中国农业科学 ›› 2020, Vol. 53 ›› Issue (9): 1890-1903.doi: 10.3864/j.issn.0578-1752.2020.09.015
白云赫,王文然,董天宇,管乐,宿子文,贾海锋,房经贵,王晨()
收稿日期:
2019-09-05
接受日期:
2019-12-25
出版日期:
2020-05-01
发布日期:
2020-05-13
通讯作者:
王晨
作者简介:
白云赫,E-mail:2017104028@njau.edu.cn。
基金资助:
YunHe BAI,WenRan WANG,TianYu DONG,Le GUAN,ZiWen SU,HaiFeng JIA,JingGui FANG,Chen WANG()
Received:
2019-09-05
Accepted:
2019-12-25
Online:
2020-05-01
Published:
2020-05-13
Contact:
Chen WANG
摘要:
【目的】研究vvi-miR160家族(vvi-miR160s)及其靶基因在‘魏可’葡萄种子发育过程中的作用,探究其应答赤霉素(GA)调控葡萄果实无核的潜在机理。【方法】采用miR-RACE、生物信息学分析、RT-qPCR、RLM-RACE等方法,鉴定vvi-miR160家族成员及其靶基因,分析vvi-miR160s及其靶基因应答GA的时空表达模式及其潜在功能。【结果】花前GA处理强烈抑制‘魏可’葡萄胚珠及种子发育,高效诱导其无核,且无核率达99.8%。克隆鉴定了VvMIR160s前体基因序列(501 bp)及成熟体序列,且它们在不同物种间具有较高的保守性;基于vvi-miR160s序列,预测到靶基因VvARF18,利用RLM-RACE技术检测到vvi-miR160s对VvARF18的裂解位点在第10和第11位之间,裂解频度9/17,证明VvARF18是vvi-miR160s的真实靶基因。该靶基因编码683个氨基酸,在398—411位存在核定位信号,且该蛋白亚细胞定位于细胞核上。VvARF18与其他物种间序列的同源保守性较高,基因结构相似,其中VvARF18蛋白与茶、烟草、梅花等物种亲缘关系较近。VvARF18启动子中包含4种作用元件,且含有较多激素相关元件。RT-qPCR结果显示,随着葡萄果实的发育,vvi-miR160c/d/e呈现‘V’形表达趋势,在硬核种子发育期表达较低,而VvARF18表现出与前者相反的表达趋势,在硬核种子发育期呈现高表达,表明vvi-miR160c/d/e对VvARF18负调控,但vvi-miR160a/b与VvARF18表达水平却不存在明显负相关。GA处理极显著地上调了vvi-miR160a/b在葡萄硬核种子发育期的表达,同时也显著抑制了VvARF18在同时期的表达,且它们的表达水平呈负相关,表明GA处理促进了vvi-miR160a/b对VvARF18的负调控作用;相反,GA减弱了vvi-miR160c/d/e对VvARF18的负调控作用。【结论】在vvi-miR160家族中,vvi-miR160c/d/e可能介导VvARF18在葡萄种子发育的特定阶段调控种子的发育形成,而vvi-miR160a/b可能主要介导VvARF18参与调控GA诱导葡萄种子败育的过程。
白云赫,王文然,董天宇,管乐,宿子文,贾海锋,房经贵,王晨. vvi-miR160s介导VvARF18应答赤霉素调控葡萄种子的发育[J]. 中国农业科学, 2020, 53(9): 1890-1903.
YunHe BAI,WenRan WANG,TianYu DONG,Le GUAN,ZiWen SU,HaiFeng JIA,JingGui FANG,Chen WANG. vvi-miR160s in Mediating VvARF18 Response to Gibberellin Regulation of Grape Seed Development[J]. Scientia Agricultura Sinica, 2020, 53(9): 1890-1903.
表1
VvMIR160s PCR扩增引物序列"
基因名称 Gene name | 正向引物序列 Forward primer sequence | 反向引物序列 Reverse primer sequence |
---|---|---|
VvMIR160a | ACACCTCCTAAAATCATTGTCTG | CTTGTGACATGAATATGGTGCG |
VvMIR160b | CTATGTATTTGTCTTGTTCTGATTGAA | TGAATGGTCACAGTTCTTTGG |
VvMIR160c | GGCCTGGCCTCTATAAATATCA | AATCGACCCACAATCAAACC |
VvMIR160d | GATGTGGTGCTTCGCCAAT | ATGTGGGTTTTCTAAATGCCTAACC |
VvMIR160e | CACTCACTCACACCCTTCC | ATATTATATTCTCTCTGCAGCCAAG |
表2
vvi-miR160s及VvARF18定量引物序列"
基因名称 Gene name | 正向引物序列 Forward primer sequence | 反向引物序列 Reverse primer sequence |
---|---|---|
vvi-miR160a | TGACCTTTGTGCTTCAGTGG | GCTATCTGGGTTGACCTCCA |
vvi-miR160b | TTCTGCAGGAGATGGAGCTT | AGTGTTTCGCCTGCTTGACT |
vvi-miR160c | CCACATTCCGTGACCTTTCT | GCACAACCCATTTCACCTTT |
vvi-miR160d | CGCCAATGCAGGAAATTTAT | GGGAGCCAGGCATGTAAGTA |
vvi-miR160e | CTGTATGCCATTTGCAGAGC | GGGGGAGAAGATTGAAGAGG |
VvARF18 | CTGAACACGCCTATGGGAAT | CCGTTTCACCCTCAGTGTTT |
图3
microR160s进化分析和成熟体序列比对 Vv:葡萄Vvtis vinifera;Ha:向日葵Helianthus annuus L;Zm:玉米Zea mays;Ta:小麦Triticum aestivum;Sb:高粱Sorghum bicolor;Os:水稻Oryza sativa;Nt:烟草Nicotiana tabacum;Sl:番茄Solanum lycopersicum;Pt:毛果杨Populus trichocarpa;Cs:柑橘Citurs sinensis;Md:苹果Malus domestica;Gh:棉花Gossypium herbaceum;Cm:甜瓜Cucumis melo;At:拟南芥Arabidopsis thaliana;Ah:花生Arachis hypogaea;Fv:草莓Fragaria vesca"
表3
VvARF18基因启动子顺式作用元件"
元件类型 Component type | 相关元件 Related component | 数量 Number | 功能注释 Functional comment |
---|---|---|---|
光响应元件 Photoresponsive element | 3-AF1 binding site | 1 | 光响应元件Photoresponsive element |
AE-box | 1 | 光反应元件Photoresponsive element | |
Box 4 | 7 | 光响应元件Photoresponsive element | |
G-box | 2 | 光响应元件Photoresponsive element | |
GT1-motif | 6 | 光响应元件Photoresponsive element | |
激素响应元件 Hormone response element | ABRE | 2 | 脱落酸响应元件Abscisic acid response element |
AuxRR-core | 1 | 生长素响应元件Auxin response element | |
CGTCA-motif | 1 | 茉莉酸甲酯响应元件Methyl jasmonate response element | |
TGACG-motif | 1 | 茉莉酸甲酯响应元件Methyl jasmonate response element | |
P-box | 1 | 赤霉素响应元件Gibberellin response element | |
胁迫相关元件 Stress related component | ARE | 3 | 厌氧诱导元件Anaerobic inducing element |
LRT | 1 | 低温诱导元件Low temperature inducing element | |
结合位点 Bonding component | CCAAT-box | 1 | MYBHv1结合位点MYBHv1 binding site |
MBS | 2 | MYB参与干旱诱导位点MYB participates in drought induction sites |
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