Severity: Warning
Message: file_get_contents(https://...@gmail.com&api_key=61f08fa0b96a73de8c900d749fcb997acc09): Failed to open stream: HTTP request failed! HTTP/1.1 429 Too Many Requests
Filename: helpers/my_audit_helper.php
Line Number: 143
Backtrace:
File: /var/www/html/application/helpers/my_audit_helper.php
Line: 143
Function: file_get_contents
File: /var/www/html/application/helpers/my_audit_helper.php
Line: 209
Function: simplexml_load_file_from_url
File: /var/www/html/application/helpers/my_audit_helper.php
Line: 3098
Function: getPubMedXML
File: /var/www/html/application/controllers/Detail.php
Line: 574
Function: pubMedSearch_Global
File: /var/www/html/application/controllers/Detail.php
Line: 488
Function: pubMedGetRelatedKeyword
File: /var/www/html/index.php
Line: 316
Function: require_once
Severity: Warning
Message: Attempt to read property "Count" on bool
Filename: helpers/my_audit_helper.php
Line Number: 3100
Backtrace:
File: /var/www/html/application/helpers/my_audit_helper.php
Line: 3100
Function: _error_handler
File: /var/www/html/application/controllers/Detail.php
Line: 574
Function: pubMedSearch_Global
File: /var/www/html/application/controllers/Detail.php
Line: 488
Function: pubMedGetRelatedKeyword
File: /var/www/html/index.php
Line: 316
Function: require_once
As a mean to cope with their potential cytotoxicity for the host plant, secondary metabolisms are often sequestered within specific cell types. This spatial organization may reach complex sequential multicellular compartmentation. The most complex example so far characterized is the sequential multicellular biosynthesis of the anticancer monoterpene indole alkaloids in Catharanthus roseus. RNA in situ hybridization has proven a key technological approach to unravel this complex spatial organization. Pioneer work in 1999 discovered the involvement of epidermis and laticifer/idioblasts in the intermediate and late steps of the pathway, respectively. The localization of the early steps of the pathway to the internal phloem-associated parenchyma later came to complete the three-tissular block organization of the pathway. Since then, RNA in situ hybridization was routinely used to map the gene expression profile of most of the nearly 30 genes involved in this pathway. We introduce here a comparison of advantages and drawbacks of in situ hybridization and more popular promoter: GUS strategies. Two main advantages of in situ hybridization are the suitability to any plant species and the direct localization of transcripts rather than the localization of a promoter activity. We provide a step-by-step protocol describing every details allowing to reach a medium throughput including riboprobe synthesis, paraffin-embedded plant tissue array preparation, prehybridization, in situ hybridization, stringent washing and immunodetection of hybridized probes, and imaging steps. This should be helpful for new comers willing to domesticate the technique. This protocol has no species limitation and is particularly adapted to the increasingly studied model, nonmodel species, nonamenable to promoter::GUS transformation, such as C. roseus.
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http://dx.doi.org/10.1007/978-1-0716-2349-7_1 | DOI Listing |
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