具有不同抗病性的鹰嘴豆基因型中 CaDRRG 基因启动子区域的硅内分析和基因组追踪

IF 3.9 3区 生物学 Q1 PLANT SCIENCES
Farhad Shokouhifar, Mojtaba Mamarabadi, Narges Sadeghi, Azam Kaseb
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引用次数: 0

摘要

鉴定早期反应基因对确定植物的病原体防御系统非常有用。从鉴定早期反应性诱导启动子及其在抗性栽培品种育种中的应用方面来看,它也具有极大的意义。在本研究中,根据鹰嘴豆基因对引起鹰嘴豆疫霉病的真菌 Ascochyta rabiei 感染的表达数据分析,确定了一个在接种该病原体 3 小时后具有高诱导性的转录本。通过比较转录组和基因组数据,确定了该转录本在鹰嘴豆基因组上的位置,并选择了相应的预测基因序列。然而,将这些蛋白质与鹰嘴豆基因组中的预测蛋白质序列进行比较后,上述转录本被命名为 CaDRRG 进行研究。为了分析该基因诱导启动子的序列,从基因组数据中检索了其上游序列,并确定了共识调控元件(如 TATA-框和 CAAT-框)的位置。此外,还确定了启动子序列中已知的可诱导病原体的顺式调控元件(如 AS-1、W-box 和 G-box)的位置和数量。此外,通过将 CaDRRG 基因的上游序列与其他对 A. rabiei 诱导水平最高的基因进行比较,在该基因的启动子序列中发现了三个新的潜在调控元件。在抗性和敏感鹰嘴豆基因型中对含有这些调控元件的片段进行了追踪和测序。通过对不同鹰嘴豆基因型中 CaDRRG 基因启动子序列的多重序列比对,在已确定的调控元件位置之外发现了几个点突变。在分析 CaDRRG 基因启动子序列诱导能力的初步实验中,克隆了该序列约 700 bp 的 beta-葡糖醛酸酶报告基因上游序列,并通过农业注射法研究了其在烟草叶片上的基础表达和对 A. rabiei 真菌提取物处理的诱导能力。通过评估真菌提取物处理后烟草叶盘中β-葡糖醛酸酶的活性并与基础表达水平进行比较,证实了该片段的诱导性,并与 CaMV 35S 组成型启动子的表达水平进行了比较。尽管初步分析结果表明,所选片段有可能用作表达抗雷伯菌抗性基因的诱导启动子,但仍有必要进行更多研究,以确定对这种真菌病原体做出反应的调控元件。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

In-Silico Analysis and Genomic Tracking of CaDRRG Gene Promoter Region in Chickpea Genotypes with Different Levels of Resistance to Ascochyta Blight

In-Silico Analysis and Genomic Tracking of CaDRRG Gene Promoter Region in Chickpea Genotypes with Different Levels of Resistance to Ascochyta Blight

Identification of early responsive genes is very useful in determining the plant's defense system against pathogens. It also has a great interest from the aspect of identifying early responsive inducible promoters and their application in the breeding of resistant cultivars. In the present study, based on the expression data analysis of chickpea genes against the infection with the fungus Ascochyta rabiei that causes chickpea Ascochyta blight, a transcript was identified with high inducibility 3 hours after inoculation with this pathogen. The position of this transcript on the chickpea genome was identified by comparing the transcriptomic and genomic data and the corresponding predicted gene sequence was selected. However, the comparison of these proteins with the predicted protein sequence in the chickpea genome led to the mentioned transcript being investigated under the name of CaDRRG. In order to analyze the sequence of this gene inducible promoter, its upstream sequence was retrieved from genomic data and the position of consensus regulatory elements such as TATA-box and CAAT-box was determined on it. Moreover, the position and number of Cis-regulatory elements known to be inducible against pathogens such as, AS-1, W-box, and G-box, were identified in the promoter sequence. In addition, by comparing the upstream sequence of CaDRRG gene with other genes with the highest induction level in response to A. rabiei, three new potential regulatory elements were identified in the promoter sequence of this gene. The fragment containing these regulatory elements was tracked and sequenced in resistant and sensitive chickpea genotypes. Multiple sequence alignment of the CaDRRG gene promoter sequence in different chickpea genotypes led to the identification of several point mutations outside the positions of the identified regulatory elements. In a preliminary experiment to analyze the induction capacity of the CaDRRG gene promoter sequence, about 700 bp of this sequence was cloned upstream of the beta-glucuronidase reporter gene and its basal expression and inducibility in response to the treatment of A. rabiei fungal extract was investigated on Nicotiana benthamiana leaves by agroinjection method. The assessment of beta-glucuronidase enzyme activity in tobacco leaf discs after treatment with fungal extract and its comparison with the basal expression level confirmed the inducibility of this fragment which was observationally compared with the expression level of CaMV 35S constitutive promoter. Although the results of the preliminary analysis showed that the selected fragment has the potential to be used as an inducible promoter for the expression of resistance genes against A. rabiei, more additional studies are necessary to identify the regulatory elements responsible for responding to this fungal pathogen.

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来源期刊
CiteScore
8.40
自引率
6.20%
发文量
312
审稿时长
1.8 months
期刊介绍: The Journal of Plant Growth Regulation is an international publication featuring original articles on all aspects of plant growth and development. We welcome manuscripts reporting question-based research on various aspects of plant growth and development using hormonal, physiological, environmental, genetic, biophysical, developmental and/or molecular approaches. The journal also publishes timely reviews on highly relevant areas and/or studies in plant growth and development, including interdisciplinary work with an emphasis on plant growth, plant hormones and plant pathology or abiotic stress. In addition, the journal features occasional thematic issues with special guest editors, as well as brief communications describing novel techniques and meeting reports. The journal is unlikely to accept manuscripts that are purely descriptive in nature or reports work with simple tissue culture without attempting to investigate the underlying mechanisms of plant growth regulation, those that focus exclusively on microbial communities, or deal with the (elicitation by plant hormones of) synthesis of secondary metabolites.
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