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Molecular cloning and characterization of a 3-hydroxy-3-methylglutaryl-coenzyme A reductase 1 (hmgr1) gene from rubber tree (Hevea brasiliensis Muell. Arg.): A key gene involved in isoprenoid biosynthesis.

ABSTRACT: Natural rubber (cis-1,4-polyisoprene) is a secondary metabolite produced in the laticiferous tissue of Hevea tree. Mevalonate synthesis, which is the first step in isoprenoid biosynthesis, is catalyzed by the enzyme 3-hydroxy-3-methylglutarylcoenzyme A reductase 1 (hmgr1). We have cloned and characterized a full-length cDNA as well as genomic DNA for hmgr1 gene from an elite Indian rubber clone (RRII 105). The nucleotide sequence of the genomic clone comprises 4 exons and 3 introns, giving a total length of 2440 bp. The sequences of 42 bp 5' UTR and 69 bp of the 3' UTR were also determined. The hmgr1 cDNA contained an open reading frame of 1838 bp coding for 575 amino acid protein with a theoretical pI value of 6.6 and the calculated protein M W was 61.6 kDa. The deduced amino acid sequence showed high identity with other plant hmgr1 sequences. The amino acid sequence of the Hevea hmgr1 revealed several motifs which are highly conserved and common to the other plant species. These sequence conservations suggest a strong evolutionary pressure to maintain amino acid residues at specific positions, indicating that the conserved motifs might play important roles in the structural and/or catalytic properties of the enzyme. Southern blot analysis of genomic DNA from Hevea probed with a genomic fragment indicated that there were at least three isoforms of hmgr in Hevea. This result reveals that hmgr1 is one of the members of a small gene family. (Northern blot analysis showed that hmgr1 mRNA transcripts were noticed in all tissues - latex, leaf, immature leaf, and seedlings), however, the abundance of transcript level was higher in latex cells. As one step towards a better understanding of the role that this enzyme plays in coordinating isoprenoid biosynthesis in plants, hmgr1 cDNA was over expressed in transgenic Arabidopsis plants. Transgenic plants were morphologically distinguishable from control wild-type plants and an increased expression level of hmgr1 mRNA was detected. These data suggest that hmgr1 gene expression is playing an important role in isoprenoid biosynthesis.

SUBMITTER: Venkatachalam P

PROVIDER: S-EPMC3550373 | biostudies-other | 2009 Apr

REPOSITORIES: biostudies-other

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Molecular cloning and characterization of a 3-hydroxy-3-methylglutaryl-coenzyme A reductase 1 (hmgr1) gene from rubber tree (Hevea brasiliensis Muell. Arg.): A key gene involved in isoprenoid biosynthesis.

Venkatachalam P P Priya P P Jayashree R R Rekha K K Thulaseedharan A A

Physiology and molecular biology of plants : an international journal of functional plant biology 20090401 2

Natural rubber (cis-1,4-polyisoprene) is a secondary metabolite produced in the laticiferous tissue of Hevea tree. Mevalonate synthesis, which is the first step in isoprenoid biosynthesis, is catalyzed by the enzyme 3-hydroxy-3-methylglutarylcoenzyme A reductase 1 (hmgr1). We have cloned and characterized a full-length cDNA as well as genomic DNA for hmgr1 gene from an elite Indian rubber clone (RRII 105). The nucleotide sequence of the genomic clone comprises 4 exons and 3 introns, giving a tot ...[more]

PMID: 23572922

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Project description:Light is one of most important factors to plants because it is necessary for photosynthesis. In this study, physiological and gene expression analyses under different light intensities were performed in the seedlings of rubber tree (Hevea brasiliensis) clone GT1. When light intensity increased from 20 to 1000 µmol m(-2) s(-1), there was no effect on the maximal quantum yield of photosystem II (PSII) photochemistry (Fv/Fm), indicating that high light intensity did not damage the structure and function of PSII reaction center. However, the effective photochemical quantum yield of PSII (Y(II)), photochemical quenching coefficient (qP), electron transfer rate (ETR), and coefficient of photochemical fluorescence quenching assuming interconnected PSII antennae (qL) were increased significantly as the light intensity increased, reached a maximum at 200 µmol m(-2) s(-1), but decreased from 400 µmol m(-2) s(-1). These results suggested that the PSII photochemistry showed an optimum performance at 200 µmol m(-2) s(-1) light intensity. The chlorophyll content was increased along with the increase of light intensity when it was no more than 400 µmol m(-2) s(-1). Since increasing light intensity caused significant increase in H2O2 content and decreases in the per unit activity of antioxidant enzymes SOD and POD, but the malondialdehyde (MDA) content was preserved at a low level even under high light intensity of 1000 µmol m(-2) s(-1), suggesting that high light irradiation did not induce membrane lipid peroxidation in rubber tree. Moreover, expressions of antioxidant-related genes were significantly up-regulated with the increase of light intensity. They reached the maximum expression at 400 µmol m(-2) s(-1), but decreased at 1000 µmol m(-2) s(-1). In conclusion, rubber tree could endure strong light irradiation via a specific mechanism. Adaptation to high light intensity is a complex process by regulating antioxidant enzymes activities, chloroplast formation, and related genes expressions in rubber tree.

