黄瓜miR156/157-SPL途径基因鉴定和表达分析

doi:10.16420/j.issn.0513-353x.2020-0952

摘要/Abstract

摘要：

以黄瓜‘9930’为试验材料,统计不同株龄黄瓜植株表型,注释黄瓜miR156/157-SPL途径基因,对基因结构、分类和表达进行分析。结果表明,黄瓜具有株龄表型特征,1/2叶期为幼苗期,3/4叶期为过渡期,5叶期为成株期。黄瓜基因组中共有7个Csa-MIR156位点,3个Csa-MIR157位点和14个Csa-SPL基因,分别位于第1、2、3、4和6号染色体。Csa-SPL基因的外显子数2 ~ 10个不等,Csa-SPL蛋白的SBP结构域由第1和第2外显子共同编码,SBP结构域含2个锌指结构域（Zn_1和Zn_2）和1个核定位信号。大分子量Csa-SPL8和Csa-SPL11还具有额外蛋白结构域。亲缘关系分析将Csa-SPL归为5个亚家族,其中11个Csa-SPL为Csa-miR156/157的靶基因,Csa-SPL5、10和14的Csa-miR156/157识别位点位于基因的3′UTR。Csa-SPL7为Csa-miR156靶基因,但Csa-miR156识别位点第11位碱基存在变异,且该变异在葫芦科植物保守存在。基因表达检测显示Csa-MIR156B和C为主效位点,Csa-miR156表达量随株龄增加而下降,靶基因Csa-SPL表达量则随株龄增加而上升。特殊的是,Csa-SPL7在黄瓜子叶期高表达,且不随株龄变化而变化,Csa-SPL7的表达特征同Csa-miR156识别位点碱基变异相吻合,表明Csa-SPL7已解除Csa-miR156的负调控作用。研究结果提示miR156/157-SPL对黄瓜营养期表型（叶形、节间长度和卷须发生）具有调控作用;此外,葫芦科植物miR156-SPL7负调控作用的解除,可能在其营养期早期发育行使特殊功能。

关键词: 黄瓜, Csa-miR156/157, Csa-SPL, 基因表达

Abstract:

In this study,Cucumis sativus‘9930’was used as the material. The cucumber phenotype during different stages,the annotation of miR156/157-SPL,the structure and expression of genes were analyzed. The results showed cucumber exhibited an age-dependent developmental pattern,the leaf 1/2,leaf 3/4 and leaf 5 stages were classified as the juvenile stage,transition stage and adult stage,respectively. In cucumber genome,seven Csa-MIR156 loci,three Csa-MIR157 loci and 14 Csa-SPL were identified and distributed on chromosome 1,2,3,4 and 6,respectively. Exon number of Csa-SPL span is from 2 to 10,and their SBP domains are coded by exon 1 and 2. SBP domains contain two zinc finger domains （Zn_1 and Zn_2）and a nuclear localization signal. In addition,the large molecular weight of Csa-SPL8 and Csa-SPL11 contain several additional domains. Csa-SPL were clustered into 5 sub-family by phylogenetic tree analysis. Eleven Csa-SPL were predicted as target of Csa-miR156/157,and three of them （Csa-SPL5,10 and 14）hold the Csa-miR156/157 binding sites within the 3′UTR. Csa-SPL7,as a miR156 target,holds a specific mutation in 11th position. Interesting,this mutation is conserved among Cucurbitaceae plants. Gene detection results showed Csa-MIR156B and C functioned as the major loci,the expression of Csa-miR156 decreased and its Csa-SPL targets increased during age development. Specifically,Csa-SPL7 was highly expressed in cotyledon stage and its abundance showed no change during plant development. The expression pattern of Csa-SPL7 was consisted with the mutation within Csa-miR156 binding site,which suggested negative regulation by Csa-miR156 was relieved in Csa-SPL7. In summary,this study suggests miR156/157-SPL pathway plays a role in regulating cucumber vegetative traits（leaf shape,internode length and tendril formation）,moreover,the releasing of SPL7 may play an unknown role during early vegetative stage in Cucurbitaceae plants.

