Capsicum Of Purple Stripe Gene CaPs In Pepper Fruit And Functional Analysis Of Candidate Genes

Pepper（Capsicum annuum）as the Solanaceae（Solanaceae Juss.）pepper（Capsicum）garden plants.It is the vegetable crop with the largest cultivated area in our country.However,the reported genes cannot fully explain all instances of purple stripes in pepper,so it is important to identify new genes or new functions that regulate the purple trait in pepper.In the present study,we used the pepper mutant line’Chen12-4’as a target to finely localize the genes for the purple stripe trait in pepper and verify their functions,with the aim of elucidating the molecular mechanism of pepper anthocyanin biosynthesis regulation.The main findings are as follows:1.Fine localization of Ca Ps locus for purple stripe trait gene.The purple striped strain’Chen12-4’was crossed with the normal green fruit strain’Z101-M’to construct a genetic mapping population,and the purple striped trait gene Ca Ps was finely localized by BSA mixed pool analysis and genetic linkage mapping.The results showed that Ca Ps was localized in the purple stripe trait.The results showed that Ca Ps were located in the 841.39 kb region on chromosome 10,and two genes,CA10g11690 and CA10g11700,were found in the region by using’C.annuum CM334’as the reference genome.CA10g11690 encodes a R2R3-MYB transcription factor involved in anthocyanin biosynthesis,and combined with q RT-PCR gene expression analysis,it was identified as the best candidate gene for Ca Ps.2.The full-length sequences,CDS regions and protein structural domains of the Ca Ps candidate genes are significantly different.Through comparing drawing group parents this CA10g11690 gene sequences,found in the’Chen12-4’second intron area has a 1926 bp insertion fragment,the eight SNPs loci detected between the two parents.In addition,there were 49 SNPs,2 deletions and 4 insertions in the promoter region.Amplification of the CDS region of the CA10g11690 gene in both parents revealed that CA10g11690 had alternative splicing and produced different transcripts.CA10g11690generated a c690^Long-Z101transcript in’Z101-M’and another c690^{Short-chen12-4}transcript in’Chen12-4’.The c690^{Short-chen12-4}transcript has a more complete R3 domain than the c690^Long-Z101transcript.3.VIGS silencing verified that gene CA10g11690 was involved in the formation of purple stripes on the exocarp of’Chen12-4’fruit by regulating the accumulation of anthocyanins in green fruits.Designed according to CA10g11690 gene sequence specific primers amplification fragment,VIGS vector is constructed,and in’Chen12-4’cotyledon flattened period using the back injection osmosis infect cotyledon,to cultivate the plant fruit 15 d observation record phenotype.After that,the exocarp of VIGS silenced fruit and normal fruit were collected for transcriptome analysis and quantitative q PCR detection of candidate genes.In the anthocyanin pathway,Ca CHS,Ca F3H,Ca F3’5’H,Ca DFR,Ca ANS,Ca UFGT,Ca3GT and Ca3RT were down-regulated,and the anthocyanin regulatory genes CA10g11690（Ca MYB）,Cab HLH and Ca WD40 were significantly down-regulated.The expression of Ca CHI was up-regulated.These results indicated that CA10g11690 transcription factor was involved in the formation of purple stripes in fruit exocarp of’Chen12-4’by regulating anthocyanin accumulation in fruit.4.Validation of the involvement of CA10g11690 in the synthesis and regulatory functions of tobacco anthocyanins.Based on the results of sequence analysis data,gene expression vectors p MV2-c690^Long-Z101and p MV2-c690^{Short-Chen12-4}driven by Ca MV35S promoter were constructed.Using the tobacco leaf disc method of transformation,transgenic plants were obtained from the T0 generation and selfed to obtain the T1generation.The exogenous gene expression was analyzed by phenotypic observation,molecular detection and q RT-PCR.The results showed that both transcript strains exhibited anthocyanin accumulation in the T0 generation of transgenic tobacco.PCR assay of CA10g11690 transcripts in transgenic tobacco revealed the presence of a 702bp amplification in Ca MV35S::c690^{Short-Chen12-4}strain and two amplifications of 702 bp and 1087 bp in Ca MV35S::c690^Long-Z101strain,respectively.t1 generation The T1generation of the transgenic tobacco strains produced a segregation of traits,with the transgenic tobacco strain Ca MV35S::c690^{Short-Chen12-4}segregating into dark purple and green strains and Ca MV35S::c690^Long-Z101segregating into light purple and green strains.c690^{Short-Chen12-4}and Ca MV35S::c690^Long-Z101,a 1087 bp amplification was detected in the transgenic tobacco purple strain Ca MV35S::c690^{Short-Chen12-4},and a 702 bp amplification was detected in the transgenic tobacco purple strain Ca MV35S:.c690^Long-Z101was detected at 702 bp and 1087 bp.Analysis of the expression patterns of anthocyanin synthesis pathway genes and MBW by quantitative real-time fluorescence q RT-PCR showed that overexpression of CA10g11690 gene in tobacco significantly upregulated the expression levels of EBGs,LBGs and MBW transcription factors,and their expressions were positively correlated with anthocyanin content.This study provides a new idea for exploring the biosynthetic mechanism of anthocyanins,and also provides a theoretical basis for the selection and breeding of purple striped pepper varieties in the future.