Editing And Study On Function Of Lycopene Cyclase Genes In Tomato

Lycopene cyclase is a key enzyme in regulating lycopene metabolism,which is closely related to the content of carotenoids.Therefore,in order to provide the target gene and molecular theoretical basis for the cultivation of high lycopene tomato,this study aimed at four lycopene cyclase genes in tomato,obtained a variety of gene editing tomatoes with high lycopene content by CRISPR/CAS9 gene editing system,and adopted molecular biology,genetics and other methods.The biological effect and mechanism of lycopene cyclase involved in the regulation of tomato plant growth and development and fruit coloration were analyzed.The main results are as follows:1.Establishment of multi-target CRISPR/CAS9 system in tomato.Four lycopene cyclase genes,Sl Lcy B1,Sl Lcy B2,Sl Cyc B and Sl Lcy E,were identified by sequence alignment.The most suitable target sequence of each gene 20 nt was screened by CRISPR-P website and other websites.The target sequence was introduced into the sg RNA expression cassette with Arabidopsis U3/U6 promoter by Overlapping PCR,and the Cas9 vector and several g RNA expression cassette fragments were assembled by Golden Gate cloning.Through sequencing identification,the multi-knock lycopene cyclase gene vector was named p SL-Pubi-H-4.The p SL-Pubi-H-4 vector was transformed into wild-type tomato M82 by Agrobacterium tumefaciens-mediated transformation,and 200 transgenic tomato seedlings with hygromycin resistance were obtained.Three tomato mutants of T0 generation with editing effect were obtained by amplifying Sl Lcy B1,Sl Lcy B2,Sl Cyc B and Sl Lcy E fragments and sequencing.The three T0 mutants were stably self-crossed to T2 generation.Combined with off-target analysis,the marker-free tomato mutants 119-6(Sl Lcy B2 knockout),10-22(Sl Cyc B knockout),10-1(Sl Lcy B2 and Sl Cyc B knockout),119-3(Sl Lcy B1 knockout),10-6(Sl Lcy B2 and Sl Cyc B knockout,Sl Lcy B1 double peaks)and 10-6-1(Sl Lcy B1,Sl Lcy B2 and Sl Cyc B knockout)without foreign genes were screened.2.Statistics of the germination rate of tomato seeds of each genotype showed that the germination rate was 119-3>119-6>10-1 ≈ 10-6>10-22>M82 after 3-5 days of sowing.Compared with the wild-type M82 plants,the tomato mutants are significantly dwarfed.The height of the main stem of the tomato at the seedling stage one month after sowing is counted,119-3,119-6,10-1,10-6 and 10-22 About 50%-60% of the wild type,of which10-6-1 is abnormally developed,the leaves are albino,and the main stem is about 30% of the wild type.The relative chlorophyll of the leaves is calculated,and each mutant has a decrease,10-6-1 Only a very small amount of chlorophyll can be detected in.Analyze the lycopene content in tomato pulp 10 days after the color of the tomato fruit is broken.Among them,the content of 119-3 is the highest,which is about 3 times that of the wild type;the content of 119-6 is the lowest,which is about 1.5 times that of the wild type,and that of other tomato mutants.The content of lycopene was significantly increased.3.On the basis of finding that the fruit of single knock Lcy B1 mutant(named LC)began to show obvious yellow in the pre-ripening stage,the premature tomato fruits of LC and M82 were selected for transcriptome sequencing,and 2655 differential genes were screened,of which 783 were down-regulated and 1872 were up-regulated.The two pathways most annotated by KEGG are metabolic pathways(47.52%)and biosynthesis of secondary metabolites(25.57%).The TOP3 of KEGG enrichment analysis is the biosynthesis of secondary metabolites,plant pathogen interaction and plant hormone signal transduction.The contents of carotenoids and ABA in fruits were determined by liquid chromatography-tandem mass spectrometry(LC-MS).The results showed that the contents of lutein,β-carotene and violaxanthin in LC were significantly lower than those in M82.γ-carotene and lycopene could not be detected in M82,but the contents of phytoene in LC were about 100 times higher than those in M82.Compared with the wild type,the proportion of lycopene and its upstream carotenoids in LC increased significantly in the total carotenoids.In addition,it was also found that the contents of ABA and ABA-GE in LC decreased significantly compared with M82,which were about 13% and 36% of those of wild type,respectively.4.Based on the above data,we selected carotenoid biosynthesis and plant hormone signal transduction in KEGG annotation for association analysis,and found that the expression of five genes such as Solyc04g040190.1 in carotenoid biosynthesis pathway was negatively correlated with the contents of lycopene,γ-carotene and phytoene in LC pre-ripening fruits.It was positively correlated with violaxanthin,neoxanthin,β-carotene,sea urchin ketene,lutein,lutein myristate acid ester and β-crypxanthin.The other nine genes are the opposite.In the plant hormone signal transduction pathway,80 differential genes are also related to the changes of carotenoid content.In this study,several genetically edited tomato materials with high lycopene yield were bred.The functions of Sl Lcy B1,Sl Lcy B2 and Sl Cyc B in regulating carotenoid biosynthesis metabolism and plant hormone signal transduction in tomato were analyzed at the transcriptome and metabolic level.It was found that Sl Lcy B1 is one of the main genes regulating tomato fruit color in the early stage,and many kinds of carotene have the potential to be used as markers of plant hormone signal transduction.To provide theoretical guidance and material support for the subsequent breeding of high lycopene and color tomato.