Key words: cucumber, Csa-miR156/157, Csa-SPL, gene expression

中图分类号:

S642.2

汪淑雯, 杨爱怡, 王华森, 徐云敏. 黄瓜miR156/157-SPL途径基因鉴定和表达分析[J]. 园艺学报, 2021, 48(11): 2227-2238.

WANG Shuwen, YANG Aiyi, WANG Huasen, XU Yunmin. Identification and Expression Analysis of miR156/157-SPL Pathway Genes in Cucumber[J]. Acta Horticulturae Sinica, 2021, 48(11): 2227-2238.

图/表 9

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基因名称 Gene name	正向引物（5′-3′） Forward primer	反向引物（5′-3′） Reverse primer
Csa-SPL1	CCCTGTCACGAAGTATTTTGTCTC	TGTTTATCAGGAAAGTGGGTTGG
Csa-SPL2	GCAGCATCAACCGACCGACTATCA	ACCAAAGGACGACCAATGGGAAT
Csa-SPL3	CCACCAAACTACCTTATCAAGCG	GAAAGCACGAGGAATGTGAACTA
Csa-SPL4	TAACGCCTCCATTTCTTTGCTCC	TAGATGGGGATTGATTTCCAGGATT
Csa-SPL5	GCGACTGGAAAAGCTGATGGAA	ATCCCCTGAAAACCCTAATTCTGC
Csa-SPL6	GAAGAACGAATCATGGGCCAACT	TCCGAAATCATCCGTCCCTCC
Csa-SPL7	CAGTCCCATCTGAGTGTTCCAGT	CTTCCTCCATTTCAAGACCCA
Csa-SPL8	GAAGGAAGGAGTAATGCGATGGA	GAACCGTGGCAGTGAAAAGAG
Csa-SPL9	TCCAGAAACCGAACGACGACT	GCATGTGAAACTTGGTGATTGAGAC
Csa-SPL10	AGAGCCAAGGGGAAAGCACAA	CTTCCTGGAGGAGCCATCTGAAT
Csa-SPL11	ATCACCTACTACTCTTGGTCGTTGT	CTTAGCCTTGGTTGCGAAGA
Csa-SPL12	TATGTTGCCCGGTTCGTTGT	CCGTTTCCGAGCACTTTCTG
Csa-SPL13	CTAACTCATCGCAACTGATTCAAAG	TGGTTAAGGCAGTCTAGTGACATTCT
Csa-SPL14	CTCAGAAGATCAAGGTTGGGAGG	ACCACCCCGAGCACTGGTTA
Csa-MIR156A	GTATTGAAAATTAGAGTAAGGGGAAG	CTTCAAGCATGAACCCTAACAT
Csa-MIR156B	GAGAGAAAAGCACAAAAGACCAAGA	CTAAGTAAAGGTATGAACTTTCAACTT
基因名称 Gene name	正向引物（5′-3′） Forward primer	反向引物（5′-3′） Reverse primer
Csa-MIR156C	GTTTATCGTTCTTGAATTTGGTTAG	ATACACTTTATAGCAACTGCCTCTG
Csa-MIR156D	TGAATGATGGTGAGTGTGTTAGGAG	CACGCACCCGCAAAGGTAT
Csa-MIR156E	AAAGGTGATTAAAATTGAGGGGAA	TGGCATATTATTATACCAAAAGCCT
Csa-MIR156F	CATACGGAAGGTAATCTCAAGGA	AATATCTAGCATAAGCGGCCATG
Csa-MIR156G	TCTCACGGTCTATTTCTAACACG	CACAAAGCTTCGAGCATTGATAT
Csa-MIR157A	GGGTTTAGAAATTTGGAGAGAGACA	TATTAAAAGTGAAGGGATGGGATATA
Csa-MIR157B	ATTTATCATGCACAAGGGAGAACTT	AATTGCTATTAGTGGTACCGATTGA
Csa-MIR157C	CCAATACGGTGATAGCTATATGTTGT	TAACAAAGTGGTGTTTACGACTCTA
Csa-Tub	CACTACACCGTTGGAAAGGAAA	CAAAAGGAGGGAGCCGAGA
Csa-U6	GGGGACATCCGATAAAATT	TGTGCGTGTCATCCTTGC
Csa-miR156	GCGGCGGTGACAGAAGAGAG	GTGCAGGGTCCGAGGT
Csa-miR156 RT	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCAC	TGGATACGACGTGCTC

