Isolation of Key Genes for Carotenoid Biosynthesis in Curcuma alismatifolia Based on Transcriptome Sequencing
LIU Jian-Xin1,2,*, MAO Li-Hui2, XU Hua1, CHEN Hai-Yan1, CAI Shu-Yu1, WU Li-Yuan1, SHEN Shu-Yu1
1 Yuanpei College, Shaoxing University, Shaoxing 312000, China; 2 Xiaoshan Cotton and Bast Fiber Crops Research Institute of Zhejiang Province, Hangzhou 311202, China
Abstract：Curcuma alismatifolia is a newly emerging tropical bulbous flower in China. However, there is very little known genetic and genetic information about this species, especially the research on bract color genes were blank, which hinders molecular breeding of C. alismatifolia flower color. Sequencing based on the PacBio platform is a third-generation sequencing technology that combines multiple advantages such as rapidness, high throughput, low cost, direct read sequence without PCR amplification, and ultra-long read length advantages. In order to obtain key genes of carotenoid biosynthesis pathway in C. alismatifolia, PacBio platform sequencing was used to sequence full-length transcriptome of C. alismatifolia 'Chiangmai pink', and key genes of the carotenoid biosynthesis pathway were isolated by re-sequencing separation and verified and real-time quantitative PCR (qPCR) method. The experiment obtained a total of 1 189 636 759 bp read bases of insert and a total of 494 242 reads of insert. The mean read length of these inserts were 1 562 bp (1~2 kb range), 2 646 bp (2~3 kb range) and 2 897 bp (3~6 kb range), respectively. Finally, 64 471 high- quality full-length transcript sequences (HQFL polished consensus) were obtained, included 22 107 in the range of 1~2 kb, 40 843 in the range of 2~3 kb, and 1 521 in the range of 3~6 kb. The obtained sequences were functionally annotated and classified using public databases including NR (non-redundant protein database), KOG (eukaryotic orthologous groups), Swiss Prot (Swissprot protein database), COG (cluster of orthologous groups), GO (gene ontology), KEGG, etc. The results showed that a total of 56 215 sequences were annotated. In the KOG classification statistics, a total of 6 608 Unigenes were annotated and divided into 25 categories; in the WEGO functional annotation analysis, 31 789 Unigenes were annotated to the GO database, and all distributed in the 3 major categories of biological process, molecular function, and cellular component with 39 functional groups. In the KEGG functional annotation, 15 785 of the 44 813 Unigenes were annotated, all of which were distributed in 334 metabolic pathway branches. According to the annotation results and re-sequencing, full-length cDNA sequences of 11 key genes in the carotenoid synthesis pathway were obtained, which were CHYB1 (beta-carotene 3-hydroxylase 1), ZDS1 (zeta-carotenedesaturase 1), ZEP1 (zeaxanthin epoxidase 1). PSY1 (phytoene synthase 1), PDS1 (phytoene desaturase 1), CRTLSO1 (prolycopene isomerase 1), LCY-B1 (lycopene beta-cyclase 1), LCYE1 (lycopene epsilon cyclase 1), GPPS1 (geranyl geranyl pyrotenoid synclease1), CCDs1 (carotenoid cleavage dioxygenase1) and carotenoid 9, 10 (9', 10') -cleavage dioxygenase 1. Finally, three genes, PSY1, LCY-B1 and ZEP1, were screened out for tissue expression function verification. PSY1, which was located upstream of the carotenoid synthesis pathway, had the highest expression in sterile bracts and the lowest expression in florets. The gene expressions of LCY-B1 and ZEP1 which were closer to the middle and lower ends of the carotenoid synthesis pathway, were basically the same, and both had the highest expression in florets, followed by sterile bracts. Acquisition of these genes is conducive to improvement of bract color breeding by using molecular regulation methods in the later stage, which will lay the molecular basis for the cultivation of new bract color varieties.
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