葡萄VvWRKY13介导乙烯生物合成调控果实发育

doi:10.7668/hbnxb.20194673

摘要/Abstract

摘要：

果实发育是决定葡萄产量和质量的关键时期,WRKY家族转录因子在调节植物生长发育和环境适应过程中发挥重要作用。乙烯是调控果实发育的重要植物激素,ACC合成酶是调节乙烯合成的关键酶。为研究葡萄WRKY家族转录因子VvWRKY13在果实发育过程中的作用及其与乙烯之间的作用关系,以葡萄品种左优红、VvWRKY13过表达葡萄愈伤组织以及转VvWRKY13番茄株系为材料,利用植物生理生化方法及分子生物学技术进行研究。结果表明,在葡萄果实发育早期VvWRKY13、ACC合成酶基因VvACS2和VvACS7表达量均显著上调;VvWRKY13过表达葡萄愈伤组织中VvACS7表达量显著高于对照,VvACS2与对照差异不显著;酵母单杂交试验证明,VvWRKY13可与VvACS7启动子直接结合,与VvACS2无直接作用。同时,异源过表达VvWRKY13番茄中乙烯含量与合成酶基因ACS家族一些成员,如 SlACS1b、SlACS4和SlACS6,表达量显著高于野生型,番茄开花—果实转色的时间比野生型缩短3~6 d。以上结果表明,VvWRKY13能够通过促进ACC合成酶基因表达调控乙烯合成,进而促进果实发育。

关键词: 葡萄, VvWRKY13, VvACS7, 乙烯, 果实发育

Abstract:

Fruit development is the key stage which determines the yield and quality of grape.WRKY family transcription factors play important roles in regulating plant development and environment adaptation.Ethylene is the important plant hormone which participates in regulation of fruit development,and ACC synthase is the key enzyme that limits ethylene synthesis.Using grape variety Zuoyouhong, VvWRKY13 overexpressing grape callus,as well as VvWRKY13 heterologous over-expressed tomato lines as materials,the role of VvWRKY13 from grape in fruit development and its relationship with ethylene were studied though plant physiological and biochemical methods as well as molecular biological techniques.The results displayed that the expressions of VvWRKY13 as well as ACC synthase genes VvACS2 and VvACS7 significantly upregulated at early stage of fruit development,and in VvWRKY13 over-expression grape callus,the expression of VvACS7C was significantly higher than control,but the expression of VvACS2 displayed no significant difference compared with control.Yeast one hybrid experiment showed that VvWRKY13 could directly bind to the VvACS7 promoter,VvWRKY13 had no direct interaction with VvACS2.We also found that the ethylene content and the expression of ACS family members,such as SlACS1b,SlACS4 and SlACS6,the ethylene synthesis genes,were significantly induced in VvWRKY13 heterologous over-expressed tomato,and the time from flowering to breaking of tomato was 3—6 days shorter than wild type;the above results indicated that,VvWRKY13 could regulate ethylene synthesis by promoting the expression of ACC synthase gene to participate in regulation of fruit development.

Key words: Vitis vinifera, VvWRKY13, VvACS7, Ethylene, Fruit development

车永梅, 陈慧婷, 张岁芳, 惠梦玲, 叶青, 侯丽霞, 刘新. 葡萄VvWRKY13介导乙烯生物合成调控果实发育[J]. 华北农学报, 2024, 39(2): 99-105. doi: 10.7668/hbnxb.20194673.

