Identification And Expression Profiling Of Critical Genes Associated With Lignin Biosynthesis In Radish(Raphanus Sativus L.)

Radish is an important root vegetable crop with high nutritional,economic and medicinal value.The taproot of radish is the edible and storage organs,which were rich in abundant nutrients affected plants development and quality.Lignin is a secondary metabolite,as an important dietary fiber,lignins widely exist in grains,fruits and vegetables with the role of promoting food digestion and numerous medicinal activities,such as anti-tumor,anti-HIV,anti-oxidant,prevention against heart disease and regulation of blood sugar level.The appropriate amount of lignin in specific cells could help to promote plant development,whereas excessive accumulation would lead to plant lignification,which has negative effect on radish nutritional value,texture and flavor.Therefore,exploring molecular mechanism underlying lignin biosynthesis is of great significance for genetic improvement of plants.To date,many lignin biosynthesis-related genes have been identified in some important plants.Nevertheless,little information underlying identification of lignin biosynthesis-related genes is available in radish.In this research,we determined lignin content in radish leaves and roots at different stages of radish development.Moreover,we identified the genes that involved in lignin biosynthesis based on transcriptome sequencing and analysed the gene expression profile at four stages(pre-cortex splitting,cortex splitting,thickening and mature stage)during taproot expanded.These results would provide the theoretical basis to explore the molecular mechanism of lignin biosynthesis.The main achievements were as follows:1.Lignin content of 'NAU-YHUA','NAU-XBC','NAU-ZQH' and 'NAU-DY' were determined during the different stages of radish taproot development.Determination of lignin content demonstrated that it gradually accumulated in leaves during radish development and reached the highest at mature stages.The lignin content accumulated was respectively 5.20%-12.28%,7.82%-15.16%,7.86%-14.75%,7.36%-14.75%.The lignin content in radish roots was significantly changed at four stages during radish development.Among them,the content was increased in root of 'NAU-XBC' at four stages,which was ranged from 6.73%to 14.30%.However,it declined at thickening stage in other varieties,'NAU-YHUA' was varying from 6.76%to 14.46%,'NAU-ZQH' was varying from 8.98%-15.37%and 'NAU-DY' was varying from 8.19%-15.24%.2.The radish root transcriptome was analysed by Illumina HiSeq 2500 sequencing platform.A total of 37.94M clean reads were generated and assembled into 71,148 unigenes with an average length of 966 nt.Among them,60,467 assembled unigenes(84.99%of total)were annotated to the public databases of NR(71.14%),GO(68.63%),COG(25.22%),KEGG(41.11%)and NT(83.21%).Analysis of GO annotation revealed that unigenes were mainly assigned to cells,cell components,cellular processes,physiological metabolism and binding processes.According to KEGG,66 assembled unigenes were identified to involve in lignin monolignol biosynthesis and 94 assembled unigenes were corresponding to peroxidase.The key enzymes were PAL(K10775,EC:4.3.1.24),C4H(K00487,EC:1.14.1311),4CL(K01904,EC:6.2.1.12),C3H(K09754,EC:1.141336),F5H(K09755,EC:1.14.-.-),HCT(K13065,EC:2.3.1.133),COMT(K13066,EC:2.1.1.68),CCoAOMT(K00588,EC:2.1.1.68),CCR(K00083,EC:1.2.1.44),CAD(K00430,EC:1.1.1.195)and POD(K00430,EC:1.11.1.7).Transcriptome sequencing provided numerous unigene sequences for the identification of lignin biosynthesis-related genes.3.Based on radish transcriptome database,genome database and NCBI database,8 genes were isolated and sequenced by T-A cloning in radish root and leaf.The DNA full length of RsPAL2,the cDNA full length of RsC3H,the DNA and cDNA full length of RsC4H,RsHCT,RsCCoAOMTl,RsCOMT,RsF5H and RsCCR2 were required respectively.RT-PCR and RT-qPCR was respectively used to analyze the expression characteristics of lignin biosynthesis-related genes.These results revealed that the expression patterns of Rs4CL5 and RsF5H were gradually increased at four stages,which were agreement with lignin content changes in radish leaves.Moreover,the expression patterns of Rs4CL,RsCCoAOMT1 and RsCOMT were consistent with the changes in root lignin content,suggesting that these candidate genes may play important roles in lignin formation of radish root.These findings would provide valuable information for the identification of lignin biosynthesis-related genes and provide insights into dissection of molecular mechanism underlying lignin biosynthesis in radish and other root vegetable crops.