Genome-wide Identification Of CONSTANS-like Gene Family And Expression Analysis On Photoperiod Induction In Kenaf

Kenaf(Hibiscus cannabinus L.)is an annual short-day photoperiodsensitive bast fiber crop.Flowering time is one of the key factors affecting the yield of kenaf fiber.To explore the photoperiod-related genes and study the mechanism of photoperiod-induced flowering is important to improve the adaptability,fiber yield and quality of kenaf.The CO(CONSTANS)gene is one of the key genes that regulates plant photoperiod flowering by promoting or inhibiting the expression of its downstream genes to achieve the regulation of plant flowering transformation.Predecessors had identified that the COL(CONSTANS-like)genes family have typical conserved domains in Arabidopsis,cotton and other plants.However,it was not clear that the identification of Actin and COL family genes and the expression pattern of COL genes induced flowering under short-day in kenaf.Therefore,kenaf Zhongfuhong No.2 was used as the material,the screened kenaf Actin and COL gene families were identified by protein sequence analysis,conservative domain prediction,etc.,and then we checked the expression patterns of Actin and COL genes in kenaf using q RT-PCR,the results were as follows:1.Selection and identification of reference genes in kenaf.In this study,37 Actin family genes were screened from kenaf genome-wide data,which were respectively distributed on 17 chromosomes: 1,2,4,5,6,7,8,9,10,11,12,13,14,15,16,17 and 18,containing the typical conserved domain of the Actin gene "NBD?sugar-kinase?HSP70?actin superfamily".The stability expression analysis of the four reference genes Hc ACT3,Hc ACT5,Hc ACT7 and Hc ACT8 showed that the sequences length were 6154,4148,2851,2713 bp respectively,which were closer to the Malvaceae plants and then to Cucurbitaceae plants,far from Euphorbiaceae plants.Four kenaf Actin genes and 18 S r RNA and PP2 A and other genes stabilities were evaluated by Ge Norm,Norm Finder,Best Keeper,Ref Finder reference evolution softwares under Zhongfuhong No.2 different development stages,abiotic stress(salt and drought treatment)and hormone stress(ABA and Me JA treatment),which were used for stability evaluation.The results showed that PP2A-1 and ACT5 were the most suitable reference genes under salt and drought abiotic stress;PP2A-2 and ACT3 were the most suitable reference genes under hormone stress;ACT7 and 18 S r RNA-3 were the most stable expressed genes.In general,PP2A-1 and ACT3 were the most stable expressed reference genes combination for all samples.2.Genome-wide identification analysis of COL gene family in kenaf.The 38 Hc COL members were identified in genome-wide using bioinformatics methods.They all have B-box and CCT conserved domains,which were localized to 15 chromosomes in kenaf: 1,2,3,4,5,6,7,8,10,11,12,13,15,16,and 18,respectively.Phylogenetic analysis showed that the members of Hc COL gene family could be divided into three groups.Class I consisted of Hc COL1,Hc COL4,Hc COL5,Hc COL6,Hc COL7,Hc COL8,Hc COL9,Hc COL10,Hc COL15,Hc COL16,Hc COL17,Hc COL18,Hc COL21,Hc COL25,Hc COL26,Hc COL28,Hc COL29,Hc COL30,Hc COL34 and Hc COL36,which characterized by one B-box domain,one differentiated zinc finger structure and one CCT domain;Class II consisted of Hc COL12,Hc COL13,Hc COL32,Hc COL37 and Hc COL38,which contained one B-box domain and one CCT domain;Class III consisted of Hc COL2,Hc COL3,Hc COL11,Hc COL14,Hc COL19,Hc COL20,Hc COL22,Hc COL23,Hc COL24,Hc COL27,Hc COL31,Hc COL33 and Hc COL35,which contained two B-box domains and one CCT domain.Hc COL11,Hc COL12,Hc COL13,Hc COL19 and Hc COL20 genes were selected for evolutionary analysis.The results showed that kenaf COL genes had the closest relationships with the dicotyledonous hibiscus and upland cotton plants.3.Expression analysis of kenaf COL genes in different period's tissues and circadian rhythms.The expression analysis of kenaf COL genes in different development periods tissues and long/short daylight circadian rhythm,the results showed that Hc COL19 had the highest expression levels in leaves and petal tissues,and the 5 Hc COL genes expression levels were higher in the leaves and differentiating in different growth periods tissues.Hc COL12 and Hc COL13 genes had the lower expression levels in different tissues,while Hc COL11,Hc COL19 and Hc COL20 had higher expression levels in sepals and petals during flowering period.Circadian rhythm expression analysis of kenaf COL genes under long-day(LD)and short-day(SD)conditions showed that the gene expression levels of Hc COL11,Hc COL12 and Hc COL13 genes increased under SD condition,while Hc COL19 and Hc COL20 genes expression levels were decreased under SD condition.The expression levels of Hc COL11,Hc COL13,Hc COL19 and Hc COL20 genes were significantly decreased under long-day and short-day(LD/SD)conditions.The expression levels of COL genes appeared a slight recovery at dawn.The expression levels of Hc COL12 gene under LD and SD conditions were increased first and then decreased,and the expression level reached the peak at 18 h,and there was an obvious circadian rhythm,indicated that the COL family genes were more involved in the flowering pathway of kenaf photoperiod regulation and played an important role in kenaf photoperiod flowering pathway.All the above results could lay a theoretical foundation for the research on the mechanism of kenaf photoperiod regulation.