GUAR LEAF TRANSCRIPTOME ANALYSIS AND EVALUATION OF GUAR GUM DRUG DELIVERY SYSTEM

Abstract

Guar or clusterbean is an economically important crop because of galactomannan gum obtained from its seed endosperm. Genetic improvement in industrially important guar (Cyamopsis tetragonoloba [L.] Taub.) crop has been hindered due to the lack of sufficient genomic or transcriptomic resources. In this study, RNA-Seq technology was employed to sequence and characterize the transcriptome of leaf tissues from two guar varieties, namely, M-83 and RGC- 1066. Approximately 30 million high-quality pair-end reads of each variety were generated by Illumina HiSeq platform and used for de novo assembly by Trinity program. A total of 62,146 non-redundant unigenes with an average length of 679 bp were obtained. The quality assessment of assembled unigenes revealed 87.50 % complete and 97.18 % partial core eukaryotic genes (CEGs). Sequence similarity analyses and annotation of the unigenes against non-redundant protein (Nr) and Gene Ontology (GO) databases identified 175,882 GO terms. The species distribution analysis of the unigenes showed highest similarity with Glycine max genes. The comparison of assembled unigenes with the sequence data of closely related sequenced species on the basis of meta annotation, gene family and functional annotation showed 63 %, 55.9 % and 56.7 % similarity of unigenes to the sequences of Glycine max, Medicago truncatula and Lotus japonicus, respectively. A total of 11,308 guar unigenes were annotated with various enzyme codes (EC) and categorized in six categories with 55 subclasses. The annotation of biochemical pathways resulted in a total of 11,971 unigenes assigned with 145 KEGG maps and 1,759 enzyme codes. The differential gene expression analysis showed ~80 % similar gene expression in both the varieties; 2,863 unigenes were found to express in variety M-83 only and 2,120 unigenes in RGC-1066 variety only. Approximately 175 and 158 unigenes overexpressed in RGC-1066 and M-83 varieties, respectively. A total of 5,773 potential simple sequence repeats (SSRs) and 3,594 high-quality single nucleotide polymorphisms (SNPs) were identified. Out of 20 randomly selected SSRs for wet laboratory validation, 13 showed consistent PCR amplification in both guar varieties. In silico analysis of SSRs resulted in identification of 145 polymorphic SSR markers in two varieties. In addition, 2,930 and 3,984 Insertion-Deletion (InDel) variations were found in the varieties M-83 and RGC-1066, respectively. Guar gum based drug delivery system was formulated by two methods and evaluated for colonic drug release using 5-fluorouracil as a model drug. The microparticles were spherical in shape with a size range of 7-30 micrometer. The study of drug release was carried out in PBS buffer (pH 7.4). The drug was found to be released after 6 h in both preparations of microparticles.