Functional Identification Of Specific High Expression Acetolactate Synthase Gene For Sugar Esters Synthesis In Tobacco Glandular Trichomes

Solanaceaes sugar esters（also known as acylsugars）are specifically synthesized in glandular trichomes.Sugar esters not only have a variety of biological functions such as antibacterial,anti-insect,anti-microbial,anti-oxidation,but also are important aroma precursors and have important contribution to aroma quality in tobacco.Therefore,excavation and identification of sugar esters synthesis genes,revealing the molecular mechanism of sugar esters synthesis regulation are of great significance for the breeding of solanaceae crops with important breeding goals to increase sugar esters content,as well as biosynthetic utilization of sugar esters.Recently,researches on the assembly of acyl chains and sugar rings and their key regulatory genes during the synthesis of solanaceous sugar esters have made great progress in tomato and petunia.Regarding the formation mechanism of acyl chains,previous studies have suggested that they are synthesized through the branched chain amino acid synthesis pathway,but still few key regulatory genes have been identified.Based on homology comparison analysis in this study,three key acetolactate synthase genes regulating branch chain amino acid anabolic metabolism were excavated.Combining transcriptome sequencing data of glandular trichomes and leaf in tobacco and tissue expression pattern analysis,we found that one of the acetolactate synthase genes（named NtALS1）was specifically expressed in glandular trichomes,and speculated that it may be involved in tobacco sugar esters synthesis.Therefore,the function of NtALS1 were systematically identified through tissue localization,subcellular localization,and CRISPR Cas9 knockout technology.The results showed that NtALS1 was specifically expressed in chloroplasts of the tip cells of glandular trichomes and was a key gene for anabolic metabolism of sugar esters in tobacco.The main results are as follows:1.Homology genes of 3 ALS large subunit（named NtALS1,NtALS2 and NtALS3）were found from K326 genome of the Solanaceae Genome Network,and the expression patterns of these genes in different tissues of flue-cured tobacco Honghuadajinyuan were analyzed by qRT-PCR.The results showed that NtALS1 was specifically expressed in glandular trichomes,NtALS2 was expressed in 7 tissues,and NtALS3 was hardly expressed.The phylogenetic tree of other homology ALSs from the Solanaceae Genome Network was constructed and the genetic origin of NtALS1 was analyzed by genes cloning.The results showed that NtALS1 was derived from ancestral N.tomentosiformis.Homologous ALSs classified into the same class with NtALS1,their corresponding species contained sugar esters and they might be specifically expressed in glandular trichomes,while Nisyl-mRNA-72505 in N.sylvestris without sugar esters was clustered into the same class with NtALS2 and NtALS3.2.NtALS1 promoter sequence（named NtALS1-P）was cloned from Honghuadajinyuan,and a tissue localization vector was constructed,with expression of GFP driven by the NtALS1-P.Agrobacterium tumefaciens was used for infection and transient expression using N.benthamiana as a receptor,and the fluorescent signal was collected using a laser confocal microscope.The results showed that the GFP signal could only be detected in the tip cells of glandular trichomes,indicating that NtALS1 was specifically expressed in these cells.3.NtALS1 was predicted through subcellular localization websites.The results showed that NtALS1was localized to chloroplasts.The CDS sequence of NtALS1 from Honghuadajinyuan was cloned and the subcellular localization fusion vector was constructed,with co-expression of the NtALS1 and GFP driven by the 35S promoter.Agrobacterium tumefaciens was used for infection and transient expression using N.benthamiana as a receptor,and the fluorescent signal was collected using a laser confocal microscope.The results showed that the GFP signal of NtALS1 was well overlaped with the autofluorescence signal of chloroplasts,indicating that NtALS1 was localized to chloroplasts.4.The CRISPR Cas9 vector containing three NtALS1 knockout target sites was constructed,and genetic transformation was performed with flue-cured tobacco variety K326 as a receptor.By identifying transgenic positive plants and sequencing the target site,fourteen plants of T₀ mutants were obtained,ntals1-1 and ntals1-2 mutants were selected for seeds and seeded into T₁ generation lines respectively.By identifying transgenic positive strains and sequencing the target site,six plants of 2 bp base insertion homozygous mutants and four plants of 40 bp base deletion homozygous mutants were obtained from the T₁ generation lines of ntals1-1 and ntals1-2,respectively.Surface secretions of tobacco leaves of the T₁ generation homologous mutants,including three ntals1-1 and two ntals1-2 plants,were detected by GC/MS technology.The results showed that,compared with the wild K326,in surface secretions of two types of mutant materials,the contents of main diterpenes had no significant difference,but the content of sugar esters was significantly lower than the control.The research results proved that NtALS1 was a key gene for anabolic metabolism of sugar esters in tobacco.