CHE Yongmei, CHEN Huiting, ZHANG Suifang, HUI Mengling, YE Qing, HOU Lixia, LIU Xin. VvWRKY13 from Vitis vinifera Regulate Fruit Development by Controlling Ethylene Biosynthesis[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(2): 99-105. doi: 10.7668/hbnxb.20194673.

http://www.hbnxb.net/CN/Y2024/V39/I2/99

图/表 8

表1 定量PCR引物序列

Tab.1 Quantitative PCR primer sequence

引物名称 Primer name	引物序列(5'—3') Primer sequences(5'—3')
VvWRKY13-FP	GGTTGCCAACAATCCCT
VvWRKY13-RP	GTCATCTCCACCGATACTTC
VvActin-FP	ATAGAAGCAGCAAGGGA
VvActin-RP	TGAGGCTCTTACTAATG
VvACS2-FP	GAGGTGGAGAACAGGAGTGC
VvACS2-RP	GAGCAAGCCTTTCACTTTGG
VvACS7-FP	GATGAAGTTTACGCTGGAACTGTG
VvACS7-RP	CCAGAGTCGGAAAGAAGGCAC
SlACS2-FP	GGAGGTTCGTAGGTGTTGAG
SlACS2-RP	ATGGTGAGGGAGGAATAGG
SlACS4-FP	GGGTTATTCAGATGGGTCTC
SlACS4-RP	TCCACCAGCCATTACTACAC
SlACS6-FP	GCCACAAATGATGGACATGG
SlACS6-RP	GAGGCTTTTGGGTTGTTGACT
SlACS1a-FP	CATTGCTCAGAGCCAGG
SlACS1a-RP	AAGACATAGGTGAATGAGGAG
SlACS1b-FP	TGTTCTGAACCTGGTTGGT
SlACS1b-RP	TAAAATCATCCAATCTTCGA
SlActin-FP	GGAATGGGACAGAAGGAT
SlActin-RP	CAGTCAGGAGAACAGGGT

图1 葡萄果实不同发育阶段VvWRKY13相对表达量不同小写字母表示差异显著(P<0.05)。图2—3,5同。

Fig.1 The relative expression level of VvWRKY13 in grape fruit at different development stage Different lowercase letters indicate significant differences at 0.05 level.The same as Fig.2—3,5.

图2 葡萄果实发育过程中乙烯合成关键酶基因相对表达量变化

Fig.2 The transcription profiels of ethylene biosynthesis genes during the development of grape fruit

图3 转VvWRKY13葡萄愈伤组织中乙烯合成基因表达变化 A.转VvWRKY13葡萄愈伤组织(OEWRKY13);B.OEWRKY13中VvWRKY13表达量;C.OEWRKY13乙烯合成基因表达变化。

Fig.3 Expression levels of ethylene biosynthesis genes in VvWRKY13 over-expressing grape callus A.VvWRKY13 over-expression grape callus(OEWRKY13);B.The relative expression of VvWRKY13 in OEWRKY13;C.Relative expression of ethylene biosynthesis genes in OEWRKY13.

图4 酵母单杂交试验验证VvWRKY13与VvACS2(A)及VvACS7(B)启动子之间的相互作用

Fig.4 The binding activity of VvWRKY13 to promoters of VvACS2 (A)and VvACS7 (B)in a yeast one-hybrid system

图5 VvWRKY13异源表达番茄果实中乙烯含量及乙烯合成关键酶基因表达变化

Fig.5 Ethylene content and expression levels of ethylene biosynthesis genes in fruit of VvWRKY13 ectopic over-expressing tomato plants

图6 VvWRKY13异源表达番茄果实 dpa.开花后天数;dpk.断裂后天数。

Fig.6 The fruit of VvWRKY13 ectopic over-expressing tomato dpa.Days post anthesis;dpk.Days post breaking.

表2 VvWRKY13异源表达番茄从开花到果实转色及转色到成熟的时间

Tab.2 The times from flowering to breaking and breaking to maturity of VvWRKY13 ectopic over-expressing tomato

株系 Lines	开花—转色时间/d Time from flowering to breaking	转色—成熟时间/d Time from breaking to maturity
WT	42.67±1.15a	10±1.15a
L3	36.33±1.52b	10±1.52a
L9	39.33±4.0ab	10±4.04a
L14	36.33±1.52b	10±1.52a

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