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Pak. J. Bot., 44(4): 1289-1296, 2012.

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IDENTIFICATION AND VALIDATION OF STABLE INTERNAL CONTROL FOR HEAT INDUCED GENE EXPRESSION OF AGAVE AMERICANA

SITWAT AMAN¹, NOOR UL HAQ¹ AND SAMINA N. SHAKEEL^1*

Abstract: 18S ribosomal RNA (18S rRNA) has been used as housekeeping gene for normalization in gene expression studies of plants. Recently, the effect of experimental conditions and nature of samples have been shown on the stability of internal control gene. Agave americana is a monocot heat tolerant plant adapted to arid conditions with Crassulacean acid metabolism (CAM). Few reports have shown the gene expression studies of this or other CAM plants due to lack of suitable reference gene. Here, we partially sequenced 18S rRNA gene of agave and evaluated its potential use as reference gene under heat stress conditions. Gene specific primers were designed from highly conserved regions of known 18S rRNA genes and amplified by using genomic DNA and transcript of Agave followed by sequencing (submitted to gene bank with accession # HM991824). To validate the potential use of Agave 18S rRNA gene for real-time PCR data normalization, we evaluated the expression stabilities of this gene in different tissues and various heat stress conditions. The plants were treated with different temperatures viz., 35ºC, 40ºC, 45ºC, 50ºC and 60ºC. The relative abundance of a heat regulated gene, Cp-sHSP (chloroplast small heat shock protein) was examined by real-time PCR. Varied levels of Cp-sHSP gene expression under different heat treatments showed the heat regulated expression. Maximum Cp-sHSP gene expression was observed in the leaves of Agave after heat stress for four hours at 45ºC. No significant difference in 18S rRNA expression was observed among control and heat treated samples. Conclusively, this 18S rRNA gene could be used as a stable internal control for normalization of real-time PCR data of A. americana. This work will help to explore many key players in the heat stress related pathways of CAM plants.

¹Department of Biochemistry, Quaid-i-Azam University, Islamabad, 45320 Pakistan.

^*Corresponding author’s e-mail: snq@qau.edu.pk