Genome-wide Identification And Function Analysis Of Heteromeric ACCase In Cotton (Gossypium)

Cotton is an important economic crop and oil crop.Cottonseed is rich in fatty acids,widely used in processing of edible oil,industrial materials and biodiesel.Heteromeric ACCase is an important and rate-limiting enzyme in de novo fatty acid synthesis.Heteromeric ACCase is composed of biotin carboxylase(BC),biotin carboxyl carrier protein(BCCP)and carboxyl transferase subunits of alpha and beta.In our study,based on the publishion of diploid cotton(G.raimondii and G.arboreum)and tetxaploid cotton(G.hirsutum and G.barbadense)genomes,we perfomed genome-wide identification and analysis of three subunits encoding heteromeric ACCase.Under low tempreture and salt stresses,the expression patterns of BCCP genes in G.raimondii,G.arboreum(Shixiya 1)and G,hirsutum(TM-1)were different,and combined with the public RNA-Seq data of TM-1,we analyzed the expression patterns of three subunit family members under different abiotic stresses.We verified the subcellular location of GhBCCPl,and cloned its promoter sequence and qualitative analyzed activities of GhBCCPI deletion expression vectors.We analyzed the expression patterns of heteromeric GhACCase in different tissues of Xu244(the receptor of transgenic plants),or under different abiotic stresses.Furthermore,we screened and determined oil content of single subunit encoding heteromeric GhACCase materials,and two two subunits hybrid materials as well.The main research results are as follows:1.In total,24 BCCP(4 GaBCCP,4 GrBCCP,8 GhBCCP4 and 8 GbBCCP),12 BC(2 GaBC,2 GrBC,4 GhBC and 4 GbBC)and 11 CTα(3 GaCTα,2 GrCTα,4 GhCTα and 2 GbCTα)were identified from G.raimondii,G.arboretum,G.hirsutum and G.barbadense.All of them were distributed on the corresponding chromosomes as a singleton.Phylogenetic analysis of BCCP gene family members showed that BCCP gene families can be divided into two groups;the lengths of BCCP genes in group Ⅰ were changed larger than group Ⅱ,the gene structures of the two groups were more complex;fragment duplication of BCCP genes caused the amplification of BCCP gene in G.hirsutum and G.barbadense.We successfully identified 20 BCCP orthologous gene pairs,6 BC orthologous gene pairs and 6 CTa orthologous gene pairs.The gene structures of 11 BCCP orthologous gene pairs,3 BC orthologous gene pairs and 3 CTa orthologous gene pairs were similar,while gene structures of the remaining of 9 BCCP,3 BC and 3 CTa orthologous gene pairs were different,the results implying that those gene pairs had not highly conserved gene structures.2.For the salt stress,in roots,six BCCP genes in cotton showed up-regulated expression after salt treatment for 24 h.In stems,BCCP genes of G.raimondii and G.arboreum were up-regulated expression under salt stress.Under cold stress,GrBCCP3,GaBCCPl,GaBCCP2,GaBCCP3,GaBCCP4 and GhBCCP7 were up-regulated expression.GrBCCP3,GaBCCP1,GaBCCP3 and GaBCCP4 were up-regulated in stems under cold stress.At the same time,under both salt and cold stresses,GrBCCP3,GaBCCP1,GaBCCP2 and GaBCCP4 were up-regulated in stems,and GaBCCPl and GaBCCP3 were induced in leaves.Based on different tissues and different abiotic stresses of TM-1 RNA-Seq data,we found 8 GhBCCP,4 GhBC and 4 GhCTa genes were expressed ubiquitously in all the tested tissues with different expression levels,and 4 GhBCCP(GhBCCP2,GhBCCP4,GhBCCP6 and GhBCCP7),2 GhBC(GhBC1 and GhBC3)and 2 GhCTα(GhCTα1 and GhCTα3)gene had very high transcriptions at the different ovule developmental stages,this implied that these genes may play a positive role in fatty acid accumulation.Under different abiotic stresses,the results showed that GhBCCP1 presented high-low-high trends under salt and drought stresses,implying that GhBCCP1 may be subject feedback regulation.GhBCCP5 and GhBCCP6 were down-regulated under salt,drought and hot stresses,implying that these genes may have negative regulation.Under hot stress,4 GhBC genes were up-regulated,this result showed that 4 GhBC genes may be had positive regulation.GhCTα1,GhCTα3 and GhCTα4 genes were up-regulated after 12 h by hot,drought and salt stresses,suggesting the three genes may have positive