荔枝LcWRKY47基因的克隆、亚细胞定位、表达及延缓果实褐变的功能初探

Preliminary Study on the Cloning,Subcellular Localization and Expression of LcWRKY47 in Litchi and its Function in Delaying Fruit Browning

荔枝(LitchichinensisSonn.)是亚热带地区重要的经济作物之一,但荔枝采后难贮存、易褐变,是目前保鲜过程中存在的最大问题之一。WRKY家族是植物中常见的一类转录因子,主要调控植物的逆境胁迫响应以及成熟衰老等生理过程。为了探究WRKY对采后荔枝果实成熟衰老的影响,本研究以妃子笑荔枝为试验对象,克隆LcWRKY47基因,长度为1077 bp;LcWRKY47编码的蛋白中含有WRKY保守结构域,二级结构以无规则卷曲为主,占比65.64%,三级结构主要为无规则卷曲和α-螺旋。通过motif分析及多序列比对,发现LcWRKY47蛋白属于第Ⅱ类WRKY转录因子;系统进化分析树表明荔枝LcWRKY47与同为无患子科的龙眼DlWRKY47亲缘关系最近;亚细胞定位试验表明,LcWRKY47蛋白定位在细胞核中;RT-qPCR分析表明,2mmol/L草酸处理后LcWRKY47基因在荔枝果皮和果肉中的表达趋势相同,均先升后降,后期处理组表达量均显著高于对照组,推测草酸处理后该转录因子在荔枝果实成熟衰老过程中起正调控作用。过表达LcWRKY47基因发现,转基因果实中LcWRKY47的表达量显著高于对照果实,且转基因荔枝衰老褐变情况明显优于处理组。综上推测,LcWRKY47转录因子可能在荔枝衰老与褐变过程中起着调节作用。

Litchi(Litchi chinensis Sonn.)is one of the important economic crops in subtropical areas.However,it is difficult to store litchi after picking and easy to browning,which is one of the biggest problems in the preservation process.WRKY family is a common transcription factor in plants,which mainly regulates physiological processes such as plant stress response and maturation.In order to research the effect of WRKY on the ripening and senescence process of litchi fruit after harvest,this study took Feiziao litchi as the experimental object,and cloned LcWRKY47 gene.The gene length was 1077 bp.The protein contains WRKY conserved domain.The secondary structure is dominated by random curling,accounting for 65.64%,and the tertiary structure is dominated by random curling andα-helix.Through motif analysis and multiple sequence alignment,LcWRKY47 protein was found to be a class II WRKY transcription factor.Phylogenetic analysis showed that LcWRKY47 in litchi was closely related to DlWRKY47 in longan,which are all classified as Sapindaceae.Subcellular localization experiments showed that LcWRKY47 protein was localized in the nucleus.RT-qPCR analysis showed that after treatment with 2 mmol/L oxalic acid,the expression of LcWRKY47 gene in the peel and pulp of litchi showed the same trend,which increased first and then decreased,and the expression level of LcWRKY47 gene in the treatment group was significantly higher than that in the control group after storage,suggesting that this transcription factor plays a positive role in the regulation of oxalic acid in the ripening and senescence process of litchi fruit.Overexpression of LcWRKY47 gene showed that the expression level of LcWRKY47 in transgenic fruits was significantly higher than that in control fruits,and the senescence process and browning of transgenic litchi was significantly better than that in treatment group.In conclusion,LcWRKY47 transcription factor may play a regulatory role in the senescence and browning process of litchi.