Cloning And Mechanism Analysis Of The Dominant Glandless Gene Gl₂^e In Cotton?Gossypium Hirsutum?

Cotton is an important strategic resource and one of the most important economic crops in the world.Cotton fiber is excellent natural material in the textile industry and cottonseed is an important nutrient resource.There are a large number of pigment glands containing plenty of toxic gossypol on plants and seeds of common cotton cultivars.The toxic gossypol makes the cottonseed fat and protein and other nutrients cannot be fully taken advantage of.However,the toxicity of gossypol in cotton plants,which playing an important role in cotton growth,can improve cotton resistance to a variety of diseases and insect pests.Therefore,the main objective of low-gossypol cotton breeding is to develop cotton cultivars with gossypol in plants and without gossypol in seeds.The number of pigment gland,which is storage organ of gossypol,is closely associated with gossypol content in cotton.Hence,it is always an important research direction of low-gossypol cotton breeding that cloning and identification gland-related genes and exploring the molecular mechanism of pigment gland formation.Single dominant glandless gene Gl₂^e is one of the important genetic resources in low-gossypol cotton breeding and is of great significance to study the mechanism of pigment gland formation.This study mainly includes two aspects: one is cloning and analysis of Gl₂^e candidate gene and functional verification;another is identification of differential expression mi RNAs and their targets between glanded and glandless cotton by small RNA sequencing of 7 gland-related materials to explore mi RNAs and signaling pathways involved in gland formation.The main results are as follows:1.According to the previous study,candidate region for Gl₂^e gene was narrow down to a 15 kb interval on 12 chromosome in which only one gene predicted.In this study,the predicted gene was cloned as Gl₂^e candidate.The gene is 1428 bp in length without intron and encodes 475 animo acids.The protein was annotated as cotton MYC2-LIKE protein including b HLH-MYC_N and b HLH conserved domains and belongs to b HLH-MYC transcription factor family,we named it as Gh MYC2.There were three base mutations in the gene sequence from glandless cotton,and the base mutation at 128 th locus caused an animo acid substitution in b HLH-MYC_N region.QRT-PCR results revealed Gh MYC2 gene expression level in glanded cotton was much higher than that in glandless cotton.Therefore,Gh MYC2 gene is likely to be the gene controlling formation of pigment gland.2.Comparing the Gh MYC2 gene promoter sequences from CCRI12 and Dgl-CCRI12,besides 10 different bases,there were two base sequence deletions “TATTACC” and “T” locating 1.5 kb and 1 kb upstream of ATG respectively in Dgl-CCRI12.But these two deletions did not change the cis-acting elements of the promoter.Meanwhile,Two Cp G islands in promoter were chosen for bisulfite sequencing and the results showed that methylation condition of these two Cp G islands had no significant difference between CCRI12 and Dgl-CCRI12.These results indicated that the gene differential expression between two materials might be regulated by other factors.3.The result of sub-cellular localization analysis showed Gh MYC2 protein was localized in the nucleus,further confirming that Gh MYC2 was indeed a transcription factor.The transcriptional activation activity analysis in yeast revealed that single amino acid mutation could affect the transcriptional activation activity but not invalidate it.Generally,activity region of MYC transcription factor is at the N-terminus.In this study,the activity of the b HLH-MYC_N region from Gh MYC2 was similar to that of full-length protein,which was also consistent with the characteristics of MYC transcription factor.4.The Gh MYC2 gene in CCRI12 was successfully silenced by virus induced gene silencing?VIGS?,and pigment gland formation was effectively suppressed in VIGS plants.The results confirm Gh MYC2 transcription factor was essential to pigment gland formation.To further validate the function of Gh MYC2 gene,we obtained RNAi plants by genetic transformation.The T0 generation RNAi plants and seeds as well as T1 generation plants showed a completely glandless phenotype.The results indicate that silencing of Gh MYC2 could result in a hereditable glandless phenotype in plants and seeds.Gh MYC2 is the first cloned gene related to pigment gland with function validation,which laying solid foundations for study of molecular mechanism of pigment gland development.5.To investigate the different mi RNA between dominant glandless and recessive glandless materials,CCRI12 vs Dgl-CCRI12 and L7 vs Dgl-L7 were regarded as dominant glandless group?Dgl group?,CCRI12 vs Rgl-CCRI12?recessive glandless line of CCRI12?and CCRI17 vs Rgl-CCRI17 were regarded as recessive glandless group?Rgl group?for differential expression analysis.Finally,58 known mi RNAs and 109 novel mi RNAs?novel₁ to novel₁09?were identified among these seven samples.Ten and nine differential expression mi RNAs were identified in Dgl group and Rgl group,respectively.Novel₁4 and novel₄5 were differential expression mi RNAs in both groups and both down-regulated in glandless cotton.6.In Dgl group,73 target genes were predicted for 8 differential expression mi RNAs.In Rgl group,111 target genes were predicted for 5 differential expression mi RNAs.However,there were no overlapped target genes between two groups except for 8 target genes of novel₁4 and novel₁5.KEGG analysis showed that target genes from two groups were both involved in phytohormone signal transduction,phagosome,metabolic pathways and MAPK signal pathway.In addition,disparate pathways were also obtained in two groups.The results indicate that the regulatory pathway of dominant glandless trait and recessive glandless trait are partly overlapped and independent.7.There were 14,17 and 34 MYC proteins identified from G.arboreum,G.raimondii and G.hirsutum,respectively.These MYC proteins can be divided into three sub-families according to the phylogenetic tree.MYC proteins in G.hirsutum were mostly homologous to the MYC proteins in G.arboreum or G.raimondii,suggesting MYC proteins have existed before the divergence of cotton species.All 8 target genes of novel₁4 and novel₄5 encoded MYC family proteins and had close relationship with Gh MYC2.Meanwhile,they are all differential expression between glanded and glandless cotton,implying that multiple MYC family proteins may play important roles in pigment gland development.