Silencing Of ATP-citrate Lyase Gene(ACLB-2) Leads To Programmed Cell Death In Cotton

Cotton is an important economic crop in the world,and the natural fiber produced is an important raw material for textile industry.Verticillium wilt,known as cancer of cotton,seriously affects the growth and yield of cotton.Gossypium hirsutum is the main cotton in China,accounting for more than 95%of cotton yield.However,due to the lack of immunity or high resistance to Verticillium wilt in Gossypium hirsutum,it has become a major problem in cotton breeding.Gossypium australe is resistance to Verticillium wilt.Therefore,it is of great significance to explore the high disease resistance genes of wild cotton such as Gossypium australe for cotton disease resistance breeding.The Gossypium hirsutum-Gossypium australe chromosome 7 translocation lines（T7）previously obtained in our laboratory has high resistance to Verticillium wilt.Using T7 as the experimental material,transcriptome sequencing technology was used to compare the transcriptome differences between T7 and CK after Verticillium wilt V991 induction in cotton root,and a total of 21 candidate genes were screened out by bioinformatics analysis.Using VIGS technique,21 candidate genes were further screened.The result indicated that when the candidate gene Gaus13 was silenced,it led to the gradual wilting of the apical meristem the plant,and finally the phenomenon of apical meristem death.The ATP-citrate lyase gene was identified by cloning the full length of the gene.In this study,we further analyse the gene function.The main results are as follows.1.Characterization of Gossypium australe ATP-citrate lyase gene GausACLB-2The total length of the gene sequence is 4056 bp,including 14 introns and 15 exons.The total length of CDS gene is 1827 bp,encoding a protein containing 608 amino acids.Amino acid sequence analysis showed that the protein encoded by this gene belonged to PLN02522,ATP citrate（pro-S）-lyase family,named GausACLB-2（Gossypium australe ATP citrate lyase subunit B 2）,including two functional domains:Ligase＿Co A（PF00549）and Citrate＿Synt（PF00285）.Blast homology search revealed that there were four copies in the genome of tetraploid Upland cotton and Island cotton,namely two copies each in the At and Dt subgenomes,named as Gh ACLB-1A,Gh ACLB-1D,Gh ACLB-2A,Gh ACLB-2D,Gb ACLB-1A,Gb ACLB-1D,Gb ACLB-2A and Gb ACLB-2D,respectively.Evolutionary analysis demonstrated that the gene belonged to the same branch as Gh ACLB-2A,Gh ACLB-2A,being closer to the At subgenome.The RNA-Seq data of 19 tissues and organs of Upland cotton and Island cotton were analyzed.It was found that the gene was expressed in root,stem,leaf,torus,petal,sepal,anther,bract,filament,ovule and fiber at different developmental stages of both Upland cotton and Island cotton.Both Gh ACLB-2A and Gb ACLB-2A are highly expressed in the whole growth period of cotton and are predominance expressed in root.To investigate the regulatory mechanism of GausACLB-2,the cis-regulatory elements of the 2000 bp promoter sequence were analysed.The result showed that its promoter region contained the expression regulatory elements（TATA-box,CAAT-box）and transcription start site（TSS）that are necessary for higher plant-based promoters.The promoter region also has salicylic acid and abscisic acid and hormone-related cis-regulatory elements（TCA-elements,ABRE）.Salicylic acid plays an important role in plant disease and stress resistance.It also includes regulatory elements associated with defense and stress response（TC-rich repeats）and a large number of regulatory elements associated with light response（ACE,AT1-motif,Box 4,and CHS-Unit 1 M1）.2.PCD induced by ACLB-2 gene silencing in cottonBy Virus induced gene silencing（VIGS）,the constructed TRV:ACLB-2 fusion vector was injected into cotton cotyledon,and TRV:00 and TRV:CLA were respectively used as negative control and positive control for target fragment silencing.It was found that after silencing Gh ACLB-2 and Gb ACLB-2 gene,the true leaf at the top of cotton gradually wilted,and finally withered and died.The content of H₂O₂ was measured.The results showed that after silencing Gh ACLB-2 and Gb ACLB-2 gene,a large amount of H₂O₂would accumulate in the leaf at the top of cotton,indicating that the silencing of ACLB-2 gene caused programmed cell death in cotton.Q-PCR analysis showed that,the three aging genes SGR,WRKY23 and OSL57 were significantly expressed after Gh ACLB-2 and Gb ACLB-2 gene silencing,leading to cotton wither and death.At the same time,the resistance gene PR1 was also significantly up-regulated.3.Ectopic expression of GausACLB-2 gene in Arabidopsis thalianaGausACLB-2 gene in Arabidopsis thaliana was further functionally studied by heterologous expression using transgenic technology.The T-DNA insertion mutant was used targeting the AT5G49460 gene（SALK＿138734.17.15.n）,named aclb-2.Overexpression vectors were constructed using p BI121 plasmid vector as the skeleton,and the vectors were introduced into aclb-2 and Col-0 by impregnation method.Verticillium wilt resistance was identified in the T2-generation positive lines of Arabidopsis thaliana overexpression and aclb-2 transgenic complement.The results showed that the disease resistance of aclb-2 transgenic complement lines decreased significantly under the background of mutant aclb-2,and the higher the expression level of the complement gene,the worse the disease resistance.Under the background of wild-type Col-0,the disease resistance of wild-type Arabidopsis thaliana overexpressed was significantly reduced,and the higher the gene expression level,the worse the disease resistance.These results indicate that GausACLB-2 gene plays a negative regulatory role in disease resistance.