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光合作用相关的核基因(photosynthesis-associated nuclear genes,Ph ANGs)可以响应包括光照、干旱、糖以及脱落酸(abscisic acid,ABA)等多种环境信号,目前已发现并报道了多种Ph ANGs启动子上关于光调控的顺式作用元件,但是关于干旱以及ABA等响应元件仍然缺乏系统性研究。本研究主要对核酮糖-1,5-二磷酸羧化酶小亚基基因(ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit,RBCS)启动子区域响应逆境胁迫的顺式作用元件进行分析。首先从中国春小麦(Triticum aestivum)基因组中分离得到Ta RBCS11基因上游1 839 bp的核苷酸序列,对该启动子进行结构分析的结果表明,转录起始位点位于起始密码子上游59 bp;经Plant CARE数据库分析,发现该启动子序列上除大量保守光调控元件外(如BoxⅠ、G-Box、I-box和GATA-motif等),还包含多种应答干旱、盐及激素等逆境因子的保守顺式作用元件(如MYB结合位点(MYB binding site,MBS)、CCAAT-box、乙烯响应元件(ethylene-responsive element,ERE)和赤霉素应答元件(gibberellin-responsive element,GARE-motif)等);从小麦基因组数据库分离得到Ta RBCS11、Ta RBCS13、Ta RBCS10和Ta RBCS14启动子序列,序列比对显示,其具有极高的同源性,同时还发现多个保守光调控元件(如MNF1、GATA-motif、I-box、G-Box和Sp1)位于这4段启动子近侧区(-200bp~ATG);根据Ta RBCS11启动子结构特征及调控元件分布情况,设计5条上游引物及1条下游引物用于启动子缺失片段扩增,长度分别为1 656、1 258、866、533和277 bp;随后对该启动子5’端进行缺失并构建了5个不同长度片段融合报告基因β-葡萄糖醛酸酶基因(β-glucuronidase,GUS)的缺失表达载体,利用农杆菌(Agrobacterium tumefaciens)介导的烟草(Nicotiana tabacum)叶片瞬时表达体系研究酶活变化情况。组织化学染色与酶活分析表明,光能明显提高GUS酶活,进一步分析表明,随启动子片段的缩短,启动子片段上光调控元件缺失,启动子活性依次下降;此外,对不同启动子缺失材料进行干旱、高盐、ABA、黑暗及恢复光照处理,发现干旱、高盐、ABA和黑暗均对全长启动子(P1656)有显著的抑制作用,-804~-474 bp的区域对该段启动子响应干旱及高盐胁迫具有重要作用,而参与ABA响应的核心调控区域位于-1 597~-1 198bp。本研究验证了Ta RBCS11基因的表达受干旱、高盐、ABA和光调控影响,并鉴定出其启动子上参与响应非生物胁迫的重要调控区域,研究结果为今后深入研究Ph ANGs表达调控机制提供了重要参考。
Photosynthesis-associated nuclear genes (Ph ANGs) can respond to various environmental signals including light, drought, sugar and abscisic acid (ABA). Up to date, many Ph ANGs have been reported and reported There are still a lack of systematic studies on response elements such as drought and ABA. In this study, the cis-acting elements of ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit (RBCS) promoter region under stress response were analyzed . First, a nucleotide sequence of 1 839 bp upstream of the Ta RBCS11 gene was isolated from the Chinese Triticum aestivum genome. The structural analysis of the promoter showed that the transcription start site was 59 bp upstream of the start codon. The results of Plant CARE database showed that the promoter sequence contained a number of stress factors such as BoxⅠ, G-Box, I-box and GATA-motif, (Such as MYB binding site (MBS), CCAAT-box, ethylene-responsive element (ERE) and gibberellin-responsive element (GARE-motif) ), Etc.); Ta RBCS11, Ta RBCS13, Ta RBCS10 and Ta RBCS14 promoter sequences were isolated from the wheat genome database. The sequence alignment showed that they have very high homology and many conserved light regulatory elements MNF1, GATA-motif, I-box, G-Box and Sp1) located in the proximal promoter region (-200bp ~ ATG). According to the structural characteristics of Ta RBCS11 promoter and its regulatory elements, five upstream primers And a downstream primer for The deletion fragments of the promoter were 1 656, 1 258, 866, 533 and 277 bp, respectively. Then, the 5 ’end of the promoter was deleted and 5 different length fragment fusion reporter genes, β-glucuronidase (GUS). The transient expression system of tobacco (Nicotiana tabacum) mediated by Agrobacterium tumefaciens was used to study the changes of enzyme activity. Histochemical staining and enzyme activity analysis showed that light could significantly increase GUS activity. Further analysis showed that with the shortening of the promoter fragment, the light regulatory elements were deleted and the promoter activity was decreased in turn. In addition, The results showed that drought, salt, ABA and dark all had significant inhibitory effect on the full-length promoter (P1656), and the region from -804 to -474 bp was sensitive to drought, salt, ABA, darkness and light restoration. The promoter plays an important role in response to drought and salt stress, while the core regulatory region involved in ABA response is located at -1 597-1198 bp. This study demonstrated that the expression of Ta RBCS11 gene is affected by drought, high salt, ABA and light regulation, and identified important regulatory regions on its promoter involved in abiotic stress. The results provide the basis for further study on the regulatory mechanism of Ph ANGs in the future Important reference.