Project description:BackgroundNatural rubber, an indispensable commodity used in approximately 40,000 products, is fundamental to the tire industry. The rubber tree species Hevea brasiliensis (Willd. ex Adr. de Juss.) Muell-Arg., which is native the Amazon rainforest, is the major producer of latex worldwide. Rubber tree breeding is time consuming, expensive and requires large field areas. Thus, genetic studies could optimize field evaluations, thereby reducing the time and area required for these experiments. In this work, transcriptome sequencing was used to identify a full set of transcripts and to evaluate the gene expression involved in the different cold-response strategies of the RRIM600 (cold-resistant) and GT1 (cold-tolerant) genotypes.ResultsWe built a comprehensive transcriptome using multiple database sources, which resulted in 104,738 transcripts clustered in 49,304 genes. The RNA-seq data from the leaf tissues sampled at four different times for each genotype were used to perform a gene-level expression analysis. Differentially expressed genes (DEGs) were identified through pairwise comparisons between the two genotypes for each time series of cold treatments. DEG annotation revealed that RRIM600 and GT1 exhibit different chilling tolerance strategies. To cope with cold stress, the RRIM600 clone upregulates genes promoting stomata closure, photosynthesis inhibition and a more efficient reactive oxygen species (ROS) scavenging system. The transcriptome was also searched for putative molecular markers (single nucleotide polymorphisms (SNPs) and microsatellites) in each genotype. and a total of 27,111 microsatellites and 202,949 (GT1) and 156,395 (RRIM600) SNPs were identified in GT1 and RRIM600. Furthermore, a search for alternative splicing (AS) events identified a total of 20,279 events.ConclusionsThe elucidation of genes involved in different chilling tolerance strategies associated with molecular markers and information regarding AS events provides a powerful tool for further genetic and genomic analyses of rubber tree breeding.

Project description:An indirect phenotyping method was developed in order to estimate the susceptibility of rubber tree clonal varieties to Corynespora Leaf Fall (CLF) disease caused by the ascomycete Corynespora cassiicola. This method consists in quantifying the impact of fungal exudates on detached leaves by measuring the induced electrolyte leakage (EL%). The tested exudates were either crude culture filtrates from diverse C. cassiicola isolates or the purified cassiicolin (Cas1), a small secreted effector protein produced by the aggressive isolate CCP. The test was found to be quantitative, with the EL% response proportional to toxin concentration. For eight clones tested with two aggressive isolates, the EL% response to the filtrates positively correlated to the response induced by conidial inoculation. The toxicity test applied to 18 clones using 13 toxinic treatments evidenced an important variability among clones and treatments, with a significant additional clone x treatment interaction effect. A genetic linkage map was built using 306 microsatellite markers, from the F1 population of the PB260 x RRIM600 family. Phenotyping of the population for sensitivity to the purified Cas1 effector and to culture filtrates from seven C. cassiicola isolates revealed a polygenic determinism, with six QTL detected on five chromosomes and percentages of explained phenotypic variance varying from 11 to 17%. Two common QTL were identified for the CCP filtrate and the purified cassiicolin, suggesting that Cas1 may be the main effector of CCP filtrate toxicity. The CCP filtrate clearly contrasted with all other filtrates. The toxicity test based on Electrolyte Leakage Measurement offers the opportunity to assess the sensitivity of rubber genotypes to C. cassiicola exudates or purified effectors for genetic investigations and early selection, without risk of spreading the fungus in plantations. However, the power of this test for predicting field susceptibility of rubber clones to CLF will have to be further investigated.

Dataset Information

Molecular cloning and characterization of a 3-hydroxy-3-methylglutaryl-coenzyme A reductase 1 (hmgr1) gene from rubber tree (Hevea brasiliensis Muell. Arg.): A key gene involved in isoprenoid biosynthesis.

Publications

Molecular cloning and characterization of a 3-hydroxy-3-methylglutaryl-coenzyme A reductase 1 (hmgr1) gene from rubber tree (Hevea brasiliensis Muell. Arg.): A key gene involved in isoprenoid biosynthesis.

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