Echinocystis Lobata or

Echinocystis Lobata or

Echinocystis Lobata

The basic technology of RNA extraction, RNA-seq analysis and informatics is similar as described elsewhere (Han et al., 2015). Fresh tissues and organs were collected from healthy P. lobata plants growing in Chiba, Japan in May 2012. Puerarin and daidzin standard substances were purchased from LC laboratories (USA). The materials were kept in RNA stabilization solution (RNAlater, Life technologies, USA) immediately after sampling. The RNAlater solution was gently removed with a Kimwipe, and the remaining sample was frozen by liquid nitrogen and powdered using Multi Beads Shocker (Yasui Kikai, Japan). TRIzol Reagent (Invitrogen, USA) was used to extract total RNA from powdered P. lobata. The RNA obtained was then treated using the RNeasy Mini Kit (Qiagen, USA).



Studies on legumes showed the isoflavones daidzein and genistein were major metabolites in all embryonic organs within the dry seeds. Seedling roots and callus cultures are known to produce daidzein, with the highest daidzein concentration to be found in mature fruits (Graham, 1991; Bouque et al., 1998). We intended collecting information regarding the nature of the genes responsible for the biosynthesis of daidzein and daidzin in P. lobata. We extracted total RNA from the leaf, mature root, root vascular cylinder (Root VC), young root and stem of the plant. Five distinct cDNA libraries were established from these five tissue samples. We will refer to the five libraries in the following manner: Library 1 (leaf), Library 2 (mature root), Library 3 (root VC), Library 4 (young root), Library 5 (stem). Wherever applicable, a uniform color scheme will be used to represent the libraries: red (Library 1), purple (Library 2), green (Library 3), blue (Library 4), and yellow (Library 5).All five libraries were processed using the Illumina HiSeq 1000 platform. Reads of poor quality, empty reads and those with unknown bases were trimmed by CLC software. In order to consolidate the available bio-information to obtain more reliable and thorough findings, we combined the resultant clean reads with P. lobata EST sequences obtained from the NCBI database to conduct de novo assembly; thus, 83,041 contigs were generated.

By adopting CD-HIT-EST with a threshold set to 0.9, duplicates were retrieved and discarded, leaving 81,508 NR contigs for downstream analysis. An overview of the experimental pipeline is shown in Figure 1. Tables 1 and 2 summarize trimming, sequencing, and assembly results. The reported GC content for unigene sequences in soybean and Arabidopsis is 0.43 and 0.44, respectively (Tian et al., 2004; Kawaguchi and Bailey-Serres, 2005). The mean GC content of P. lobata transcripts was found to be 39.9% (Supplementary S1). In eukaryotes, average GC content covers the range from ∼20 to 60% (Serres-Giardi et al., 2012). Our values are in the middle of this range, slightly lower than those reported for Glycine max (43%) but very close to those reported for Sophora flavescens (39.3%) and Medicago truncatula (40%) (Tian et al., 2004; Han et al., 2015). (Source:www.frontiersin.org)



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