三孢布拉霉crgA启动子的克隆和活性分析

Cloning and activity of the crgA promoter from Blakeslea trispora

摘要:

【背景】CrgA是三孢布拉霉(Blakeslea trispora, Bt)中调控类胡萝卜素合成的关键负调控因子，其表达水平会影响类胡萝卜素的合成。【目的】克隆三孢布拉霉crgA启动子并分析其活性，为进一步解析CrgA表达调控机制奠定基础。【方法】通过综合微生物基因组(integrated microbial genomes, IMG)数据库提供的基因组序列，克隆crgA翻译起始位点上游2 000 bp序列，分析其顺式调控元件和转录起始区域预测，通过RT-qPCR分析不同光照时间对三孢布拉霉crgA相对转录水平的影响；构建4个不同长度的crgA启动子截短序列驱动的GUS-mGFP5重组表达载体p1303-procrgAF、F1、F2和F3，利用农杆菌侵染整合到三孢布拉霉基因组中，在黑暗和光照条件下测定β-d-葡萄糖苷酸酶(β-d-glucuronidase, GUS)酶活性并观察荧光信号。【结果】crgA启动子不仅包含基础的TATA-box和CAAT-box元件，还包括多个与光响应相关的元件。观察荧光结果显示CaMV35S和构建的4个突变启动子均能在三孢布拉霉体内驱动下游基因表达，检测GUS酶活性发现procrgAF3活性最强，且截短的启动子活性均强于CaMV35S，btcrgA启动子序列在1 499−989 bp范围内有激活下游基因表达的光响应元件。【结论】三孢布拉霉的crgA表达具有“光激活”和“光适应”现象，crgA启动子核心区位于procrgAF3序列，btcrgA启动子上具有响应光调控的顺式作用元件位于1 499−989 bp。

Abstract:

[Background] CrgA is a key negative regulator of carotenoid synthesis in Blakeslea trispora (Bt). [Objective] To clone and determine the activity of the crgA promoter from B. trispora, laying a foundation for deciphering the regulatory mechanism of CrgA expression. [Methods] According to the genome sequence in the integrated microbial genomes (IMG), we cloned a sequence of 2 000 bp upstream of the translation initiation site of crgA, identified the cis-regulatory elements, predicted the transcription initiation region, and then Analysis of crgA transcription level of B. trispora under different light time by RT-qPCR. Furthermore, we constructed four recombinant expression vectors p1303-procrgAF, F1, F2, and F3 driven by truncated sequences of crgA promoters with different lengths, and integrated them into the genome of B. trispora via Agrobacterium tumefaciens. Under dark and light conditions, the fluorescence signals were observed and the β-d-glucuronidase (GUS) activity was determined. [Results] The crgA promoter contained not only the basic elements TATA-box and CAAT-box but also multiple elements related to light response. Both CaMV35S and the constructed four mutant promoters could drive the expression of downstream genes in B. trispora. The GUS activity indicated that procrgAF3 had the strongest activity, and the truncated promoter had stronger activity than CaMV35S. The btcrgA promoter sequence has a light-responsive element at 1 499−989 bp that activates downstream gene expression. [Conclusion] The transcription of crgA in B. trispora presented the phenomena of light activation and light adaptation. The core region of the crgA promoter was identified as procrgAF3, and the cis-acting elements on the btcrgA promoter that responded to light regulation were located in the region of 1 499−989 bp.