辣椒MADS-box转录因子CaRIN基因克隆、表达与功能分析

Cloning,expression,and functional analysis of the CaRIN gene,encoding a MADS-box transcription factor from pepper

【目的】探究MADS-box家族基因RIN的表达特征和功能,解析其对辣椒类胡萝卜素代谢的影响。【方法】基于辣椒果实发育转录组,通过RT-PCR克隆辣椒MADS-box转录因子CaRIN基因CDS全长,分析其生物信息学、表达模式、亚细胞定位、转录活性等,并探讨VIGS诱导CaRIN基因沉默对类胡萝卜素代谢的影响。【结果】(1)CaRIN基因CDS全长732 bp,编码243个氨基酸,蛋白分子质量为27.95 kD,等电点(pI)为7.06,其编码蛋白具有典型的MEF2_like MADS结构域,属MICK型转录因子。(2)CaRIN基因主要在花和果实中表达,具有组织特异性;CaRIN定位在细胞核,并且具有转录激活活性。(3)CaRIN基因启动子具有ABRE等多个激素应答元件,外源脱落酸和乙烯利均能加速果实转红,诱导CaRIN及相关基因高表达。(4)VIGS诱导沉默CaRIN基因后,类胡萝卜素代谢途径基因PSY1、CCS、PDS、CRTZ、LCYB和NCED1表达水平降低为对照组的0.27~0.59倍,且果实总类胡萝卜素含量(0.379 mg/g)较对照(0.650 mg/g)显著降低。【结论】CaRIN是辣椒果实类胡萝卜素代谢过程的重要调控因子。

[Objective]The study aims to explore the expression and function of the RIN gene,encoding a MADS-box,and analyze its impact on the metabolism of carotenoids in pepper.[Methods]Based on the fruit development transcriptome of pepper,the full-length CDS of the MADS-box transcription factor CaRIN gene related to chili ripening was cloned.The bioinformatics,expression pattern,subcellular localization,and transcriptional activation activity were examined,and the effects of VIGS-induced CaRIN gene silencing on carotenoid metabolism were investigated.[Results](1)The CDS of CaRIN gene was 732 bp in length.CaRIN gene encoded a protein containing 243 amino acids with a molecular mass of 27.95 kD and an isoelectric point(pI)of 7.06.CaRIN protein had a typical MEF2_like MADS domain,belonging to the MICK type transcription factor.(2)CaRIN gene was mainly expressed in flowers and fruits with tissue specificity.CaRIN was located in the nucleus and had transcriptional activation activity.(3)The promoter of CaRIN gene had several hormone response elements such as ABRE,and exogenous ABA and ethephon accelerated the fruit to turn red and induced the high expression of CaRIN and related genes.(4)After VIGS-induced gene silencing of CaRIN,the expression level of PSY1,CCS,PDS,CRTZ,LCYB,and NCED1,genes involved in carotenoid metabolic pathway,was decreased by 0.27-0.59 times,and the total carotenoid content in fruits(0.379 mg/g)was decreased compared with the control(0.650 mg/g).[Conclusion]CaRIN may be an important regulatory factor of carotenoid metabolism in capsicum fruits.