基因名称 Gene name	正向引物（5′-3′） Forward primer	反向引物（5′-3′） Reverse primer
Csa-SPL1	CCCTGTCACGAAGTATTTTGTCTC	TGTTTATCAGGAAAGTGGGTTGG
Csa-SPL2	GCAGCATCAACCGACCGACTATCA	ACCAAAGGACGACCAATGGGAAT
Csa-SPL3	CCACCAAACTACCTTATCAAGCG	GAAAGCACGAGGAATGTGAACTA
Csa-SPL4	TAACGCCTCCATTTCTTTGCTCC	TAGATGGGGATTGATTTCCAGGATT
Csa-SPL5	GCGACTGGAAAAGCTGATGGAA	ATCCCCTGAAAACCCTAATTCTGC
Csa-SPL6	GAAGAACGAATCATGGGCCAACT	TCCGAAATCATCCGTCCCTCC
Csa-SPL7	CAGTCCCATCTGAGTGTTCCAGT	CTTCCTCCATTTCAAGACCCA
Csa-SPL8	GAAGGAAGGAGTAATGCGATGGA	GAACCGTGGCAGTGAAAAGAG
Csa-SPL9	TCCAGAAACCGAACGACGACT	GCATGTGAAACTTGGTGATTGAGAC
Csa-SPL10	AGAGCCAAGGGGAAAGCACAA	CTTCCTGGAGGAGCCATCTGAAT
Csa-SPL11	ATCACCTACTACTCTTGGTCGTTGT	CTTAGCCTTGGTTGCGAAGA
Csa-SPL12	TATGTTGCCCGGTTCGTTGT	CCGTTTCCGAGCACTTTCTG
Csa-SPL13	CTAACTCATCGCAACTGATTCAAAG	TGGTTAAGGCAGTCTAGTGACATTCT
Csa-SPL14	CTCAGAAGATCAAGGTTGGGAGG	ACCACCCCGAGCACTGGTTA
Csa-MIR156A	GTATTGAAAATTAGAGTAAGGGGAAG	CTTCAAGCATGAACCCTAACAT
Csa-MIR156B	GAGAGAAAAGCACAAAAGACCAAGA	CTAAGTAAAGGTATGAACTTTCAACTT
基因名称 Gene name	正向引物（5′-3′） Forward primer	反向引物（5′-3′） Reverse primer
Csa-MIR156C	GTTTATCGTTCTTGAATTTGGTTAG	ATACACTTTATAGCAACTGCCTCTG
Csa-MIR156D	TGAATGATGGTGAGTGTGTTAGGAG	CACGCACCCGCAAAGGTAT
Csa-MIR156E	AAAGGTGATTAAAATTGAGGGGAA	TGGCATATTATTATACCAAAAGCCT
Csa-MIR156F	CATACGGAAGGTAATCTCAAGGA	AATATCTAGCATAAGCGGCCATG
Csa-MIR156G	TCTCACGGTCTATTTCTAACACG	CACAAAGCTTCGAGCATTGATAT
Csa-MIR157A	GGGTTTAGAAATTTGGAGAGAGACA	TATTAAAAGTGAAGGGATGGGATATA
Csa-MIR157B	ATTTATCATGCACAAGGGAGAACTT	AATTGCTATTAGTGGTACCGATTGA
Csa-MIR157C	CCAATACGGTGATAGCTATATGTTGT	TAACAAAGTGGTGTTTACGACTCTA
Csa-Tub	CACTACACCGTTGGAAAGGAAA	CAAAAGGAGGGAGCCGAGA
Csa-U6	GGGGACATCCGATAAAATT	TGTGCGTGTCATCCTTGC
Csa-miR156	GCGGCGGTGACAGAAGAGAG	GTGCAGGGTCCGAGGT
Csa-miR156 RT	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCAC	TGGATACGACGTGCTC