regulation under those three stresses.These results suggested that GhBCCPs,GhBCs and GhCTas might participate in different abiotic stresses response with different response patterns.3.GhBCCPl was located on chloroplast,the strain and GUS activity of deletion expression vectors of GhBCCP1 showed that between 1449-1195 bp of GhBCCP1 promoter exsited important DOF core element and MYB element,these elements had important effect on the activities of GhBCCP1 promoter.4.The four subunits of heteromeric GhACCase expressed ubiquitously in all the tested tissues with different expression levels,including leaves,stems,roots and fiber at different development stages.GhCTβ had highly expression level in leavea,which could be attributed to the fact that leaf cells are rich in chloroplast.The expression levels of GhBCCP1,GhBC1,GhCTα2 and GhCTβ were up-regulated after 12 h by ABA treatment,implying the four genes might response to ABA treatment as a coordinated pattern after 12 h ABA treatment,while the four genes had different expression patterns under low temperature and MeJA treatments,suggesting heteromeric ACCase had different response patterns under low temperature and MeJA treatments.5.Oil content determination of 8 non-transgenec materials in ovules showed that oil accumulation rates were fast at 20-30 DPA,subsequnently decreased at 30-35 DPA,and had four materials oil contents had decline trends at 35-40 DPA.Expression patterns of heteromeric GhACCase showed that four subunits had the highest peaks in the same period of high oil content materials,such as 10H1041,which oil content was 35.29%,four subunits genes encoding heteromeric GhACCase had most abundant transcripts at 25 DPA of ovules;three subunits had the highest peaks in the same period of slight high oil content materials,such as 11-0514 and 10H1007,which oil contents were 29.51%and 29.69%,all of GhBCCP1,GhBC1 and GhCTa2 had the highest transcripts at 35 DPA of ovules,and 10H1014 oil content was 33.55%,all of GhBCCP1,GhBC1 and GhCTα2 had the highest transcripts at 25 DPA of ovules;two subunits had the highest peaks in the same period(the peaks of GhBCCP1 and GhBC1、GhCTα2 and GhCTβ presented in the different ovule developmental stages)or four subunits had the highest peaks in different developmental stages of low oil content material,such as Xu244,11-0711,11-0509 and 11-0512.These results suggested that high oil content cotton materials had coordinated expression of four subunits encoding heteromeric ACCase,while low oil content materials had no significant coordinated expression in the four subunits of heteromeric ACCase.6.Among the overexpression of single subunit of heteromeric GhACCase in cotton,GhBCCPl transgenic plants of T3 generation showed a significant increase of 21.92%in oil content in cotton seeds,while about a 17%increase was noted for GhBCl and GhCTβ,respectively.So overexpression of each subunit of heteromeric GhACCase may effectively increase seed oil content in Upland cotton.The oil contents of T5 generation of overexpression GhCTβ materials had 4.27 percentage points higher than control on average,the T5 generation of overexpression GhBCCPl and GhBCl materials had 1.01 and 1.67 percentage points higher than control on average,respectively,the T5 generation of overexpression GhCTa2 materials had 0.51 percentage points lower than control on average,and the trends in oil contents of T6 and T9 generations of the four genes overexpression materials were consistent with their corresponding T5 generation.These results suggested that GhCTβ,GhBCCP1 and GhBC1 had certain roles in increasing cottonseed oil content as well.The oil contents of GhBCCP1×GhCTβ transgenic materials had no significant different compared with their both parents,while GhCTβ×GhBC1 and GhBCCP1×GhCTa2 transgenic materials were lower than one of their corresponding parent materials.The result showed that these two two hybrid materials hasd no significantly improved oil content of cottonseed,implying the activity of ACCase was not imprived in two two hybrid transgenic materials,and recombined the four subunit genes may be increase oil content of cottonseed,but this could be need further performed study.