Identification And Functional Analysis Of Upland Cotton ?-Ketoacyl-ACP Synthetase ?(KAS?) Family Genes

?-ketoacyl-ACP synthase II(KASII)catalyzes the 16:0-ACP to produce 18:0-ACP in plastids.It is a key enzyme responsible for the ratio of 16C/18C fatty acid in plant cells,and also an important target for plant oil metabolism engineering.Cultivated upland cotton(Gossypium hirsutum Linn)is an important economic crop which could produce fiber,oil and protein.Its genome is allotetraploid(AADD).The information of the enzyme characteristics,gene structure and biological function of cotton GhKASII proteins has not been fully reported yet.Based on the genomic data of upland cotton,this study was conducted to perform a genome-wide characterization of GhKASII gene family.Bioinformatics tools were employed to analyze the physicochemical properties,gene structure and phylogenetic evolution of GhKASII family members.The temporal-spatial expression patterns of GhKASII gene family members were also detected by qRT-PCR.Five GhKASII genes were cloned,and their ORFs were inserted into the expression vector for constructing their overexpression vectors.These five GhKASII genes were transiently expressed in heterologous tobacco leaves by Agrobacterium-mediated infiltration.Phenotypic identification on those leaves transient-expressed the GhKASII was employed for fanctional analysis of these GhKASII members.This study will enrich the understanding of oil biosynthesis and regulatory mechanisms in plant seeds,providing scientific basis for improving the quality and yield of cotton oil by genetic engineering.The main findings are described as follows:(1)A total of 21 GhKASII members were obtained from the whole genome of upland cotton.The amino acid length of those proteins was between 152 and 580 aa,and the molecular weight ranged from 62.34 kDa to 16.67 kDa.The enzyme proteins were divided into four types:hydrophilic-stable,hydrophilic-unstable,hydrophobic-stable and hydrophobic-unstable.There were four forms of secondary structures in those proteins,and their frequency was:a-helix>random coil>extended strand>?-turn.The proportion of a-helix exceeded 50%in six protein members.None of the 21 GhKASII protein members had a signal peptide,and most of them were located in the chloroplast.Phylogenetic tree analysis of 21 GhKASII members showed that they can be divided into three subfamilies:GhKASIIA,GhKASIIB and GhKASIIC.(2)Primers for RT-PCR were designed by using the transcript sequences of GhKASII family genes as templates.The expression profiles of GhKASII members were examined by quantitative PCR in five organs/tissues including roots,stems,leaves,flowers and ovules.Of the 21 GhKASII members,four members were expressed at very low levels,12 members at moderate levels.Importantly,5 members were expressed at high levels in all 5 tissues tested.The expression of each member was tissue-specific.(3)Further quantitative PCR analysis was performed for five highly expressed GhKASII genes.The result showed that the KASII8,KASII18 and KASII21 had the highest expression levels in flowers,while KASII13 and KASH20 were highly expressed in stems leaves,respectively.The lowest expression levels of the five genes were detected,respectively,in stems,leaves,ovules,roots and ovules.The highest expression levels of five highly expressed genes were 3.12,1.76,3.58,2.91 and 2.47 times that of the lowest expression levels,respectively.The expression differences were extremely significant.Of the five genes,the expression level of KASII13 was the highest,and relatively expression levels of KASII8 and KASII20 were the lowest,with the KASII8 and KASII20 's expressions were 50%less than the KASII13's expression.(4)Five highly-expressed GhKASII genes were isolated and cloned into the intermediate vector pBluescript.Their ORFs were separately cloned into the expression vector pCAMBIA1303-N to form the overexpression vectors.These expression vectors were transferred into Agrobacterium.Tobacoo(Nicotiana benthamiana)leaves were infected with positive Agrobacterium for transient expression of those five GhKASIIs in the tissues.The infected tobacco leaves were used to extract oil for examining fatty acid composition and their contents by GC.The results showed that the content of palmitic acid was decreased,and correspondingly,the stearic acid was increased in different level.This proved that these five GhKASII enzyme proteins have higher KASII activity.They can catalyze 16:0 to generate 18:0 in heterologous expression tissues.