{"title":"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","authors":"Farhad Shokouhifar, Mojtaba Mamarabadi, Narges Sadeghi, Azam Kaseb","doi":"10.1007/s00344-024-11493-7","DOIUrl":null,"url":null,"abstract":"<p>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 <i>Ascochyta rabiei</i> 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 <i>CaDRRG</i>. 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 <i>CaDRRG</i> gene with other genes with the highest induction level in response to <i>A. rabiei</i>, 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 <i>CaDRRG</i> 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 <i>CaDRRG</i> 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 <i>A. rabiei</i> fungal extract was investigated on <i>Nicotiana benthamiana</i> 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 <i>A. rabiei</i>, more additional studies are necessary to identify the regulatory elements responsible for responding to this fungal pathogen.</p>","PeriodicalId":16842,"journal":{"name":"Journal of Plant Growth Regulation","volume":null,"pages":null},"PeriodicalIF":3.9000,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"In-Silico Analysis and Genomic Tracking of CaDRRG Gene Promoter Region in Chickpea Genotypes with Different Levels of Resistance to Ascochyta Blight\",\"authors\":\"Farhad Shokouhifar, Mojtaba Mamarabadi, Narges Sadeghi, Azam Kaseb\",\"doi\":\"10.1007/s00344-024-11493-7\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p>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 <i>Ascochyta rabiei</i> 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 <i>CaDRRG</i>. 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 <i>CaDRRG</i> gene with other genes with the highest induction level in response to <i>A. rabiei</i>, 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 <i>CaDRRG</i> 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 <i>CaDRRG</i> 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 <i>A. rabiei</i> fungal extract was investigated on <i>Nicotiana benthamiana</i> 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 <i>A. rabiei</i>, more additional studies are necessary to identify the regulatory elements responsible for responding to this fungal pathogen.</p>\",\"PeriodicalId\":16842,\"journal\":{\"name\":\"Journal of Plant Growth Regulation\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":3.9000,\"publicationDate\":\"2024-09-16\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of Plant Growth Regulation\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://doi.org/10.1007/s00344-024-11493-7\",\"RegionNum\":3,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"PLANT SCIENCES\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Plant Growth Regulation","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1007/s00344-024-11493-7","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"PLANT SCIENCES","Score":null,"Total":0}
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.
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.