基因名称 Gene name	基因编号 Gene ID	长度/bp Length	染色体 Chromosome	位置 Location
Csa-SPL1	Csa1G001450	379	Chr1	237765..239802 (+)
Csa-SPL2	Csa1G015680	550	Chr1	2088067..2093861 (+)
Csa-SPL3	Csa1G039890	548	Chr1	3934043..3940006 (-)
Csa-SPL4	Csa1G051590	314	Chr1	6001183..6004194 (-)
Csa-SPL5	Csa1G074980	162	Chr1	7700146..7703682 (+)
Csa-SPL6	Csa3G117960	340	Chr3	6445761..6448246 (+)
Csa-SPL7	Csa3G567830	344	Chr3	22275377..22277440 (+)
Csa-SPL8	Csa3G664550	1 013	Chr3	25953081..25964518 (+)
Csa-SPL9	Csa3G809420	382	Chr3	30985575..30991777 (+)
Csa-SPL10	Csa4G631590	202	Chr4	20657444..20658877 (-)
Csa-SPL11	Csa4G664590	1 031	Chr4	23212471..23218465 (+)
Csa-SPL12	Csa6G094760	328	Chr6	6509944..6513210 (+)
Csa-SPL13	Csa6G109120	297	Chr6	7313479..7317016 (-)
Csa-SPL14	Csa6G517960	141	Chr6	27284755..27286747 (+)
Csa-MIR156A		127	Chr1	13072567..13072694 (+)
Csa-MIR156B		112	Chr2	1701701..1701813 (+)
Csa-MIR156C		121	Chr2	19844384..19844505 (+)
Csa-MIR156D		175	Chr4	22021976..22022151 (+)
Csa-MIR156E		138	Chr4	22620675..22620813 (-)
Csa-MIR156F		89	Chr1	22345006..22345095 (+)
Csa-MIR156G		148	Chr6	16464371..16464519 (-)
Csa-MIR157A		165	Chr1	2522433..2522598 (+)
Csa-MIR157B		177	Chr3	22790035..22790212 (-)
Csa-MIR157C		85	Chr2	20893540..20893625 (-)

基因名称 Gene name	基因编号 Gene ID	长度/bp Length	染色体 Chromosome	位置 Location
Csa-SPL1	Csa1G001450	379	Chr1	237765..239802 (+)
Csa-SPL2	Csa1G015680	550	Chr1	2088067..2093861 (+)
Csa-SPL3	Csa1G039890	548	Chr1	3934043..3940006 (-)
Csa-SPL4	Csa1G051590	314	Chr1	6001183..6004194 (-)
Csa-SPL5	Csa1G074980	162	Chr1	7700146..7703682 (+)
Csa-SPL6	Csa3G117960	340	Chr3	6445761..6448246 (+)
Csa-SPL7	Csa3G567830	344	Chr3	22275377..22277440 (+)
Csa-SPL8	Csa3G664550	1 013	Chr3	25953081..25964518 (+)
Csa-SPL9	Csa3G809420	382	Chr3	30985575..30991777 (+)
Csa-SPL10	Csa4G631590	202	Chr4	20657444..20658877 (-)
Csa-SPL11	Csa4G664590	1 031	Chr4	23212471..23218465 (+)
Csa-SPL12	Csa6G094760	328	Chr6	6509944..6513210 (+)
Csa-SPL13	Csa6G109120	297	Chr6	7313479..7317016 (-)
Csa-SPL14	Csa6G517960	141	Chr6	27284755..27286747 (+)
Csa-MIR156A		127	Chr1	13072567..13072694 (+)
Csa-MIR156B		112	Chr2	1701701..1701813 (+)
Csa-MIR156C		121	Chr2	19844384..19844505 (+)
Csa-MIR156D		175	Chr4	22021976..22022151 (+)
Csa-MIR156E		138	Chr4	22620675..22620813 (-)
Csa-MIR156F		89	Chr1	22345006..22345095 (+)
Csa-MIR156G		148	Chr6	16464371..16464519 (-)
Csa-MIR157A		165	Chr1	2522433..2522598 (+)
Csa-MIR157B		177	Chr3	22790035..22790212 (-)
Csa-MIR157C		85	Chr2	20893540..20893625 (-)