCT
MdMLP6	CCTTCAGGTCATCCAAGGTG	TTTGGTGTGACTTGAACCGT
MdMLP7	CGAAGGTGATTGGGAGACTG	CGCTTCAACCGTCTCCTTTA
MdMLP810	TTCTAGCAAGGTGGAGACGA	CCACAAGAAGACACAACCCA
MdMLP9	GGAAACACACGGCTCTGTTA	CCGACATGATCCCGATTGTT
MdMLP11	CAAGGCTGCAACTCTGAAAG	TATTCGATCCACAGGTTGGC
MdMLP12	CCCACCTCATCCCAAACATT	GCAATGCAGGTGCTTTCATC
MdActin	ATTCAAGTATGCCTGGGTGC	CAGTCAGCCTGTGATGTTCC

表2 苹果中鉴定出的MdMLP家族基因及其基本特性

Table 2 The MdMLP gene family in Malus × domestica and their basic characteristics

基因名称 Gene name	基因组登录号 Gene accession No.（GDR）	编码区/bp CDS	染色体位置 Chromosome location	蛋白质 Protein
基因名称 Gene name	基因组登录号 Gene accession No.（GDR）	编码区/bp CDS	染色体位置 Chromosome location	信号肽 Signal peptide （Sec/SPI）	长度/ aa Length	分子量/ kD Molecular weight	pI	不稳定指数 Instability index	亲水性 Hydropa- thicity
MdMLP1	MD00G1063600	468	Chr00:11768127..11768939（+）	0.0011	155	17.62	5.32	27.52	-0.576
MdMLP2	MD13G1022700	498	Chr13:1627661..1629214（-）	0.0021	165	18.68	5.52	22.52	-0.444
MdMLP3	MD13G1022900	468	Chr13:1645481..1646431（-）	0.0011	155	17.62	5.32	27.52	-0.576
MdMLP4	MD13G1023000	468	Chr13:1657247..1658331（-）	0.0016	155	17.45	5.32	26.36	-0.497
MdMLP5	MD13G1088200	462	Chr13:6177671..6178671（+）	0.0006	153	16.83	5.26	22.52	-0.137
MdMLP6	MD16G1024000	474	Chr16:1729158..1730220（-）	0.0012	157	17.58	8.43	37.24	-0.258
MdMLP7	MD16G1024100	462	Chr16:1731391..1732218（-）	0.0011	153	17.27	4.88	30.74	-0.381
MdMLP8	MD16G1024300	468	Chr16:1742688..1743368（-）	0.0007	155	17.57	4.88	30.00	-0.338
MdMLP9	MD16G1024900	558	Chr16:1792284..1793136（-）	0.0022	185	21.01	6.71	28.99	-0.334
MdMLP10	MD16G1025000	420	Chr16:1802244..1802758（-）	0.0007	139	15.78	5.33	27.31	-0.394
MdMLP11	MD16G1025800	453	Chr16:1839602..1840796（-）	0.0017	150	17.05	5.47	11.39	-0.386
MdMLP12	MD16G1026200	387	Chr16:1862558..1863354（-）	0.0022	128	14.68	6.14	20.19	-0.303
MdMLP13	MD16G1088600	462	Chr16:6094690..6095895（+）	0.0006	153	16.92	4.97	23.39	-0.145

图1 不同物种MLP蛋白序列比对 Md:苹果;At:拟南芥;Pg:人参;Op:罂粟。

Fig. 1 Multiple sequence alignment of MLP protein from different species Md:Malus × domestica;At:Arabidopsis thaliana;Pg:Panax ginseng;Op:Papaver somniferum.

图2 MdMLP蛋白质进化树及22种氨基酸残基含量

Fig. 2 Phylogenetic tree and 22 amino acid residues of MdMLP protein

图3 不同物种MLP家族蛋白序列系统进化树（A）、基因结构（B）和蛋白质保守结构域motifs（C） Md:苹果;At:拟南芥;Fv:野草莓;Vv:葡萄;RcH:蔷薇。

Fig. 3 Phylogenetic relationships,gene structure,architecture of conserved protein motifs in MLP genes from 5 species Md:Malus × domestica;At:Arabidopsis thaliana;Fv:Fragaria vesca;Vv:Vitis vinifera;RcH:Rosa.

图4 苹果与蔷薇、葡萄、拟南芥和草莓MLP家族基因的共线性分析背景中灰色线条表示基因组中的共线区域,红色线条突出显示共线的MLP基因对。

Fig. 4 Synteny analysis of MLP genes between apple and rose,grape,Arabidopsis and strawberry Gray lines in the background indicate the collinear blocks within genomes,while the red lines highlight the syntenic MLP gene pairs.

图5 MdMLP互作蛋白功能预测网络图利用String蛋白数据库基于拟南芥同源蛋白分析MdMLP1 ~ MdMLP13蛋白功能关系网络图,包括AtMLP28（AT1G70830）、AtMLP31（AT1G70840）、AtMLP34（AT1G70850）、AtMLP43（AT1G70890）、AtMLP328（AT2G01520）和AtMLP423（AT1G24020）。

Fig. 5 Prediction of functional network of interacting proteins of MdMLP Based on Arabidopsis thaliana homologous proteins,String protein database was used to analyze the functional relationship network diagram of MdMLP1-MdMLP13 proteins,including AtMLP28（AT1G70830）,AtMLP31（AT1G70840）,AtMLP34（AT1G70850）,AtMLP43（AT1G70890）,AtMLP328（AT2G01520）and AtMLP423（AT1G24020）.

图6 MdMLP在‘新疆1号’苹果不同器官中基因表达情况将叶中MdMLP的表达量设为1,以MdActin作为内参对照,2-??Ct法处理数据,3个重复。

Fig. 6 Expression levels in different organs of‘Xinjiang 1’of MdMLP in apple The expression of MdMLP in leaves was 1,and the data was calculated based on the 2-??Ct method,and three replicates were set.

图7 MdMLP在非生物胁迫处理‘新疆1号’苹果组培苗中表达的qRT-PCR分析 MdActin作为内参对照,MdMLPs在0 h的表达水平设为1,2-??Ct法处理,设置3个重复,同一基因不同处理时间之间进行显著性差异分析,置信区间为5%（P < 0.05）。

Fig. 7 Expression of MdMLP genes in apple cultured under different abiotic stress The expression level of MdMLPs at 0 hour was set as 1,and the data was calculated based on the 2-??Ct method,and three replicates were set. The difference between the same gene and different treatment time was analyzed with a confidence interval of 5%（P < 0.05）.

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